Identification of Ryanodine Receptor 1 (RyR1) Interacting Protein Partners Using Liquid Chromatography and Mass Spectrometry

by

Timothy Ryan

A thesis submitted in conformity with the requirements for the degree of Masters of Science

Department of Physiology University of Toronto

© Copyright by Timothy Ryan 2010

Identification of Ryanodine Receptor 1 (RyR1) Interacting Protein Partners Using Liquid Chromatography and Mass

Spectrometry

Timothy Ryan

Master of Science

Department of Physiology University of Toronto

2010

Abstract

Ryanodine receptor 1 (RyR1) is a homotetrameric calcium channel located in the

sarcoplasmic reticulum (SR) of skeletal muscle. We employed metal affinity

chromatography followed by liquid chromatography mass spectrometry from HEK-293

cells to purify affinity tagged cytosolic RyR1, with interacting proteins. In total, we

identified 703 proteins with high confidence (>99%). Of the putative RyR1 interacting

proteins, five candidates [calcium homeostasis endoplasmic reticulum protein (CHERP),

ER-golgi intermediate compartment 53kDa protein (LMAN1), T-complex protein (TCP),

phosphorylase b kinase (PHBK) and four and half LIM domains protein 1 (FHL1)], were

selected for interaction studies. Immunofluorescence analysis showed that CHERP co-

localizes with RyR1 in the SR of rat soleus muscle. Calcium transient assays in HEK293

cells over-expressing RyR1 with siRNA suppressed CHERP or FHL1, showed reduced

calcium release via RyR1. In conclusion, we have identified RyR1 interacting proteins in

CHERP and FHL1 which may represent novel regulatory mechanisms involved in

excitation-contraction coupling.

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Acknowledgments

Firstly, I would like to thank my supervisor, Dr. Anthony Gramolini. Since I

began my work as a Master’s student, Tony has shown me a great deal of support and

provided me with all of the resources necessary to succeed in the lab.

I would also like to thank my colleagues in the lab. Parveen, your mentorship and

patience from day one in the MacLennan lab, as well as your friendship, have made this

process infinitely better. Shaan (styll), Wen-Ping, Thiru, Melissa, and Vijay - thanks for

your endless support and all the laughs.

Most importantly, I need to thank my family as this process has had its ups and

downs. Dad, thanks for the objective advice and constant support. Mom, thanks for the

“secret” $20 bills, the soy milk, the cereal, the flattened chickens, the Delissio pizzas, and

most importantly, your intense love. Lyndsey, thank you for putting the rye bread in my

school bag, it was delicious. Finally, thank you Justin. You have been there (with a bottle

of scotch) for the good times, and absorbed the bad with a tolerance and support a kin to

that of a true best friend.

I would also like to extend my thanks to the Margaret J. Santalo and the Heart and

Stroke/Richard Lewar Scholarships for providing me with funding for the course of my

Masters degree.

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Table of Contents

Abstract.............................................................................................................................. ii Acknowledgments ............................................................................................................ iii List of Common Abbreviations...................................................................................... vii Table of Figures.............................................................................................................. viii List of Tables .................................................................................................................... ix Chapter 1: Introduction ................................................................................................... 1

General Overview ........................................................................................................... 1 I. Skeletal Muscle.................................................................................................... 2

A. Different muscle fiber types.............................................................................. 2 B. Myofibrils ......................................................................................................... 3 C. Sarcoplasmic reticulum and T-tubule system................................................... 4

II. Ryanodine receptors and muscular disorders ...................................................... 5 A. Role of ryanodine receptor in skeletal muscle in SR calcium release .............. 5 B. Structure of ryanodine receptor calcium channel ............................................. 6 C. Modulation of RyR activity .............................................................................. 8 D. Endogenous modulators.................................................................................... 9 E. Exogenous modulators.................................................................................... 10 F. Calcium homeostasis ...................................................................................... 11

III. Neuromuscular disorders ................................................................................... 12 A. General Introduction ....................................................................................... 12 B. Malignant hyperthermia.................................................................................. 14 C. Central core disease ........................................................................................ 14

IV. Protein-protein interactions................................................................................ 15 A. General Introduction ....................................................................................... 15 B. Nickel chelate affinity chromatography.......................................................... 16 C. Ryanodine receptor protein interacting partners............................................. 16

V. Proteomic identification of protein complexes .................................................. 19 A. Proteomic techniques ...................................................................................... 19

VI. Statement of Intent ............................................................................................. 20 Chapter Two: Materials and Methods.......................................................................... 21

I. Generation of RyR1 cDNA................................................................................ 21 A. Cytoplasmic RyR1 and N-terminally tagged RyR1 D9 fragment .................. 21 B. Chemical Transformation of DH5-α cells ...................................................... 21 C. Amplification and Maxi Preparations of DNA............................................... 21

II. Ni-NTA chromatography purification ............................................................... 22 A. Culturing HEK293 cells.................................................................................. 22 B. Transfection of Cells....................................................................................... 23 C. Harvesting HEK293 cells and preparing protein extract ................................ 23 D. Purification of RyR1 complexes using Ni-NTA resin.................................... 24

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III. Alternative strategies for purifying functional RyR1 proteins .......................... 26 A. Size exclusion chromatography ...................................................................... 26 B. ELISA Analysis of SEC fractions................................................................... 27 C. Complexiolyte buffer solubilization of FL-RyR1........................................... 27 D. Pull-down of mouse skeletal muscle proteins using bound D9 fragment....... 27

IV. Mass Spectrometry Analysis.............................................................................. 29 A. Protein sample preparation ............................................................................. 29 B. Solid-Phase Extraction of Tryptic Peptides .................................................... 30 C. MS and proteomic analysis of HEK293 cells over-expressing RyR1 ............ 30 D. MS analysis of mouse skeletal muscle lysate on bound D9 fragment ............ 31

V. SDS-PAGE and Immunoblot analysis ............................................................... 31 A. SDS-PAGE and immunoblot analyses of RyR1 expression........................... 31 B. Immunoblot analysis in HEK293 cells and C2C12 mouse myocytes ............ 32

VI. Subcellular localizations of RyR1 and interactions ........................................... 33 A. Plating and fixing slides of HEK-293 cells..................................................... 33 B. Slide Preparations of Fixed Isolated Skeletal Soleus Muscle Fibers.............. 33 C. Co-Immunofluorescent Staining of Fixed Tissue ........................................... 33

VII. Calcium transient analysis of RyR1 expressing HEK293 cells with candidate siRNA knockdowns ...................................................................................................... 34

A. Plating slides of HEK293 cells ....................................................................... 34 B. Calcium transient analysis .............................................................................. 34 C. Statistical analysis of data............................................................................... 35

Chapter Three: Results .................................................................................................. 36

I. Purification of RyR1 contained protein complexes ........................................... 36 A. Expression and solubilization of tagged RyR1 proteins ................................. 36 B. Purification of bait and potential interactors................................................... 37

II. Identification of RyR1 interacting protein partners ........................................... 37 A. Proteins identified from RyR1 purifications by mass spectrometry............... 37 B. Filtering and Comparing Subsets of Proteins Identified from Purifications... 38 C. Prioritization of Potential Interactors.............................................................. 39 D. Screening for Known RyR1 Interacting Proteins ........................................... 39 E. Selection of Potential Interactors .................................................................... 40

III. Alternative strategies for purifying ryanodine receptors ................................... 41 A. Size exclusion chromatography ...................................................................... 42 B. Complexiolyte buffer system.......................................................................... 43 C. Purification of N-terminal D9 fragment of RyR1........................................... 44

IV. Identification of D9 binding protein partners .................................................... 44 A. Proteins identified from RyR1 purifications by MS analysis ......................... 44 B. Filtering and Comparing Subsets of Proteins ................................................. 45 C. Prioritization of Potential Interactors.............................................................. 46 D. Selection of Potential Interactors .................................................................... 46

V. Validation of Interactions .................................................................................. 47 A. Immunoblot Analysis of RyR1 Potential Interactions .................................... 47 B. Subcellular Localization ................................................................................. 48

i. CHERP Co-localization with RyR1 in Rat Soleus Muscle .......................... 48

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ii. FHL1 Co-localization with RyR1 in Rat Soleus Muscle.......................... 49 VI. Functional analysis of interaction candidates .................................................... 50

A. Calcium transient analysis of HEK293 cells expressing RyR1 with suppression of interacting candidates – 340 nm acquisition..................................... 50 B. Calcium transient analysis of HEK293 cells expressing RyR1 with suppression of interacting candidates – 340/380 ratio acquisition ........................... 51

Chapter Four: Discussion............................................................................................... 74

I. Protein Complex Isolations................................................................................ 74 II. The Biochemistry-Mass Spectrometry Interface ............................................... 76

A. Post-purification identification of RyR1 peptides via LC-MS ....................... 76 B. Identification of non-specific binding proteins............................................... 77

III. Interactions with RyR1 ...................................................................................... 79 A. CHERP............................................................................................................ 79 B. FHL1 ............................................................................................................... 81

Chapter Five: Limitations.............................................................................................. 84 Chapter Six: Future Directions ..................................................................................... 86 Chapter Seven: References ............................................................................................ 88

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List of Common Abbreviations

ARDV2 arrhythmogenic right ventricular dysplesia ATP adenosine tri-phosphate BD brody disease CaM calmodulin CaMK calmodulin-dependent protein kinase II CCD central core disease CHERP calcium homeostasis endoplasmic reticulum protein CICR calcium induced calcium release CO-IP co-immunoprecipitation CPVT catecholaminergic polymorphic ventricular tachycardia DHPR dihydropyridine receptor DPYSL3 dihydropyrimidinase-like 3 protein ER endoplasmic reticulum ERGIC-53 endoplasmic reticulum golgi intermediate compartment protein 53 FHL1 four and a half LIM domains protein 1 FKBP fk506 binding protein FL-RyR full-length ryanodine receptor GFP green fluorescent protein HIS histidine LC-MS liquid chromatography mass spectrometry MH malignant hyperthermia Mrf4 myogenic regulatory factor 4 Myf5 myogenic factor 5 NCX Na+/Ca2+ exchanger Ni-NTA nickel nitrilotriacetic acid PHKB phosphorylase B kinase regulatory beta PKA protein kinase A PKC protein kinase C PKG cGMP-dependent protein kinase PMCA plasma membrane calcium ATPase RyR ryanodine receptor RyR1 ryanodine receptor type 1 SEC size exclusion chromatography SERCA1a sarco(endo)plasmic reticulum calcium ATPase 1a SR sarcoplasmic reticulum TAP tandem affinity purification TCP T-complex protein TM transmembrane T-tubule transverse tubule WT wild-type Y2H yeast two-hybrid

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Table of Figures Figure 1 – Structure of a Skeletal Muscle........................................................................... 4 Figure 2 - Predicted Transmembrane Topology of the RyR1............................................. 8 Figure 3 - Calcium Homeostasis in Skeletal Muscle Cells............................................... 12 Figure 4 – RyR1 MH and CCD Domains......................................................................... 13 Figure 5 – Ni-NTA purification of cytosolic RyR1 followed my LC-MS ....................... 25 Figure 6 - Pull-down of mouse skeletal muscle proteins using bound D9 fragment ........ 29 Figure 7 - Expression Analysis of Cytosolic RyR1 .......................................................... 53 Figure 8 - Analysis of Cyto-RyR1 Purification Products ................................................. 54 Figure 9 – Overview of the MS data filtering process and candidate acquisition ............ 56 Figure 10 - Proteins Identified by Mass Spectrometry from RyR1 Purifications............. 58 Figure 11 - Heat-map of Proteins Found in >1 MS Run and Proteins Whose Average Spectral Count in RyR1 Runs had a Four-Fold Increase over Controls ........................... 61 Figure 12 - ELISA and MS Analysis of SEC Fractions with FL-RyR1 Proteins............. 63 Figure 13 – Immunoblot and MS Analysis of Elutant Fractions of Complexiolyte Buffer Solubilized RyR1 .............................................................................................................. 64 Figure 14 – MS Identification of Mouse-D9-Cobalt Purification Products ..................... 65 Figure 15 - FHL1 Tandem Affinity Purification LC-MS Detection Data ........................ 67 Figure 16 - Preliminary Confirmation of RyR1 interacting candidates in HEK293 cells 68 Figure 17 - Subcellular Co-localization of CHERP and RYR1 in Skeletal Muscle......... 69 Figure 18 – Subcellular Co-localization of FHL1 and RYR1 in Skeletal Muscle............ 70 Figure 19 – 340/380 Ratio Calcium Transients of RyR1 Expressing HEK293 Cells ...... 72 Figure 20 – Percentage Increase in 340/380 nm Ratio of RyR1 Expressing HEK293 Cells........................................................................................................................................... 73

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List of Tables Table 1 - 33 Potential RyR1 Interactors Found in >1 MS Run ........................................ 59 Table 2 – Enrichment of Proteins in Cyto-RyR1 Purifications in Transfected HEK293 Cells .................................................................................................................................. 60 Table 3 - Final List of Candidate Proteins Identified in Cyto-RyR1 Purifications in Transfected HEK293 Cells ............................................................................................... 62 Table 4 - Enrichment of Proteins in D9-RyR1 Purifications Incubated with Mouse Skeletal Muscle Lysate ..................................................................................................... 66

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Chapter 1: Introduction

General Overview

Skeletal muscle is a highly organized and sophisticated striated tissue responsible

for postural maintenance, movement and heat production. In humans, muscles account

for over 40% of total body mass and can be subdivided into four major muscle types,

namely skeletal, cardiac, smooth and myoepithelial. Skeletal, cardiac and smooth develop

during embryogenesis as they are derived from the mesoderm while myoepithelial are

similar to epithelium and ectodermic in origin.

In striated muscle, contraction is governed by the process of excitation-

contraction coupling, broadly defined as the linking of action potential to contraction. It is

the process that couples surface membrane depolarization to Ca2+ release from the

sarcoplasmic reticulum (Kim et al. 1983). Excitation-contraction coupling depends on a

large macromolecular protein complex or 'calcium release unit', which spans the

transverse tubule (T-tubule) surface membrane into the cytoplasm where it continues

across the SR membrane and into the lumen of the SR. The central element of this protein

complex is the Ca2+ release channel, ryanodine receptor (RyR), located in the SR

membrane.

In mammalian tissues, there are three isoforms of RyR: RyR1 is expressed

predominately in skeletal muscle, RyR2 is expressed primarily in cardiac muscle, RyR3,

is expressed more widely, with greatest levels seen in the brain. Mutations have been

found in RyR1 and RyR2 genes resulting in a number of neuromuscular disorders.

The mechanisms, by which RyR1 regulates the movement of Ca2+ ions across the

SR membrane, thus regulating muscle contraction and relaxation, are still widely

unknown. To date, there has been no large scale protein-protein interaction analysis of

RyR1. Here, we hypothesized that RyR1 is regulated via a number of protein interacting

partners and that defects in these proteins lead to genetic diseases in skeletal muscle. It is

known that RyR1 is activated by physical interaction with DHPR and there are a host of

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other proteins that interact, and may regulate RyR1 such as FK506 binding protein,

triadin, junction, calumenin, calmodulin and protein kinases. Using nickel

chromatography purification of histidine affinity tagged RyR constructs followed by

mass spectrometry analysis; we pursued the goal of elucidating protein binding partners

to provide insight into the function and regulation of RyR1 at a molecular level.

I. Skeletal Muscle

A. Different muscle fiber types

Histological studies in skeletal muscle have revealed the existence of two major

types of muscle fibers: type I and type II fibers. Type 1 fibers possess slow myosin

ATPase activity and are also known as slow-twitch fibers. They contain large amounts of

myoglobin which confer them their red colour, many mitochondria and many blood

capillaries (Ruegg et al. 1992). These fibers are very resistant to fatigue and have a high

capacity to generate ATP by oxidative metabolic processes. Type II fibers have a fast

contraction velocity, and in particular type IIB has a low content of myoglobin, few

mitochondria and blood capillaries. They are white muscles and mainly use anaerobic

metabolic processes to generate ATP which produces lactic acid. Even if they possess

large amounts of glycogen, because of rapid glycogen consumption and subsequent lactic

acid accumulation, type IIB fibers are predisposed to fatigue. They are mainly used

during short exercises requiring a lot of force. Type IIA, also called fast oxidative fibers,

have an intermediate biochemical and functional pattern between type I and IIB. The

relative proportion of fiber types differs from one muscle to another and underlines each

muscle’s identity; in addition, specific properties are controlled by stimulation of motor

neurons. The recruitment of fibers depends on the duration and intensity of the effort. For

instance, the thigh muscles of marathon runners develop 80% type I and 20% type II

muscle fibers because of the prolonged exercise required, whereas the thigh muscles of

sprinters have the inverse ratio because they require very short bursts of intensive

exercise (Ruegg et al. 1992).

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B. Myofibrils

Myofibrils are parallel cylinders lengthened in the direction of the cell, made up

of regular arrays of identical cylinders called sarcomeres which are considered the

fundamental contractile element of the skeletal muscle. Each sarcomere is composed of

thick (myosin-containing) and thin (actin-containing) myofilaments assembled in parallel

along their axis. This particular arrangement gives muscles the cross-striated pattern

observed by light and electron microscopy and hence striated muscles their name. Under

a light microscope, using polarized light, skeletal muscle shows alternating light

(anisotropic) and dark (isotropic) bands whose colour depends on the refractive index.

The dark bands, known as A-bands, are formed by polymers of myosin. The center of

each A-band is crossed by a pale area, the H-band, where creatin kinase is the main

component. H-bands are bisected by a thin M line, the anchoring point for thick filaments

and myosin-binding proteins. The light bands known as I-bands contain mainly actin. At

the center of each I-band, is a thin dark line, the Z line. The region of myofilamentous

structure between two successive Z-lines defines the sarcomere and is 2.5 μM in length.

Myosin is a protein of 200kDa that produces the contractile force. Each myosin is

composed of two globular heads and a tail domain. The heads are the sites of myosin

ATPase and actin binding. The thin filaments are composed of several proteins: actin,

troponin and tropomyosin. Actin is a 42kDa globular protein (G-actin) which can

polymerize into a filamentous polymer known as F-actin. Tropomyosin and troponin are

regulatory proteins; tropomyosin covers the myosin binding site on actin in the absence

of Ca2+ and troponin itself binds Ca2+ and regulates the structure of tropomyosin

(Jagatheesan et al. 2010).

These primary components (myosin, actin, tropomyosin and troponin) represent

75% of the proteins present in myofibers. The remaining proteins (nebulin, titin, α-

actinin, dystrophin and others) form the cytoskeletal network and are necessary for the

regulation, spacing, and precise architecture of myofilaments. The global organization of

the striated structure is probably stabilized by a large protein called titin. Titin is an

elastic filament and the biggest single protein (almost 3000kDa) found in nature. It

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connects the Z-line to the M-line in the sarcomere and provides binding sites for several

proteins (Granzier et al. 2007).

Thick (myosin) filament

Thin (actin) filament Z-disc Z-disc

M-line

I-band A-band I-band

H-zone

Elastic (titin) filament

Figure 1 – Ultrastructure of a myofilament Arrangement of a myofilament, within the contractile unit of skeletal muscle, the sarcomere.

C. Sarcoplasmic reticulum and T-tubule system

The SR is a sub-specialized form of the smooth endoplasmic reticulum and is

exclusively found in skeletal and cardiac muscles. It forms an intracellular membrane

network specialized in the sequestration and release of calcium. SR surrounds and runs

parallel to each individual myofibril. It widens at its ends forming terminal sacs, called

terminal cisternae transversally orientated with respect to the long fiber axis. The

transverse-tubules (T-tubules) are deep invaginations of the sarcolemma, permitting

membrane depolarization to quickly propagate to the interior of the fiber. Their number

differs among muscles and species.

Muscle fibers respond to the electrical signal (called an action potential) by

changing the concentration of calcium ions. T-tubules carry the action potential which is

the signal underlying the Ca2+ release from the SR. The combination of one transverse

tubule and two adjacent terminal cisternae defines a triad, the anatomical site for

excitation-contraction coupling. When a junction is only composed by one cisternae and

one tubule, it is called a dyad (Franzini-Armstrong et al. 1991; Delbono et al. 1996). In

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skeletal muscle, triads are located at the A-I junctions and in mammals there are two

triads per sarcomere. A particular portion of the sarcoplasmic reticulum facing the T-

tubule system, called the junctional face membrane assumes an important role in the

contraction mechanism, since it contains important proteins such as ryanodine receptor,

dihydropyridine receptor, calsequestrin, triadin, junctin (Costello et al. 1986).

II. Ryanodine receptors and muscular disorders

A. Role of ryanodine receptor in skeletal muscle in SR

calcium release

Excitation-contraction coupling is broadly defined as the process linking the

action potential to contraction in striated muscle. More specifically, it is the process that

couples surface membrane depolarization to Ca2+ release from the SR. Excitation-

contraction coupling requires a large macromolecular protein complex or 'calcium release

unit', which spans the transverse tubule (T-tubule) surface membrane into the cytoplasm

where it continues across the SR membrane and into the lumen of the SR (Du et al.

2002). The central element of this protein complex is the Ca2+ release channel, ryanodine

receptor (RyR), located in the SR membrane.

In skeletal muscle, contraction is initiated by the activation of RyR1 in the

junctional terminal cisternae of the SR. Activation of RyR1 occurs through a physical

interaction with the surface membrane L-type calcium channel, dihydropyridine receptor

(DHPR), located in the T-tubular membrane where it is directly opposed to RyR1 serving

as a ‘voltage sensor’ to detect action potentials (Fleischer et al. 1989; Franzini-Armstrong

et al. 1997). DHPRs form groups of four, or "tetrads," and tetrads are located in exact

correspondence to the four subunits of RyRs (Franzini-Armstrong et al. 1983; Takekura

et al. 1994; Protasi et al. 1997). This arrangement suggests an e-c coupling mechanism of

the type postulated by Schneider and Chandler (Schneider et al. 1973), in which voltage

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sensing by the DHPRs results in opening of RyRs by some direct molecular interaction

between these two components of the junction.

When muscles are relaxed, the myoplasmic Ca2+ levels are low and the

tropomyosin in the thin filaments is disposed in such a way that the myosin binding site

of actin is obstructed. Upon muscle stimulation, the myoplasmic Ca2+ levels increase, the

released Ca2+ binds to troponin inducing a conformational change that removes

tropomyosin from the thin filament. Myosin can access its binding site on actin and the

cross-bridges can form. Myosin hydrolyzes ATP to ADP and inorganic phosphate. As a

consequence, myosin in high-energy state changes its conformation and myosin heads

bind to and rotate against actin filaments resulting in shortening of the fibers: Contraction

occurs. As Ca2+ levels lower during muscle relaxation, Ca2+ detach from the troponin

complex, which in turn blocks tropomyosin and the myofilaments slide back to the

resting configuration: Contraction stops.

B. Structure of ryanodine receptor calcium channel

In parallel to IP3Rs, ryanodine receptors define a second important family of

intracellular Ca2+ release channels. Three mammalian isoforms have been identified and

named according to where they were first identified: RyR1 also called skeletal type

(Takeshima et al. 1989; Zorzato et al. 1990), RyR2 the dominant form in cardiac muscle

(Nakai et al. 1990) and RyR3 which is expressed in many tissues but originally purified

from the brain (Hakamata et al. 1992). However, the actual tissue repartition is not as

simple as suggested in this nomenclature; for instance RyR1 are also expressed in some

immune cells as will be detailed below and RyR2 is also present in the cerebellum. RyRs

are encoded by 3 different genes located on human chromosomes 19(RYR1), 1 (RYR2)

and 15 (RYR3) (Otsu et al. 1990; Sorrentino et al. 1993). Amino acid sequence

comparison has revealed that the three isoforms share an overall homology of 66%.

RyR1, 2, and 3 types have been predicted to be structurally similar. They were

first purified from skeletal muscle, heart and neuronal tissues with an apparent

sedimentation coefficient of 30S. RyRs are large homotetrameric proteins made up of

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four subunits (560kDa), each composed of about 5000 amino acids; each subunit can

bind one molecule of the 12kDa protein FKBP12. Accessory proteins, including as CaM,

calcineurin, S100 (Meissner 1994; MacKrill 1999), have been shown to form a complex

with RyRs giving rise to a huge macromolecular complex with a total molecular mass

greater than 2 million Da. The first 4000 amino acids are hydrophilic and are thought to

form a large “foot-like” structure while the last 1000 are hydrophobic and contain the

pore-forming domain.

The fine topology of the C-terminal region has not yet been fully elucidated.

Depending on the model, the exact number of transmembrane (TM) domains range

between 4 and 12. Primary sequence and hydropathy plot analysis by Takeshima et al.

(Takeshima et al. 1989) suggest an arrangement of four transmembrane spanning α-

helices and a final tail facing the SR lumen. In a second model, Zorzato et al., proposed

10 transmembrane domains. In 2002, Du et al. expressed RyR1 proteins containing

complete or progressively deleted C-terminal sequences fused in frame with enhanced

GFP in HEK293 cell lines (figure 2). After saponin-permeabilization of the cells, the

subcellular localization of the fusion proteins was observed by confocal microscopy.

Their results predict the presence of eight transmembrane helices and two domains not

membrane-associated. The fragment that connects M8 and M10 is predicted to constitute

the pore-forming region. However, determination of the exact number TM segments will

require further investigations at higher resolution.

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N

C 1

Figure 2 - Predicted Transmembrane Topology of the RyR1 This drawing illustrates the proposed RyR1 transmembrane organization according to the model of Du (rabbit skeletal muscle). It contains eight transmembrane helices organized as four hairpin loops.

C. Modulation of RyR activity

Since ryanodine receptors participate in intracellular calcium regulation, they are

involved in many physiological processes. RyRs conduct monovalent and divalent

cations and can interact with many other molecules. In vivo, RyRs are modulated by the

T-tubule potential, as well as by a number of endogenous modulators and diverse

proteins. As a result of multiple ligand interactions, RyRs also constitute an interesting

target for pharmacological investigations. In vitro, regulation of RyR function has been

mainly investigated by three complementary methods. First, Ca2+ uptake and release on

actively or passively loaded SR vesicles using rapid mixing and filtration techniques.

Second, single channel recording performed on isolated SR vesicles or purified RyRs

incorporated into planar lipid bilayers. Third, [3H]-ryanodine binding experiments to

evaluate the functional state of the channel.

This section focuses on compounds affecting the RyR calcium function, activators

and inhibitors. This overview distinguishes between endogenous and exogenous effectors

(Sutko et al. 1997; Zucchi et al. 1997). Protein-protein interactions will be covered later.

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D. Endogenous modulators

Calcium: In the absence of others regulators, calcium efflux studies have revealed a bell-

shaped activation curve of Ca2+ release dependent on extracellular Ca2+ concentration

(Kirino et al. 1983; Nagasaki et al. 1983). Such a relationship results from RyR activation

at low (1-10μM) Ca2+ levels and RyR inactivation at high (500μM to 10mM for RyR1)

Ca2+ levels; interestingly, RyR2 shows a small inactivation at high [Ca2+] (over 100mM).

This biphasic Ca2+-dependent behaviour of RyR1 suggests the existence of at least two

different Ca2+ binding sites: a high-affinity specific site, which stimulates Ca2+ release

and a low-affinity less selective site, which inhibits Ca2+ release. Putative Ca2+ activation

sites have been identified in the cytoplasmic C-terminal domain of the RyR (Bhat et al.

1997).

An additional calcium binding site was found on a luminal loop of the channel

suggesting that luminal Ca2+ may modulate Ca2+ release (Ching et al. 2000). Little is

known about the inactivation site(s). In the skeletal muscle, Ca2+ is not required to initiate

contraction, although it may contribute to activity. On the other hand, Ca2+ is necessary to

induce cardiac contraction, where the phenomenon of calcium induced calcium release

(CICR) plays the major role.

Mg2+ ions have an inhibitory effect on calcium release (Kim et al. 1983; Pessah et al.

1987; Lamb et al. 1993). The three RyR isoforms do not show the same sensitivity to

Mg2+ inhibition, skeletal muscle RyR being more sensitive than cardiac or brain isoforms.

Therefore, it has been suggested that the Mg2+ binding site is localized in different

regions of the protein. Various mechanisms could explain this inhibition, maybe Mg2+

compete with Ca2+ for the Ca2+ activator site or it binds to the low-affinity Ca2+ binding

site. Alternatively, Mg2+ may physically obstruct the conduction pathway.

Adenine nucleotides, including ATP and ADP, are RyR activators (Pessah et al. 1987;

Galione et al. 2000). The action of ATP is isoform specific: the skeletal muscle channel

activity is strongly activated whereas in cardiac muscle, ATP enhances the activation by

Ca2+; RyR3 also appears to be less sensitive to ATP. The putative nucleotide ATP-

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binding domain GXGXXG is repeated two to four times in the primary sequence of RyR

with variations between isoforms. Since ATP activates CICR without modifying Ca2+

dependence, ATP may act on the kinetics rather than on the modulation of the Ca2+

activation site by facilitating the gating activity or by increasing the open probability of

the channel. Most of the cytosolic ATP is bound to Mg2+, thus MgATP is the major

effective activator, but it is still unclear if the MgATP complex form is as potent as free

ATP (Murayama et al. 2000).

E. Exogenous modulators

Ryanodine: In 1948, the plant alkaloid ryanodine was purified, from Ryania speciosa.

Ryanodine specifically binds to RyRs and gives the receptor its name. Ryanodine has two

opposite effects on Ca2+ release: at submicromolar concentrations, ryanodine increases

the channel’s activity whereas at high micromolar concentrations, it decreases the SR

Ca2+ permeability. Consequently, ryanodine has been proposed to bind at multiple (high-

and low-affinity) sites on the ryanodine receptor; the number and location of the sites

however are still unknown. The high-affinity site may be located on the carboxy-terminal

domain of the channel. Since RyR monomers are not able to bind ryanodine, the

tetrameric structure is necessary for ligand binding. Binding of ryanodine favours the

open RyR conformation and modifies the conductance properties of the channel (Fryer et

al. 1989; Fill et al. 2002).

Caffeine, a methylxanthine, supports Ca2+ release and CICR at millimolar concentrations

(Pessah et al. 1987). Caffeine appears to increase the sensitivity of the Ca2+ activator site

for Ca2+ and even to reverse Mg2+ inhibition. RyR2 is more sensitive to caffeine than

RyR1. Caffeine and adenosine nucleotides seem to have an additive effect and to function

in synergy, suggesting that their respective binding sites are in close proximity or even

overlap with one another.

Volatile anaesthetics: In skeletal muscle, halothane increases Ca2+ efflux from the RyR

by increasing the open probability of the channel at 0.002-3.8% gas concentrations (Kim

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et al. 1984). The response of the channel to halothane stimulation is pH and Ca2+-

dependent but adenosine nucleotide-independent. Effects similar to those of halothane

have been observed with isoflurane and enflurane (2.5 to 4%).

F. Calcium homeostasis

Calcium is the most abundant metallic element present in the human body (2% of

total body mass, e.g. bone are deposits of calcium phosphate. Calcium ion is also a

universal second-messenger playing a crucial role in many biological processes including

regulation of gene expression, cell proliferation, metabolism, secretion, neuronal

excitability, muscle contraction, apoptosis (Clapham 1995; Berridge et al. 1998). The

signal generated by calcium inside a cell encodes spatial, amplitude and frequency

information which can be decoded by cellular proteins yielding information required to

regulate physiological cellular events. In skeletal muscle calcium mobilization promotes

contraction (Ebashi et al. 1968) and muscle cells have developed a specialized system to

handle repetitive Ca2+ release and re-uptake events in a finely regulated way. Under

resting conditions, muscle cells have a low free myoplasmic [Ca2+] of about 100nM

which can increase to the micromolar range during tetanic stimulation. On the other hand,

[Ca2+] in the extracellular space is around 1.3mM and around 0.1-2mM in the lumen of

the SR. A Ca2+ gradient thus exists between extracellular space and free myoplasm;

numerous organelles and proteins are involved in maintaining this gradient. Three distinct

but complementary mechanisms are involved in the regulation of intracellular [Ca2+]: (i)

net flux across the plasma membrane; (ii) release and re-uptake in intracellular

compartments; (iii) binding to cytoplasmic proteins.

Figure 3 illustrates the major ion channels responsible for calcium homeostasis.

Calcium mobilization across the plasma membrane is maintained by the Na+/Ca2+

exchanger, DHPR (or IP3R) and the plasma membrane calcium ATPase (PMCA), while

the mitochondria exchanges calcium ions across its outer mitochondria membrane.

12

Figure 3 - Calcium Homeostasis in Skeletal Muscle Cells The schematic drawing represents the major proteins involved in the Ca2+ regulation which are located either in the plasma membrane, in the cytoplasm or in intracellular organelles.

III. Neuromuscular disorders

A. General Introduction

In muscle, calcium homeostasis results from a fine orchestration of the excitation-

contraction coupling by specialized organelles and devoted proteins. Given the

importance of communication and spatial organization between calcium regulatory

partners, it is not surprising that abnormalities in their operation may lead to diseases.

Thus, defects in genes encoding key proteins of the Ca2+ signaling machinery have been

found to cause several pathologies (MacLennan 2000). Brody disease (BD) was the first

13

described disorder of skeletal muscle and is associated with a dysfunction in SERCA1a

activity (Brody 1969; Odermatt et al. 1996). Subsequent studies revealed that other

proteins of the SR involved in the Ca2+ release can lead to neuromuscular diseases upon

activation. For example, RyR2 mutations are causal features for catecholaminergic

polymorphic ventricular tachycardia (CPVT), congestive heart failure and a form of

arrhythmogenic right ventricular dysplesia (ARDV2) (Tiso et al. 2001; Marks et al. 2002;

Wehrens et al. 2003). RyR1 mutations are the underlying features of some cases of

Malignant Hyperthermia, Central Core Disease, Multi-minicore disease and Nemaline

myopathy. The mutations leading to MH and CCD lie in three distinct regions of the

RyR1 gene, as illustrated in figure 4.

MH/CCD Domain

Figure 4 – RyR1 MH and CCD Domains The RyR1 gene is represented above (grey) with MH/CCD domains (red), DHPR (beige) and FKBP (green) binding sites and the transmembrane region (pink). Mutations in the RYR1 gene associated with Malignant Hyperthermia and Central Core Disease are located in three domains (red), marked as I, II and III above.

14

B. Malignant hyperthermia

The first description of clinical manifestations of malignant hyperthermia (MH)

dates to the beginning of the 20th century. In 1960, Denborough and colleagues reported

a young man with a tibia fracture, who was afraid of receiving general anesthesia since

10 of his relatives had died during general anesthesia. In 1962, autosomal dominant

predisposition to this disorder was first suggested. It is now known that MH (OMIM

#145600) is a potentially fatal pharmacogenetic disorder which manifests itself in

genetically predisposed individuals when they are exposed to trigger agents such as the

commonly used volatile anesthetics (halothane, enflurane, isoflurane, desflurane,

sevoflurane) or depolarizing neuromuscular relaxants (e.g. succinylcholine). In their daily

life, such individuals are apparently normal and free of symptoms. The triggering agents

cause an uncompensated elevation in [Ca2+]i which produces a chain of events related to

hypermetabolism and elevated muscle activity. If not rapidly treated, an MH reaction has

a mortality rate of 80%.

C. Central core disease

Central core disease (CCD) was the first congenital muscle disorder described

involving structural changes of the muscle fibers (Magee et al. 1956; Greenfield et al.

1958). It is a rare congenital myopathy usually identified by an autosomal dominant

mode of inheritance, though several recessive forms have recently been confirmed

(Jungbluth et al. 2002; Romero et al. 2003). Affected individuals often present with

infantile hypotonia (floppy infant syndrome) and a delay in achieving motor milestones.

Later in life, the predominant symptom is a generalized hypotonia and proximal muscle

weakness. Additional variable clinical features include congenital hip dislocation, pes

cavus, kyphoscoliosis, foot deformities and joint contractures (Shuaib et al. 1987). Both

clinical and histological variability is observed, but the clinical course is usually slow or

non-progressive in adults. Although symptoms may be severe, up to 40% of patients

demonstrating central cores may show normal clinical performance. Some patients with

CCD are also susceptible to episodes MH. Diagnosis is made through histochemical

identification of amorphous central areas (cores) that can extend almost along the full

15

length of muscle fibers; predominantly, type I fibers are affected. The core regions can be

central, peripheral or eccentric, are circumscribed, and lack mitochondria and oxidative

enzymatic activity. Electron microscopic analysis of core regions reveals disintegration of

the contractile machinery and alterations in the structure and content of SR and T-tubule

membranes (Isaacs et al. 1975; Hayashi et al. 1989).

IV. Protein-protein interactions

A. General Introduction

Within the cell, proteins contribute to the control and execution of cellular

activities. One important level of functional organization of the corresponding complex

proteome is the establishment of multimeric protein complexes (Bauer et al. 2003).

Furthermore, protein interactions and the formation of complexes are governed in a time-

and space-dependent manner (Bauer et al. 2003). Within a complex, each component

contributes to the overall function, which can further be regulated by neighboring

proteins and complexes (Bauer et al. 2003). Studying protein interactions thus provides

an opportunity to gain insight into the functional roles of poorly characterized proteins.

Since proteins involved in the same cellular processes often interact, functional

associations can be predicted by elucidation of interactions with better annotated proteins,

in addition to characterizing the subcellular distribution patterns (Bauer et al. 2003; Zhu

et al. 2003; Guan et al. 2008).

Several different techniques are available for the discovery of novel protein

interactions, such as the widely accepted Y2H screening method. Another screening

method developed more recently is the protein microarray. These two protein interaction

methods have limitations however, when dealing with a protein as large as RyR1. In the

case of the Y2H screening method, one would require the binding region of the potential

interactor within RyR1 to allow reconstitution of the reporter gene. A protein microarray,

also has size limitations in which one can effectively identify the interactor to a protein of

16

interest. In contrast to these, affinity purification coupled to MS analysis offers an

alternative method whereby protein complexes can be purified from cells and identified

(Zhu et al. 2003; Guan et al. 2008).

B. Nickel chelate affinity chromatography

Originally developed in yeast, metal chelate affinity chromatography was

developed after the prior over-expression of recombinant proteins as a high-purity,

single-step purification method (Porath et al. 1975). During the development of the

method, a nitrilo-tri-acetic acid (Ni-NTA) resin was introduced (Hochuli et al. 1987). In

this resin, the metal ion is held by four chelating sites, resulting in a stronger binding to

the matrix compared to the former matrices containing only three sites. The Ni-NTA

purification method takes advantage of the high affinity of histidine residue for the nickel

ions and depends on the expression of a recombinant protein of interest with a 6x-

histidine affinity tag. The general procedure consists of affinity capturing of a desired

protein in combination with its interacting partners. Nonspecific proteins are removed

with multiple gentle washes and the remaining complexes are eluted using acidic

conditions or imidazole, both of which bind with higher affinity to the Ni-NTA resin at

appropriate concentrations. To identify the co-purified proteins, the method is often

coupled to MS analysis (Zhu et al. 2003; Giannone et al. 2007).

C. Ryanodine receptor protein interacting partners

The macromolecular composition and the ultrastructure of proteins assembling at

the triad junctions is a challenging topic for structural biologists as RyR is both an

integral membrane protein and very large (figure 6). The elucidation of the different

protein-protein interactions is a key determinant in understanding the molecular

mechanism underlying EC but this has not been accomplished yet. Nevertheless, the

known protein-protein interactions between the ryanodine receptors and other

polypeptides within the triad are presented in this section (Franzini-Armstrong et al.

1997).

17

Dihydropyridine receptor: As described before, the DHPR is a complex of 5 subunits

where the alpha1 subunit of the voltage sensor is the pore-forming region of the channel.

Eight α1s isoforms encoded by different genes have been identified. In skeletal muscle,

the coupling between DHPR and RyR is bidirectional: an orthograde signal from the

DHPR to the RyR occurs when depolarization of the membrane triggers a conformational

change of α1sDHPR and this is translated into the opening of RyR and generation of

retrograde (RyR1 specific) signal enhancing the Ca2+ channel activity of DHPR. The loop

between the second and third repeats of the α1sDHPR (particularly residues 720-765) is

required for direct physical interaction between the DHPR and the RyR1 hydrophilic

domain (Beam et al. 1989; Proenza et al. 2002).

Calmodulin has a bimodal action on channel activity depending on its association with

Ca2+ (Samso et al. 1998). The Ca2+-bound form (CaCaM) inhibits all three RyR isoforms

in the absence of ATP, whereas at low free Ca2+ concentrations (<1μM), ApoCaM

activates RyR1 and RyR3 (but not RyR2). Sequence analysis and peptide binding studies

have revealed the presence of three candidate CaM binding sites in the C-terminal part of

the RyR1 in the skeletal muscle (Zorzato et al. 1990) and RyR2 (Otsu et al. 1990).

FK506-Binding Proteins are 12-kDa immunophilins (and the cytosolic receptors for the

immunosuppressant drug FK506 also known as tacrolimus). One FKBP12 binds to each

RyR monomer. FKBPs stabilize the homotetrameric structure and facilitate the

coordination of channel opening to full conductance (Brillantes et al. 1994). FKBPs may

also synchronize the channel activity of adjacent RyRs. Binding sites for FKBPs

(FKBP12 for RyR1 and RyR3 and FKBP12.6 for RyR2) have been localized by

cryoelectron microscopy and this approach has yielded a model of four symmetrically

related binding sites about 10 nm apart from the cytoplasmic assembly of the skeletal

RyR (Wagenknecht et al. 1996).

Calsequestrin, triadin and junctin: Calsequestrin is the major Ca2+ storage protein in the

SR of all striated muscles. Calsequestrin has a large number (about 40) of acidic amino

acids that permit it to coordinately bind 40-50 Ca2+ ions per protein molecule. It has been

suggested that Ca2+- and pH-dependent conformational changes in calsequestrin may

18

modulate RyR channel activity. Furthermore, some reports indicate the action of

calsequestrin on the RyR channel depends on the presence of triadin and junctin, the

proteins which provide an anchoring point. Triadin (94kDa) is an abundant membrane-

bound protein of the junctional sarcoplasmic reticulum, where it co-localizes with the

RyR1. Triadin contains a single transmembrane domain that separates this protein into

cytoplasmic and luminal segments. Only 47 amino acids of triadin are cytoplasmic, with

the bulk of this protein including the carboxyl terminus are located in the lumen of the

sarcoplasmic reticulum. The luminal domain of triadin contains a high concentration of

positively charged amino acids. Triadin binds both DHPR and RyR and is thought to

stabilize the complex responsible for EC coupling. In addition, recent studies have

revealed that triadin also interact with calsequestrin, and in this way would help to

maintain Ca2+ close to its release site. Junctin was first identified in cardiac muscle as a

26kDa calsequestrin-binding protein. In skeletal muscle, junctin is abundantly localized

in the junctional face membrane. Junctin is thought to have a structural role, similar to

that of triadin and anchors calsequestrin to the junctional face membrane (Zhang et al.

1997).

Protein kinases and phosphatases which modulate RyRs include cAMP-dependent

protein kinase A (PKA), cGMP-dependent protein kinase (PKG), protein kinase C

(PKC), and calmodulin-dependent protein kinase II (CaMK). According to the sequence

analysis, serine and threonine residues have been identified as possible phosphorylation

sites on the RyR1. Phosphorylation of Ser2809 by CaMKII is thought to be involved in

RyR2 activation (Witcher et al. 1991). A homologous serine residue (Ser 2848) in RyR1

has been reported to be phosphorylated by PKA, PKG and CaMKII (Suko et al. 1993).

Other proteins interact with the RyR macromolecular complex (Costello et al. 1986).

These include junctate (Treves et al. 2000), JP-45 (Zorzato et al. 2000), junctophilin

(Takeshima et al. 2000), 90kDa JFP (Froemming et al. 1999), sorcin (Meyers et al. 1995),

S-100 (Treves et al. 1997), homer (Feng et al. 2002), mistugumin-29 (Takeshima et al.

1998). Several less abundant proteins with a molecular weight ranging from 20 to

120kDa have been found in the junctional face membrane but have not been

characterized at the molecular level. A global characterization of the junctional proteins,

19

including the identification of novel proteins, is required to clarify the fine details of

RyR1 regulation and the subsequent excitation-contraction mechanism.

V. Proteomic identification of protein complexes

A. Proteomic techniques

There are several techniques available to carry out proteomics-based studies

including, for example, 2D gel electrophoresis, Reverse Phase Arrays, and Forward Phase

Arrays. Each technique has various advantages, along with inherent limitations. Here we

used Liquid Chromatography coupled to Mass Spectrometry (LC-MS) as our method of

investigation. We employed LC-MS because of its availability and our labs expertise with

this methodology (Kislinger et al. 2001; Washburn et al. 2001; Gramolini et al. 2008). In

general terms, complex protein samples are subjected to trypsin digestion to obtain

soluble peptides. These peptides are purified, and then separated on the basis of

hydrophobicity through liquid chromatography. Peptides are analyzed through mass

spectrometry and eventually mapped to proteins. This entire procedure has been

collectively termed MultiDimensional Protein Identification Technology (MuDPIT).

20

VI. Statement of Intent

It has been demonstrated that protein binding partners, as well as calcium and

other intercellular ions, which regulate RyR1, act on the large cytosolic domain of RyR1.

However, the mechanisms, by which RyR1 regulates the movement of Ca2+ ions across

the SR membrane, thus regulating muscle contraction and relaxation, are still widely

unknown. To date, there has been no large scale protein-protein interaction analysis of

RyR1. We hypothesize that RyR1 is regulated via a number of protein interacting

partners and that defects in these proteins lead to genetic diseases in skeletal muscle. The

goal of elucidating these protein binding partners and their sites of interaction will

provide insight into the function and regulation of RyR1 at a molecular level.

Objectives

1. Express and purify cytosolic RyR1 molecules to identify RyR1 interacting protein

partners.

2. Express and purify N-terminal RyR1 fragments produced in bacteria to identify

interacting partners in mouse skeletal muscle.

3. Verify interactors using immunoblot analysis and confocal microscopy.

4. Test potential interactors for functional effect on RyR1 activity by measuring

calcium release from the ER via RyR1 after suppression of potential interactor

expression.

21

Chapter Two: Materials and Methods

I. Generation of RyR1 cDNA

A. Cytoplasmic RyR1 and N-terminally tagged RyR1 D9

fragment

The FL-RyR1 rabbit cDNA and the truncated, cytosolic RyR1 cDNAs were sub-

cloned into the Qiagen pQE expression vector system, containing the dual tandem affinity

tags 6x-histidine and streptavidin binding peptide (SBP).

The N-terminal fragment between amino acids 204-613 (named D9) was sub-

cloned into the bacterial expression vector pET28a-LIC (Novagen), containing the N-

terminal affinity tag 6x-histidine in order to obtain highly purified D9 from bacteria.

B. Chemical Transformation of DH5-α cells

The cDNA vectors were then introduced into Escherichia coli strain DH5-α cells

by chemical transformation. Briefly, 3 µL of plasmid DNA was added to 50 µL of DH5-

α cells and incubated on ice for 30 minutes and then heat shocked for 45 seconds at 42oC.

The cells were then shaken for 1 hour at 37oC with 950 µL of SOC media (Sigma).

Finally, the transformed cells were pelleted by centrifugation (10000 rpm, 1 minute),

resuspended in 150 µL of SOC media, and spread onto LB agar plates with ampicillin

resistance (50 µg/mL) under sterile conditions. The plates were incubated overnight at

37oC.

C. Amplification and Maxi Preparations of DNA

To amplify the cDNA, 250 mL of sterile 2x YT liquid culture (BioShop) with ampicillin

(50 µg/mL) was inoculated with a sample of DH5-α cells picked from an individual

colony from the LB agar plates. The cultures were shaken overnight at 37oC. To isolate

the plasmid DNA, the maxi preparation method was performed using Qiagen’s Plasmid

22

Maxi Kit. Approximately after 16-18 hours of shaking, the bacterial culture was

centrifuged at 4100 rpm for 15 minutes at 4oC and the supernatant discarded. The pellet

was completely resuspended in 10 mL of cold P1 Resuspension Buffer containing

RNAse, with vortexing. 10 mL of P2 Lysis Buffer was then added and thoroughly mixed

by gently inverting the tube several times. Immediately after, the reaction was terminated

with the addition of 10 mL of P3 Neutralization Buffer and mixing by inverting. The

samples were incubated on ice for 20 minutes and then centrifuged at 4100 rpm for 20

minutes at 4oC. The resulting supernatant was applied to a QIAGEN-tip 500 column,

which was already equilibrated by allowing 20 mL of Buffer QBT to drain through by

gravity flow. Three successive washes were performed with 30 mL of Buffer QC to

remove all contaminants. 15 mL of Buffer QF was then added to elute the bound DNA

and collected in a centrifuge tube. To precipitate the DNA, 10.5 mL of isopropanol was

added and mixed by inverting and centrifuged immediately at 12000 rpm for 30 minutes

at 4oC. The supernatant was discarded slowly and the DNA pellet was washed by adding

20 mL of 70% ethanol and centrifuging at 12000 rpm for 15 minutes at 4oC. The

supernatant was again removed slowly and the pellet was allowed to air-dry to remove all

traces of ethanol. Finally, the pellet was dissolved in 1 mL of TE Buffer and the resulting

DNA concentration was determined using an Ultrospec™ 2100 pro UV/Visible

Spectrophotometer (GE Healthcare).

II. Ni-NTA chromatography purification

A. Culturing HEK293 cells

The cytosolic RyR1 and N-terminal D9 cDNA constructs were subsequently

introduced, via Ca2+-phosphate transfection, into HEK293 cells, which grow rapidly and

do not contain endogenous RyR1. HEK-293 cells were cultured in Dulbecco’s Modified

Eagle’s Medium (DMEM) H21 (Tissue Culture Media Facility at University Health

Network, Toronto) in a 37oC, 5% CO2, humidified incubator. The DMEM H21 media

was supplemented with 10% fetal bovine serum (Gibco), 1x MEM Non-Essential Amino

23

Acids Solution (Gibco), and 2.5µg/mL amphotericin-β (Sigma-Aldrich). Stock cultures

were maintained in 75 cm2 cell culture flasks (BD Falcon), in 12 mL of media. Confluent

(80-100%) flasks of HEK-293 cells were plated at a dilution of 1 into 5 to ensure 50-70%

confluency of cells the next day for transfection. For affinity purification, ten 100 mm

plates of cells were plated in 10 mL of media.

B. Transfection of Cells

Transfection of cells was performed the day after plating using the calcium

phosphate transfection method. For 10 plates of HEK-293 cells, at 50-70% confluency, a

5 mL solution was prepared with 620 µL of 2M CaCl2, 100 µg of DNA, and 4.3 mL of

sterilized water. The solution was lightly mixed before being added drop wise to 5 mL of

2x HEPES (274 mM NaCl, 1.4 mM Na2HPO4-7H2O, 54 mM HEPES) and let stand 20

minutes at room temperature. 1 mL of the final solution was then added drop wise to

each plate. The cells received fresh media 18-24 hours later.

C. Harvesting HEK293 cells and preparing protein extract

40-48 hours following calcium phosphate transfection, cells were harvested from

their plates with an ice cold solution of 1x PBS (137 mM NaCl, 10 mM Phosphate, 2.7

mM KCl, pH of 7.4) with 5 mM EDTA. The cells were then pelleted by centrifugation

(4100 rpm, 5 minutes, 4oC) and washed in ice cold 1x PBS. Following centrifugation

(4100 rpm, 5 minutes, 4oC) and removal of the supernatant, the cells were resuspended in

2 mL of Lysis Buffer supplemented with 1 mM PMSF and 1x protease inhibitor. The

lysis buffer contained 5% sucrose, 150mM NaCl, 50mM Tris-Hepes, ph. 7.0, 0.01%

Triton X-100 or 0.1% CHAPS detergents, 5mM β-mercaptoethanol (alternatively

dithiothretiol for cobalt purifications) and protease inhibitors. The cells were lysed using

25 strokes in a loose-fitting Dounce homogenizer. To remove the cell debris, the lysate

was then centrifuged at 8000 rpm for 20 min at 4oC. The supernatant was retained for

analysis.

24

D. Purification of RyR1 complexes using Ni-NTA resin

While preparing the protein extract, 2 μL of 50% Ni-NTA resin slurry per 1 plate

of transfected HEK293 cells, was prepared by washing twice with 1 mL of ice cold Ni-

NTA Binding Buffer (BB) with centrifugation (4100 rpm, 5 minutes, 4oC). The BB was

supplemented with 1 mM PMSF, and 1x protease inhibitor prior to use. These washes

were essential to remove any trace of ethanol present in the resin slurry storage buffer.

The protein extract was then added to the washed resin and rotated for 2 hours at 4oC to

allow the tagged proteins to bind the Ni2+ via the 6x-His tag. Protein bound to the Ni-

NTA resin were then separated from the flow-through by centrifugation (4100 rpm, 5

minutes, 4oC) and washed three times in 1 mL of BB with 25mM imidazole by rotating

for 5 minutes at 4oC followed by centrifugation (4100 rpm, 5 minutes, 4oC). Next, bound

protein were eluted twice with 100 µL for 1 hour with ice cold Elution Buffer (EB); BB

with 500mM imidazole, 1 mM PMSF, and 1x protease inhibitor.

25

Figure 5 – Ni-NTA purification of cytosolic RyR1 followed my LC-MS Expressed RyR1 constructs (purple) and interacting proteins (orange) were purified by metal affinity chromatography on Ni-NTA resin. After washing away non-specific interactors (green), RyR1 containing protein complexes were subjected to on-bead tryptic digest to generate peptide mixtures. Extracts are separated by liquid chromatography and mass spectrometry (LC-MS). Mass spectra were correlated by database algorithms.

26

III. Alternative strategies for purifying functional

RyR1 proteins

A. Size exclusion chromatography

HEK293 cells were transfected with cytosolic RyR1 rabbit cDNA by the calcium

phosphate method using 10 ug of DNA per 100 mm plate as previously described. 40-48

hours following calcium phosphate transfection, cells were harvested from their plates

with an ice cold solution of 1x PBS (137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, pH

of 7.4) with 5 mM EDTA. The cells were then pelleted by centrifugation (4100 rpm, 5

minutes, 4oC) and washed in ice cold 1x PBS. Following centrifugation (4100 rpm, 5

minutes, 4oC) and removal of the supernatant, the cells were resuspended and solubilized

on ice for 1 h in 2 mL of SEC solubilization buffer (200 mM NaCl, 25mM PIPES, pH

7.4, 0.15mM CaCl2, 0.1mM EGTA, 20mM sucrose, 0.4% CHAPS, and 2mg/ml

phosphatidylcholine) at a protein concentration of 2mg/ml and in the presence of protease

inhibitors (Sigma Mammalian Cocktail). Solubilized protein was separated from

insoluble material by centrifugation at 100,000g for 30 min.

Isolation of RyR was carried out in a refrigerated cold-room maintained at 4oC. A

25ml chromatography column was packed with sephacryl S-300 resin (GE Healthcare)

and connected to a peristaltic pump and a fraction collector. Solubilized RyR1 protein

extract was loaded onto the column and eluted with solubilization buffer at a flow rate of

1 ml/min. Eluate fractions were collected at 1 min intervals. Elution of RyR1 was

monitored by calibration of the column with Blue dextran (2 MD). The blue dextran

marks the void volume of the column. Since the pore size of the sephacryl is slightly

smaller than 2 MD, the blue dextran, and subsequently RyR1, elute in the void volume as

the sephacryl resin impedes cellular proteins under 2 MD. Aliquots of each collected

fraction were reserved for ELISA analysis to determine which fractions contained the

RyR1.

27

B. ELISA Analysis of SEC fractions

Polystrene microtiter 96-well plates were incubated with a solution of 50mM

sodium bicarbonate and 50 mM sodium carbonate (pH 9.6). 50uL of sample were added

to the wells and allowed to bind to the wells for 2 h at 4oC while shaking. After this,

200uL of 5% non-fat skim milk in PBS-Tween was added for 1 hour at room

temperature. ELISA wells were incubated with a 1:2500 dilution of anti-RyR1 antibody

(34C) in 5% non-fat milk-PBS-T, for 1 h, followed by three washes with PBS-T for 15

min. Wells were then incubated with HRP-conjugated secondary antibody diluted in 5%

milk-PBS-T solution for 1 hour at room temperature. Following three 15 minutes washes

with PBS-T, blots were treated with SuperSignal West Pico Chemiluminescent Substrates

(Pierce) for 5 minute and then exposed to film in a dark room setting, which was

subsequently developed.

C. Complexiolyte buffer solubilization of FL-RyR1

HEK293 cells were transfected with cytosolic RyR1 rabbit cDNA by the calcium

phosphate method using 10ug of DNA per 100 mm plate as previously described. 40-48

hours following calcium phosphate transfection, cells were harvested from their plates

with an ice cold solution of 1x PBS (137 mM NaCl, 10 mM Phosphate, 2.7mM KCl, pH

of 7.4) with 5 mM EDTA. The cells were then pelleted by centrifugation (4100 rpm, 5

minutes, 4oC) and washed in ice cold 1x PBS. The cells were then lysed on ice in 1 ml of

complexiolyte buffer (48, 51, 71, 76, 81, 88, 99 and 140, Logopharm Biotech) per 10

plates of cells in the presence of protease inhibitors (Sigma Mammalian Cocktail) before

30 strokes in a loose Dounce homogenizer. Lysates were then spun at 8000 rpm for 20

minutes prior to application to Ni-NTA resin for affinity purification as described above.

D. Pull-down of mouse skeletal muscle proteins using

bound D9 fragment

The D9-RyR1 fragment was introduced into E. coli and grown up in 5 L of media.

The E. coli was pelleted by centrifugation (30 min @ 12 000 r.p.m.) and lysed in 10 mL

28

of lysis buffer (150 mM NaCl, 50mM Tris-Hepes, ph. 7.0) using loose dounce

homogenization. The lysate was applied to a 10 mL column containing 5 mL of cobalt

resin. The column was then washed with 4 column volumes of wash buffer (lysis buffer

with 25 mM imidazole) and eluted with 10 mL of elution buffer (lysis buffer with 300

mM imidazole).

Cobalt resin was prepared by washing twice with 1 mL of ice cold Binding Buffer

(BB) with centrifugation (4100 rpm, 5 minutes, 4oC). The BB was supplemented with 1

mM PMSF, and 1x protease inhibitor prior to use. These washes were essential to

remove any trace of ethanol present in the resin slurry storage buffer. The purified D9-

RyR1 fragment was then dialyzed against lysis buffer with no imidazole and then added

to the washed resin and rotated for 2 hours at 4oC to allow the tagged proteins to bind the

Co2+ via the 6x-His tag. Protein bound to the cobalt resin were then separated from the

flowthrough by centrifugation (4100 rpm, 5 minutes, 4oC) and washed three times in 1

mL of BB with 25mM imidazole by rotating for 5 minutes at 4oC followed by

centrifugation (4100 rpm, 5 minutes, 4oC). Next, mouse muscle lysate was added for 1

hour. 2 g of hind leg muscle was harvested from WT mice and lysed using an electric

homogenizer with ice cold lysis buffer with 1 mM PMSF, and 1x protease inhibitor.

The cobalt resin with D9-RyR1 and potentially bound interactors was then

washed and eluted as previously described before and subjected to LC-MS analysis.

29

Figure 6 - Pull-down of mouse skeletal muscle proteins using bound D9 fragment Expressed D9 constructs (purple) were purified by cobalt chromatography and washed extensively of bacterial proteins (grey) before the addition of mouse skeletal muscle soluble fraction lysate. After a second round of cobalt purification to remove unbound proteins (green), extracts containing D9 protein complexes were separated by liquid chromatography and mass spectrometry (LC-MS). Mass spectra were correlated by database algorithms.

IV. Mass Spectrometry Analysis

A. Protein sample preparation

Aliquots containing 100 µg of total protein from the elution fraction were

prepared for trypsin digestion by diluting the sample to 50 mM ammonium bicarbonate,

pH 8.5, supplemented with CaCl2 to a final concentration of 1 mM. Samples were

incubated overnight at 37oC with 0.02 mg/ml of proteomics grade trypsin (Roche)

(Kislinger et al. 2006; Gramolini et al. 2008).

30

B. Solid-Phase Extraction of Tryptic Peptides

Solid-phase extraction was performed using OMIX C-18 pipette tips (Varian Inc.)

to purify the peptides from potential contaminants prior to subjecting trypsin digested

samples to mass spectrometry analysis,. First, to condition the OMIX C-18 pipette tip,

110uL of Solution 1 (50% Acetonitrile) was passed through the tip twice. The column

was then equilibrated twice with 110uL of Solution 2/3 (0.1% TFA). 40 uL of 2.5% TFA

was added to the tryptic digest samples, and then the samples were loaded onto the

column. The pass-through was collected by gravity and then reapplied to the column.

This step was repeated a total of four times. Next, the tryptic peptide bound column was

washed once with 110uL of Solution 2/3 and then eluted twice with 110 uL of Solution 4

(70% Acetonitrile, 0.1% formic acid). To concentrate the pooled eluate to about 30 uL, I

performed a quick speed vacuum evaporation at 45oC. Following these steps, peptide

samples were ready for our shotgun proteomic analysis.

C. MS and proteomic analysis of HEK293 cells over-

expressing RyR1

Mass spectrometry analysis was performed in the laboratory of Dr. Thomas

Kislinger, at the University of Toronto. Briefly, for peptide separation, individual

samples were first loaded onto microcapillary fused silica columns with an internal

diameter of 75 μm, packed with 7cm of reversed phase C18 resin (Magic C18). The

columns were aligned with an LTQ linear ion trap mass spectrometer, and the peptides

were eluted using a 2 hour water/acetonitrile gradient and ionized via electrospray

ionization. Next, the generated tandem mass spectra were matched to peptide sequences

in the human IPI protein sequence database (http://www.ebi.ac.uk/IPI) using the

X!Tandem algorithm (http://gpmdb.thegpm.org/). Since the resulting proteins were

represented with different accession codes, to ensure comparability, gene names were

determined for each protein and used throughout this study. In addition, only proteins

identified with at least two unique peptides were accepted for further experimental

considerations.

31

D. MS analysis of mouse skeletal muscle lysate on bound

D9 fragment

Elutant samples of the D9 fragment with bound mouse skeletal muscle lysate

proteins were treated as described above with regard to protein sample preparation for

LC-MS analysis, including tryptic digest and solid-phase extraction of peptides. LC-MS

analysis differed in that mouse IPI protein sequence database using the X!Tandem

algorithm was used. As before, since the resulting proteins were represented with

different accession codes, to ensure comparability, gene names were determined for each

protein and used henceforth. In addition, only proteins identified with at least two unique

peptides were accepted for further experimental considerations.

V. SDS-PAGE and Immunoblot analysis

A. SDS-PAGE and immunoblot analyses of RyR1

expression

HEK293 and C2C12 cells lines were cultured and transfected with 6x-His affinity

tagged cytosolic and FL RyR1, then harvested as described previously. 30 μg of protein

from the cellular lysate, purification flowthrough, first wash, final wash and elution

fractions from RyR1 and D9 Ni-NTA purifications were subjected to standard western

blotting techniques, via SDS-Polyacrylamide Gel Electrophoresis. Briefly, 10ml of

resolving gel was first added and allowed to solidify, 37.5:1 Acrylamide/Bis Mix (BIO-

RAD) (0.2M), 1.5 M Tris (pH 8.8) (VWR International) (1.5M), 10% SDS (EMD) (0.4

M), 10% Ammonium Persulfate (VWR International), TEMED (EMD)]. Stacking gel

was then added and solidified [H20, 37.5:1 Acyrlamide/Bis Mix (BIO-RAD), 1.5 M Tris

(pH 6.8) (VWR International), 10% SDS (EMD), 10% Ammonium Persulfate (VWR

International), TEMED (EMD). Next, samples to be resolved were denatured by the

addition of 6x Protein Loading Dye and boiling for 10 minutes before being loaded onto

32

gels and resolved by electrophoresis. PageRuler™ Prestained Protein Ladder

(Fermentas) was used as a protein standard to gauge molecular weights.

For samples being probed against RyR1, voltage was run through the SDS-PAGE

gel until the 70 kDa protein marker ran through the bottom of the gel to ensure the RyR1

(500 kDa when denatured) entered the separating gel. In probing for all other proteins,

SDS-PAGE gels were run until the dye front reached the bottom of the apparatus.

B. Immunoblot analysis in HEK293 cells and C2C12

mouse myocytes

For immunoblotting, proteins were first transferred from the polyacrylamide gel

to a nitrocellulose membrane. Nitrocellulose membranes were blocked with 5% non-fat

milk in 0.05% Tween/PBS (PBS-T). Membranes were incubated with a 1:2500 dilution

of anti-RyR1 antibody (34C) in 5% non-fat milk-PBS-T, for 1 h, followed by three

washes with PBS-T for 15 min. Membranes were then incubated with HRP-conjugated

secondary antibody diluted in 5% milk-PBS-T solution for 1 hour at room temperature.

Following three 15 minutes washes with PBS-T, blots were treated with SuperSignal

West Pico Chemiluminescent Substrates (Pierce) for 5 minute and then exposed to film in

a dark room setting, which was subsequently developed.

The following commercial antibodies were used to target specific proteins: mouse

monoclonal against ryanodine receptor 1 (RyR1) (Affinity Bioreagents), rabbit

polyclonal against calcium homeostasis endoplasmic reticulum protein (CHERP)

(Abcam), goat polyclonal against Four and a half LIM domain (FHL1) (Imgenex), rabbit

polyclonal against endoplasmic reticulum golgi intermediate complex 53 (ERGIC-53)

(SIGMA), goat polyclonal against T-complex protein (TCP) (Abcam), mouse monoclonal

against dihydropyridine receptor (DHPR) (Affinity Bioreagents), rabbit monoclonal

against dysferlin (Affinity Bioreagents), rabbit polyclonal against phosphorylase kinase

alpha subunit (PHK-α) (Abcam), and mouse polyclonal against penta-histidine epitope

(Qiagen).

33

VI. Subcellular localizations of RyR1 and interactions

A. Plating and fixing slides of HEK-293 cells

Sterile glass cover slips were placed in individual wells of a 6-well cell culture

plate and coated with warm gelatin (Sigma-Aldrich) and incubated at 37oC for 30 minutes

with the lid on. An 80-100% confluent flask of HEK-293 cells was plated at a dilution of

1:18 to ensure 50-70% confluency of cells the next day for transfection. HEK-293 cells

were grown in 3 mL of media. Transfection of cells was performed the next day using the

calcium phosphate method, as previously described. Each well though was treated with

only a third of the transfection solution used for one 100 mm plate.

40-48 hours after transfection, following a 30 minute incubation in 2 mL of ice-

cold 1x PBS at 4oC, the cells were fixed with 2 mL of 2% paraformaldehyde (made in 1x

PBS, pH 7.4) for 30 minutes at 4oC. Two 1 mL washes were then performed with freshly

prepared Permeabilization Buffer (0.2% Tween-20, 0.5% Triton X-100 in 1x PBS) at 4oC

for 15 minutes each.

B. Slide Preparations of Fixed Isolated Skeletal Soleus

Muscle Fibers

Skeletal slow twitch soleus muscle fibres were isolated from the hind limbs of

adult female Spragley-Dawley rats upon euthanization via CO2 asphyxiation.

Immediately upon isolation, the tissue was incubated in excess ice-cold 1x PBS for 30

minutes before fixation in 2% paraformaldehyde for 30 minutes at 4oC. Two 1 mL

washes were then performed, in 1.5 mL microcentrifuge tubes, with freshly prepared

Permeabilization Buffer at 4oC for 15 minutes each.

C. Co-Immunofluorescent Staining of Fixed Tissue

Upon paraformaldehyde fixation, washed tissues were then incubated in 1 mL of

Blocking Buffer (5% FBS, 0.2% Tween-20, 0.5% Triton X-100 in 1x PBS) for 30

34

minutes at room temperature. For co-labeling, tissues were then treated with multiple

primary antibodies diluted in 100 µL of Blocking Buffer, overnight at 4oC. This was

followed by three 15 minute washes with 1 mL of Permeabilization Buffer at room

temperature. Next, tissues were incubated in the dark for 1 hour at room temperature

with fluorescent secondary antibodies diluted in 100 µL of Blocking Buffer.

Subsequently, three 15 minute washes were performed with 1mL of 1x PBS in the dark at

room temperature, before mounting in Fluoromount™ medium (Sigma). Dilutions for

the various commercially available antibodies used are as such: anti-CHERP (1:500),

anti-FHL1 (1:500, Aviva Systems Biology), anti-RYR1 (1:500). The fluorescent Alexa

488 and Alexa 633 secondary antibodies, anti-mouse and -rabbit (Invitrogen), were

diluted at 1:500 and 1:200, respectively.

VII. Calcium transient analysis of RyR1 expressing

HEK293 cells with candidate siRNA knockdowns

A. Plating slides of HEK293 cells

Sterile glass cover slips were placed in individual wells of a 6-well cell culture

plate. An 80-100% confluent flask of HEK-293 cells was plated at a dilution of 1:25 to

ensure 20-40% confluency of cells the next day for transfection. HEK-293 cells were

grown in 3 mL of media. Transfection of RyR1 cDNA was performed the next day using

the calcium phosphate method, as previously described. Media was changed 24 hours

later, and two hours after this, a second transfection was performed, incorporating the

selected siRNA into the cells via lipofectamine. Cells were left 48 hours to allow for

expression of RyR1 and for suppression of the selected protein of interest.

B. Calcium transient analysis

When samples were ready for calcium transient analysis, they were treated with

Fura-2 (Invitrogen) for 1 hour. 50 mg of Fura-2 was diluted in 100 uL of DMSO (Sigma-

35

Aldrich) and Fura-2 was added to each well to a final concentration of 1 uM. After 1 hour

of Fura-2 treatment, media was changed and 20 minutes later readings were taken using

the Olympus IX87 inverted microscope with Quorum XCite system.

For the 340/380 nm ratio acquisitions, calcium transients were read over a period

of 60 seconds with fluorescence measurements taken every 3 seconds, with at least 5 cells

measured. At 30 seconds, 5 mM caffeine was added to the system to induce calcium

release.

C. Statistical analysis of data

For each reading, a graph was generated, plotting 340/380 nm fluorescence ratio

versus time. From this we measured the amplitude of the peak representing the 5 mM

addition of caffeine. This value is proportional to total cytosolic calcium levels, which is

increased upon induction by caffeine. For each condition, four replicates were performed

and the amplitudes were averaged and subjected to an unpaired t-test to generate

corresponding p-values. In addition to individual t-tests comparing RyR1 to the each

knockdown experiments, an ANOVA (analysis of variance) was performed to determine

whether the sample means are within sampling variability of each other.

36

Chapter Three: Results

I. Purification of RyR1 contained protein complexes

Metal affinity chromatography purification was initially developed to purify

complex protein aggregates from yeast, such as ribosome, spliceosome, or transcription

complexes (Rigaut et al. 1999). This purification method, using a 6xHIS tagged protein

of interest, can be combined with subsequent mass spectrometry analysis to identify the

co-purified proteins (Zhu et al. 2003; Giannone et al. 2007). Here, we predicted that

6xHIS affinity tagged cytosolic RyR1 could be purified with interacting partners from

HEK293 cells. To identify artifacts inherent to the purification process, negative controls

were performed under identical conditions as the RyR1 purifications. These negatives

controls were untransfected HEK293 cells, and tagged controls transfected with 6xHIS

tagged dihydropyrimidinase-related protein 3 (DPYSL3). DPYSL3 is a protein with few

GO terms associated with calcium signaling and EC coupling making it a good candidate

for a tag-control.

A. Expression and solubilization of tagged RyR1 proteins

One of the obstacles in the purification of tagged cytosolic RyR1 is its insoluble

nature. To ensure that the cytosolic domain was in fact expressed in the cytosol we

performed an immunoblot analysis of the cytosolic and microsomal fractions of over-

expressing HEK293 cells (figure 7, left). It showed that the majority of the tagged bait

existed in the cytosol and was thus effectively solubilized so that purification was

possible.

We also performed confocal microscopy imaging transfected HEK293 cells

(figure 7, right) using the 34C antibody against RyR1. HEK293 cells have been shown to

endogenously express trace levels of RyR2, but contain no endogenous RyR1. In

combination with the fact that untransfected control showed no signal; the proteins

visualized in figure 8 was generated from the cDNA construct and contain the 6xHIS

affinity tag.

37

B. Purification of bait and potential interactors

Figure 8 illustrates the efficiency of the metal affinity chromatography procedure.

For example, a considerable amount of tagged bait remained in the flow-through

following Ni-NTA resin binding. This may have been due to saturation of the volume of

beads used, incomplete solubility of the bait protein, and in the case of the C2C12

myocyte expression, because the bait protein may have been retained by the complex

cytoskeletal network within the differentiated myotubes. Some bait protein was also lost

during the subsequent washes, though progressively less with each additional wash. The

washes were however a necessary component to improve the purity of the final product.

In addition, analysis of the eluted beads revealed recovery of the bound bait was not

complete. Regardless, a significant amount of the expressed recombinant protein could be

purified and concentrated. The procedure also proved to be functional when applied to

the tag control protein DPYSL3 (not shown).

The RyR1 proteins were not isolated alone as evidenced in figure 8. In the

Coomassie blue stained gel (left), the purified RyR1 in the elution indicated by the red

arrow is considerably less dense when compared to the cell lysate. In the elution

however, remain multiple protein bands in addition to the purification and concentration

of the bait protein. The protein bands (black arrows) detected in addition to the RyR1

bait, are representative of co-purified potential RyR1 interactors.

II. Identification of RyR1 interacting protein partners

A. Proteins identified from RyR1 purifications by mass

spectrometry

A total of nine purifications were performed from HEK-293 cells and analyzed by

MS (figure 10). Of these, three were RyR1 purifications, with three negative control bead

purifications and three tag control purifications. From the MS analysis, the generated

38

collection of peptide tandem mass spectra were searched against a protein sequence

database using the X!TANDEM algorithm. Since the resulting proteins were represented

with different accession codes, to ensure consistency and comparability, gene names were

acquired for each protein and used henceforth for identification. For every experimental

run, only proteins which were supported by at least two unique peptides were accepted

for further consideration. Thus, when combined together, a total of 703 different proteins

were identified from all six purifications.

B. Filtering and Comparing Subsets of Proteins Identified

from Purifications

Of the 703 proteins detected from all six purifications, 323 were identified in any

of the three RyR1 purifications. In order to identify potential RyR1 interactors of high

confidence, the subsets of identified proteins were filtered and compared according to

Figure 10. Figure 10 (left) depicts the 703 co-purified and identified proteins via heat-

map representation. Firstly, proteins found only in the negative and tagged control runs

were removed. Next, based solely on identical gene names, proteins found in the RyR1

runs as well as in the negative control or the DPYSL3 tag control purifications were

removed. The major contaminating proteins external to the applied cell lysate were

keratin and ribosomal proteins. Throughout the experimental process, all necessary steps

were taken to minimize keratin contamination, including the use of filtered buffers and

keratin free tips, in addition to working in a laminar flow hood. Untransfected cells were

used as negative controls to identify proteins with a capacity to bind non-specifically to

the resin. To account for technical artifact proteins, inherent to the purification process,

tag control purifications were performed to identify “sticky” proteins. Following these

eliminations, 195 proteins were identified as belonging uniquely to the RyR1

purifications (figure 10, middle). Next, the filtered RyR1 MS datasets were compared to

identify proteins present in multiple purifications, thereby strengthening confidence in the

potential interactors. Aside from the bait RyR1 protein, 33 proteins were found in at least

two RyR1 purification experiments (figure 10, right). Table 1 list these 33 proteins and

their corresponding total spectral counts, as detected in each purification.

39

C. Prioritization of Potential Interactors

RyR1 potential interactors of interest were then prioritized in two ways to

improve confidence and validate further experimental pursuit. The first method had the

proteins ordered in terms of frequency and strength of detection. In figure 10 (right), the

33 RyR1 potential interactors are prioritized firstly based on the number of MS runs they

were found, and secondarily by how many spectral counts were found in the respective

runs. In this initial treatment of the MS data, we found few candidates that met the very

rigorous criteria set forth. Upon further review of the entire dataset, we noticed many

proteins that were highly enriched in the RyR1 runs, were filtered out because of one

spectra found in one of the six control runs. In addition, the microcapillary fused silica

columns used for MS analysis are generally eluted and re-used, meaning there is

opportunity for single spectra contaminations (i.e. carry-over). Considering this, we re-

analyzed the data by averaging the spectral counts for each of the three RyR1 runs, and

for the six control runs for every protein, to generate an enrichment value over controls.

For example, CHERP was found in two of the three experimental runs (2 and 11 unique

peptides respectively), yet one spectra was found in one control run. Proteins that saw a

four-fold enrichment in RyR1 runs over controls are listed in table 2.

In Table 2, the proteins were ranked based on the average enrichment over control

values. To do this, the spectral counts in the three experimental runs were averaged and

compared to the average spectral count in the control runs. This was done for each

individual protein generating enrichment value or fold increase. Proteins that were

enriched by at least a factor of 4 were included in table 2. Again, common contaminant

proteins and proteins deemed biologically irrelevant were not further considered. For the

purposes of this project, the availability of antibodies was also factored into the selection

process.

D. Screening for Known RyR1 Interacting Proteins

To assess the validity of performing Ni-NTA purifications in combination with

MS, the 323 proteins detected uniquely in RyR1 purifications were screened to identify

40

any known RyR1 interacting partners. As a result, one putative RyR1 interactor was

discovered amongst the 323 proteins, FKBP1A was able to make it past the strict filtering

criteria as it was enriched over controls by a factor of 4.67. For instance, DHPR and

calsequestrin are known RyR1 interactors, but in our experiments they only were found

in one experimental run. In addition, neither was enriched significantly over control

values and thus these proteins did not make it into the shortlist (Table 2).

E. Selection of Potential Interactors

The proteins in tables 1 and 2 were consolidated, and represented by heat-map in

figure 11. These 48 proteins were then filtered for functionally irrelevant proteins and

eight proteins were selected for further consideration in table 3.

From the proteins listed in table 3, we then analyzed the merits of each potential

interactor based on their known functional relevance to RyR1 and calcium signaling in

general. Proteins belonging to families of common contaminants of purification

procedures, and deemed to not be of biological relevance, were not considered moving

forward. This included ribosomal, mitochondrial, spliceosomal, histone, and heat shock

associated proteins.

MYCBP2 (MYC binding protein 2) is a large multi-domain E3 ubiquitin ligase;

since ubiquitin pathway proteins are not obvious candidates for playing a role in calcium

homeostasis via RyR1, it was not pursued further. PHKB (phosphorylase b kinase) is a

kinase that is activated by increased cytosolic Ca2+ levels. Since RyR1 undergoes

extensive post-translational modifications, including phosphorylation, this candidate was

selected for further analysis by western blot.

As before, we removed proteins which fell under the previously listed groups and

this left (in addition to MYCBP2 which was previously addressed), and the remaining

proteins included numerous T-complex protein 1, LMAN1 protein (ERGIC-53 protein),

FKBP1A peptidyl-prolyl cis-trans isomerase, and CHERP (calcium homeostasis

endoplasmic reticulum protein). T-complex protein 1 is a known chaperone protein

complex with up to 8 subunits that mediates protein folding in the cytosol; because RyR1

41

is a 2 MDa homotetramer, the majority of which lies in the cytosol, it is plausible that it

requires chaperones for correct assembly and folding. T-complex protein 1 was selected

for further investigation. LMAN1 protein (or endoplasmic reticulum-golgi intermediate

complex protein 53) is a type 1 integral membrane lectin which cycles between the ER

and the golgi. It shares organelle location with RyR1 and there was easy access to an

antibody against ERGIC-53, thus it was selected for further investigation. FKBP1A is a

known RyR1 interactor which was further evidence for successful pull-down of

interactors. CHERP is an integral membrane protein that resides in the ER and whose

suppression results in impaired intracellular calcium mobilization. This function is clearly

relevant to a calcium release channel residing in the same location of the cell, thus

CHERP was selected for further investigation.

III. Alternative strategies for purifying ryanodine

receptors

Throughout our initial purification strategy, over-expression of tagged RyR1

followed by detergent-mediated solubilization and Ni-NTA chromatography, we

experienced a significant degree of difficulty in attaining high bait spectral counts.

Fortunately, we obtained three MS runs with high RyR1 spectral counts as previously

described. However, those three experimental repeats were from a larger group of 32. In

the remaining runs, some were performed under identical conditions as those in figure 10,

and some had a number of conditions varied including CHAPS and Triton X-100

concentrations, using the two detergents in combination, adjusting the salt concentrations

and the imidazole concentrations in the wash and elution steps. In these additional runs,

there were negligible bait spectra observed.

These findings led to the pursuit of alternative strategies for purifying functional

ryanodine receptors as a way of increasing observed bait spectral counts. SEC and the

42

Complexiolyte buffer system were two methods that were found in the literature to be

potentially effective in isolating intact RyR1 proteins.

A. Size exclusion chromatography

In 2002, West et al. showed that intact RyR1 proteins could be solubilized and

purified from skeletal muscle using size exclusion chromatography. Using the high

molecular weight of RyR1, greater than 2MDa in tetrameric form, they employed a size

exclusion resin with a pore size just under 2MDa. This meant that any soluble proteins

smaller than the resin pore size would be impeded from travelling through the column,

meanwhile RyR1 and the few other cellular protein larger than 2MD, would exit the

column in the void volume. Essentially, the column acted as a filter, absorbing any

soluble contaminating proteins smaller than 2MD (>93% efficiency in the literature).

Dextran Blue (MW = 2MD) was used to determine the void volume of the

column, which was 8 mL. HEK293 cells over-expressing tagged FL-RyR1 were

solubilized using the same conditions as described by West et al. (West et al. 2002).

Following this the soluble fraction was applied to the column at a flow rate of 0.75

mL/min. Fractions were taken in 1mL samples and aliquots of each fraction were

analyzed for RyR1 using ELISA as seen in figure 12 A. The presence of GAPDH, a

smaller cytosolic protein with no known relationship to calcium signaling and RyR1 was

used as a control and it eluted in fractions 23-26. This confirmed that smaller cytosolic

proteins, which compose the majority of contaminants in this type purification method,

are separated from the larger proteins and hence RyR1 proteins.

The RyR1 eluted in the void volume, in theory, purifying it from any other

soluble protein under 2MDa. Fractions 7-10 were combined and subjected to Ni-NTA

chromatography to further purify the tagged RyR1. The elution of this purification

yielded no RyR1 peptides via MS analysis. Hence, to judge if the RyR1 was there, we

simply TCA precipitated fractions 7-10 to collect all protein in the samples before

resolubilization in 8M urea and MS analysis. RyR1 was found in one MS run (figure 12

43

B), identified with three unique peptides. Fractions 23-26 were also analyzed and as

expected no RyR1 was found.

Although this biochemical method could isolate RyR1 peptides, in transitioning to

the proteomic analysis, RyR1 was not identified and enriched effectively to consider

moving forward and analyze potential interacting partners using this method.

B. Complexiolyte buffer system

Complexiolyte buffers are optimized detergent solutions for solubilization of

membrane proteins and protein complexes of up to 2MD. Logopharm Biotechnology has

shown the buffers to have been successfully applied in affinity purifications, native gel

electrophoresis (Schulte 2008). Thus, we attempted to isolate and solubilize RyR1

proteins from C2C12 myotubes over-expressing 6x-His FL-RyR1. In theory, the

Complexiolyte buffers would accomplish two key objectives. Firstly, they would

solubilize the tagged RyR1 making purification possible. Secondly, since they are

marketed as MS compatible (i.e. containing no detergents or compounds that

interfere/generate spectra in MS), there should be a seamless transition from the

biochemistry to proteomic portion of the experimental protocol.

We over-expressed tagged FL-RyR1 in C2C12 myocytes and then used a series of

complexiolyte buffers as a lysis/solubilization buffer. The lysate was then applied to Ni-

NTA resin and the elutions from those samples were run on SDS-PAGE and probed for

purified RyR1 (figure 13 A). Complexiolyte buffer 76 clearly enabled the solubilization

and purification of RyR1, thus, we subsequently subjected the remaining elutant to tryptic

digest before MS analysis. In comparison to the control lane, unpurified lysate, there is a

significant loss in tagged bait from the lysis to the elution steps. A percentage of the

protein visualized in the control lane may have been untagged, endogenous RyR1. In

addition, there may have been loss throughout the affinity purification protocol (the

efficiency of the Ni-NTA purification method will be discussed in the next chapter).

However, the MS analysis of the complexiolyte yielded no RyR1 spectra. In

addition, we performed an in-gel digest of the band visualized and only two unique

44

peptides were observed (figure 13 B). As such, similar to the previous purification

method--SEC--although we were able to isolate and purify the RyR1 proteins, the

transition to proteomic analysis was difficult and yielded no significant MS results. The

transition from biochemical isolation of the bait protein complexes to their proteomic

analysis will be discussed in the next chapter.

C. Purification of N-terminal D9 fragment of RyR1

After several attempts at purifying large full-length and cytosolic RyR1 peptides

with varied success, we chose to pursue the purification of smaller, more manageable

fragments of the RyR1 cytosolic domain. We chose RyR1 amino acids 204-613 (D9)

because we were interested in identifying proteins that interact within this region given

that this N-terminal region contains the MH/CCD domain I. In addition, the D9 cDNA

was accessible in a bacterial vector containing an N-terminal 6xHis affinity tag and was

selected as the most soluble fragment tested in the MH/CCD domain I by colleagues

performing crystallization pre-screening experiments (data not shown).

After attaining the tagged D9 cDNA, E. coli were transfected and grown up in

large volumes. An advantage to using bacteria to over-express proteins is that they can be

grown in suspension allowing for higher volumes in comparison to mono-layer cell types.

After growing the D9 proteins, they were purified on a cobalt column and eluted in 10mL

of concentrated elution buffer. The D9 was re-incubated with cobalt resin and mouse

skeletal muscle was then passed over the D9-cobalt resin. The following mixture was

purified, eluted and analyzed by MS.

IV. Identification of D9 binding protein partners

A. Proteins identified from RyR1 purifications by MS

analysis

45

A total of six purifications were performed using mouse skeletal muscle lysate on

the D9-cobalt preparation and subsequently analyzed by MS. Of these, three were mouse

lysate incubated D9-cobalt purifications, with two negative control bead purifications

containing a mouse lysate passed over empty cobalt resin and one tag control

purification. From the MS analysis, the generated collection of peptide tandem mass

spectra were searched against a protein sequence database using the X!TANDEM

algorithm. As with the 31-RyR1 MS data, the resulting proteins were represented with

different accession codes, and to ensure consistency and comparability, gene names were

acquired for each protein and used henceforth for identification. For every experimental

run, only proteins which were supported by at least two unique peptides were accepted

for further consideration. Thus, when combined together, a total of 153 different proteins

were identified from all six purifications.

B. Filtering and Comparing Subsets of Proteins

As expected with a smaller bait D9 fragment, there were vastly fewer total

proteins detected in the combined MS runs for the D9 experiments. Thus, fewer filtering

steps were required to reach a manageable list of potential interacting partners in

comparison to the previously described RyR1 data.

Figure 14 depicts the 153 co-purified and identified proteins via heat-map

representation. Proteins found only in the negative and tagged control runs were

removed. Next, based solely on identical gene names, proteins found in the RyR1 runs as

well as in the negative control or the DPYSL3 tag control purifications were removed. Of

the 153 proteins detected from all six purifications, 34 were identified in any of the three

RyR1 purifications and not in any control experiments. The remaining 34 proteins are

depicted and listed in the second heat-map.

The major contaminating proteins external to the applied cell lysate were keratin

and ribosomal proteins. As with the RyR1 purifications, throughout the experimental

process, all necessary steps were taken to minimize keratin contamination, including the

use of filtered buffers and keratin free tips, in addition to working in a laminar flow hood.

46

Untransfected cells were used as negative controls to identify proteins with a capacity to

bind non-specifically to the resin. To account for technical artifact proteins, inherent to

the purification process, tag control purifications were performed.

C. Prioritization of Potential Interactors

D9 potential interactors of interest were prioritized in two ways to improve

confidence and validate further experimental pursuit. The first method had the proteins

ordered in terms of frequency of detection. In figure 14, the 34 D9 potential interactors

are prioritized firstly based on the number of MS runs they were found, and secondarily

by how many spectral counts were found in the respective runs. Next, proteins belonging

to families of common contaminants of purification procedures, and were also deemed to

not be of biological relevance, were demoted. This included ribosomal, mitochondrial,

spliceosomal, histone, and heat shock associated proteins.

As with the RyR1 data, this initial method left few proteins of potential biological

relevance with RyR1 function and thus the MS data was analyzed using a second method.

In Table 4, the proteins were ranked based on the average enrichment over control values.

To do this, the spectral counts in the three experimental runs were averaged and

compared to the average spectral count in the control runs. This was done for each

individual protein generating enrichment value or fold increase. Proteins that were

enriched by at least a factor of 4 were included in table 4. Again, common contaminant

proteins and proteins deemed biologically irrelevant were not further considered.

To assess the validity of performing Ni-NTA purifications in combination with

MS, the 34 proteins detected uniquely in the D9 purifications were screened to identify

any known RyR1 interacting partners. As a result, one known RyR1 interactor,

calsequestrin, was discovered amongst the 34 proteins. It was identified in one D9

purification and found in no controls.

D. Selection of Potential Interactors

47

In figure 15, the list of potential interactors offered few biologically relevant

candidates. After the removal of non-relevant candidates, calsequestrin, SERCA, PHBK,

and a T-complex protein subunit remained. PHBK and T-complex protein were

previously investigated following the RyR1 purifications. Calsequestrin is a known RyR1

binding partner, and SERCA carries out the antagonist function of RyR1, in the SR

membrane. Thus, as done after the RyR1 purifications, we reanalyzed the data by

generating an enrichment value, averaging the spectral counts over the bait and control

runs for each protein identified. Proteins enriched by a factor of four were considered and

of those three proteins listed in table 4, FHL1 was the only one that could have functional

relevance in cellular calcium mobilization.

In addition to pulling down FHL1 with immobilized D9-RyR1, our colleague

Thiru Shathasivam identified RyR1 after affinity-tagged FHL1 purifications as seen in

figure 15. FHL1 and RyR1 co-purified in reversed experimental protocols which lead to

its inclusion in subsequent experiments investigating the validity of potential candidates.

V. Validation of Interactions

A. Immunoblot Analysis of RyR1 Potential Interactions

Four proteins were identified from multiple RyR1 purifications as potential

interactors. To be accepted as authentic, however, further validation was necessary. The

most commonly used techniques are co-IP and immunodetection.

Co-IP was not an option for validation of potential interactions for a number of

reasons. Firstly, there is no currently available commercial antibody against RyR1

capable of co-immunoprecipitation experiments. Secondly, performing reverse co-IP

experiments with RyR1 (i.e. pulling down RyR1 via a potential interactor target) is highly

difficult because of the size and poor solubility of RyR1 as well as the possibility of a

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weak interaction. We did attempt these experiments with no success, i.e., RyR was never

seen in any blots as either bait or prey, regardless of the direction of the co-IP.

For immunodetection, a Ni-NTA pull down assay was performed from HEK-293

cells transiently expressing RyR1-6xHIS. Immunoblot detection verified the presence of

RyR1 interacting candidates in the purified sample with RyR1, seen in figure 16.

ERGIC-53, CHERP, TCP and PHBK were all detected by immunoblot after purification

of affinity-tagged RyR1 on Ni-NTA resin. However, when the same experiments were

performed in untransfected HEK293 cells, ERGIC-53, TCP and PHKB were all found in

the elution fractions of the control runs, meaning there was binding of those proteins non-

specifically to the Ni-NTA resin, negating them as potential RyR1 interacting candidates.

CHERP however, was not found in the control runs and thus, was selected to undergo

localization and functional experiments.

B. Subcellular Localization

In order for proteins to interact, either directly or indirectly, they must be in close

proximity and capable of being visualized together. Although interacting proteins can

possess different overall subcellular expression patterns, they must share some co-

residence. Immunofluorescence studies allow for visualization of the subcellular

localization of proteins, and regions of co-expression. Co-localization thus strengthens

validation of in vivo interactions.

i. CHERP Co-localization with RyR1 in Rat Soleus Muscle

In skeletal muscle RyR1 is retained in the SR membrane at the terminal cisternae.

During immunofluorescence staining, RyR1 can typically be resolved as double rows of

dot-like immunosignals, with each spot representing a single triad structure. The Z-line

lies between the RyR1 doublets (Salanova et al. 2008). In skeletal muscle co-stained

with CHERP and RyR1, a partial co-localization pattern was evident (Figure 17). Partial

co-localization was observed, at both of the RyR1 double rows and consistently on the

side facing the Z-line throughout the longitudinal section. This gave the appearance of

two RyR1 rows facing each other, lined with CHERP rows on their inside.

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It should be noted that the staining of RyR1 in rat skeletal muscle by the anti-

RyR1 ‘34C’ antibody is highly specific. Western blots with 30 μg of protein sample from

HEK293 cells expressing RyR1 shows only one single band at 565 kDa (data not shown).

Figure 17 B and C shows the 3-dimensional analysis of mouse skeletal muscle

tissue co-stained with RyR1 (green) and CHERP (red). The Imaris analysis software

allowed us to calculate the percentage of co-localization observed in a 3D image. As

previously described, the Imaris imaging software employed a computational algorithm

to determine the presence of each signal in one voxel (a 3D pixel), thus determining the

percentage of co-localized voxels containing both green and red signal. In the

colocalization analysis, we used the automatic threshold function to objectively determine

how much of the overall signal for each channel (green and red) should be included in the

colocalization calculation. From this, 22.2% of the voxels within the region of interest

were found to ‘colocalize’. To ensure that colocalization results were not affected by the

non-specific binding of fluorescent secondary antibodies, we incubated rat soleus muscle

with Alexa 488 and 633 secondary antibodies (used in both the CHERP and FHL

colocalization experiments), followed by confocal imaging. Almost zero staining was

observed, and 3D reconstitution from a Z-series of images was not possible (data not

shown).

ii. FHL1 Co-localization with RyR1 in Rat Soleus Muscle

Like the previous images, during immunofluorescence staining, RyR1 was

resolved as double rows of dot-like immunosignals, with each spot representing a single

triad structure. FHL1 is localized at the I-band, encompassing the Z-line, and transiently

at the M-line where it extends partially into the C-zone of the A-band. The resulting

transverse banding pattern is of alternating thick and thin bands, corresponding to the I-

band and the center of the A-band respectively. In skeletal muscle co-stained with FHL1

and RyR1, a particular co-localization pattern was evident (Figure 18 A). The two

signals were found in the same region, but whether co-localization was observed, was to

be determined by 3D analysis. It appears that FHL1 and RyR1 are in close proximity only

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at one of the RyR1 double rows and consistently on the same side throughout the

longitudinal section.

3-dimensional analysis of co-stained FHL1 and RyR1 rat skeletal muscle tissue is

seen in figure 18 B and C. It is clear from figure 18 that RyR1 and FHL1 in fact do not

co-localize but lie adjacent to each other. In fact, co-localization analysis was unable to

recognize any co-localizing voxels, meaning there is no co-localization between FHL1

and RyR1. Thus, any interaction between FHL1 and RyR1 would be through an

intermediate protein as a part of a multi-protein complex.

VI. Functional analysis of interaction candidates

A. Calcium transient analysis of HEK293 cells expressing

RyR1 with suppression of interacting candidates – 340 nm

acquisition

To study the effect of interactor suppression on calcium transients in HEK293

cells expressing RyR1, we firstly analyzed these calcium transients by acquisition of 340

nm fluorescence upon treatment with caffeine, a RyR1 activator. We found that

suppression of either CHERP or FHL1 in cells expressing RyR1 reduced the intensity of

the fluorescence peak observed upon treatment with caffeine when compared to cells

expressing RyR1.

The acquisition of 340 nm Fura-2 emissions to study calcium transients has

inherent confounding variables. These include variable dye concentrations across

different cells within one sample and cell thickness. These confounding variables are

resolved by acquiring 340 and 380 nm emissions and then calculating the 340/380 nm

ratio, which is directly correlated to the amount of intracellular calcium. Previously, 340

nm acquisitions were the only option on our calcium transient equipment. With the

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addition of new hardware to our calcium imaging equipment, we were able to acquire

both 340 and 380 nm emissions.

B. Calcium transient analysis of HEK293 cells expressing

RyR1 with suppression of interacting candidates – 340/380

ratio acquisition

Figures 19 and 20 show the calcium transients analyzed by 340/380 nm ratio,

acquired from HEK293 cells expressing RyR1, under a series of secondary, siRNA

knockdowns targeting CHERP and FHL1, potential RyR1 interacting candidates. Each

condition was repeated four times. The amplitudes seen in figure 19 represent the

increase in the fluorescence ratio over baseline values, upon stimulation with 5 mM

caffeine. The fluorescence ratio is directly proportional to the level of intracellular

calcium within the cell. Each condition was repeated four times with at least 5 cells

measured per repeat. These amplitudes were quantified in figure 20. The amplitude value

was calculated by dividing each point throughout the curve by Rmin, the minimum

fluorescence value, thus generating a baseline value of approximately 1.0. The amplitude

then represents the percentage change in the fluorescence ratio.

The percentage increase in the fluorescence ratio for RyR1 expressing HEK293

cells with suppressed CHERP (n=4, 6.5 ± 1.3) was significantly lower than that of

HEK293 cells expressing RyR1 (n=4, 26.5 ± 11.4) with a p-value of 0.0127. There was a

similar decrease observed in samples with suppressed FHL1 levels (n=4, 10.0 ± 3.3)

versus those expressing RyR1 (n=4, 26.5 ± 11.4), and the difference was also significant

(p = 0.0320). ANOVA analysis of the sample means generated an F-value of 9.391 which

corresponds to a p-value of 0.002.

In all runs, after the caffeine-induced calcium release event, there was an

observed decrease in the 340/380 nm ratio followed by return to an elevated baseline.

This represents a subsequent calcium uptake by the ER after the calcium release event,

and in every run the uptake, or decrease in 340/380 nm ratio, was in the range of 25-40%

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of the initial increase in 340/380 nm ratio. This secondary response to caffeine-induced

calcium release is consistent with previous studies.

To ensure that the lipofectamine transfection was not the cause for the decrease in

total calcium release between the RyR1 samples and the candidate knockdown samples,

scrambled siRNA was transfected into HEK293 cells expressing RyR1. The RyR1

samples with transfected scrambled siRNA yielded minimal change in percentage

increase of 340/380 nm fluorescence ratio over baseline (n=4, 27.5 ± 7.9) compared to

the RyR1 samples (n=4, 26.5 ± 11.4). In addition, HEK293 cells that were transfected

with lone RyR1 were identically treated with lipofectamine containing no siRNA to

mirror the secondary treatment received by all other samples. This data suggests that both

candidate proteins play a role in calcium homeostasis regulated by RyR1.

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Figure 7 - Expression Analysis of Cytosolic RyR1 (Left) Immunoblot analysis showing RyR1 soluble construct 1-4243 aa over-expressed in HEK293 cells is predominantly cytoplasmic. The Western Blot is probed with anti-RyR1 antibody. (Right) Confocal microscopy images with antibody raised against RyR1 show that RyR1 construct 1-4243aa is cytoplasmic (left panel). Right panel is phase contrast imaging of the same image.

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Figure 8 - Analysis of Cyto-RyR1 Purification Products (Left) 6% Coomassie stained protein gel analysis of several washes and elution obtained during the purification process of RyR1 construct 31. (Right) Immunoblot analysis of purification products from HEK293 and C2C12 cell lines probed with an anti-RyR1 antibody. Arrows indicate high molecular weight interacting proteins. Red arrow indicates RyR1 construct 31. Note, RyR1 can be purified and eluted in both HEK293 and C2C12 cells.

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Ni-NTA purification of cytosolic RyR1

Figure 5

Proteins ranked two ways: 1. Ranked by abundance in RyR1 runs and absence from controls (Table 1) 2. Ranked by spectral enrichment ratio from RyR1 runs vs. controls (Table 2)

Removal of functionally irrelevant candidates classified in literature as keratins, structural, ribosomal, mitochondrial, spliceosomal, histone and heat shock associated proteins

Analysis

Remaining Proteins: MYCBP2 TCP PHBK-α ERGIC-53 FKBP1A CHERP GYS1

If the candidate was not a known RyR1 interactor (Calsequestrin, FKBP1A), one of the following criteria had to be met: 1. The protein is effected by, or causes effect on, cytosolic calcium levels (CHERP, PHBK-α) 2. The protein is located in, or near, the SR membrane (CHERP, ERGIC-53) 3. The protein is a known chaperone, as it is unknown how RyR1 is organized into the SR (TCP, FHL1)

Remaining protein for immunoblot analysis: CHERP, TCP, ERGIC-53, PHBK-α , and FHL1

(Figure 16)

Co-localization and functional analysis of CHERP and FHL1

Proteins ranked two ways: 1. Ranked by abundance in RyR1 runs and absence from controls (Figure 14) 2. Ranked by spectral enrichment ratio from RyR1 runs vs. controls (Table 4)

Remaining Proteins: Actin FHL1 Calsequestrin PHBK-α SERCA1 USP9X

LC-MS

Cobalt purification of skeletal mouse

bound to D9 RyR1 proteins Figure 6

56

Figure 9 – Overview of the MS data filtering process and candidate acquisition From the two methods originally used to purify RyR1 peptides, each data set was treated two ways in order to generate potential interacting candidates. From these, proteins were removed based on functional relevance, and performance in immunoblot verifications for pull-down with RyR1. The remaining proteins, CHERP and FHL1 were selected for co-localization and functional studies.

57

58

Figure 10 - Proteins Identified by Mass Spectrometry from RyR1 Purifications (Left) Heat-map representations of proteins identified by mass spectrometry in all tandem affinity purified samples from HEK-293 cells. Color intensities depict total spectral counts (as a function of log10). (Middle) Original RyR1 MS datasets were filtered to remove proteins common to either negative or tag control purifications. Heat-map represents proteins detected only in RyR1 purifications. Proteins were arranged based on frequency of detection and intensity. (Right) Specific cluster from B, identifying proteins detected in multiple RyR1 experiments, and thus absent from controls.

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Protein Run 1 Run 2 Run 3 RYR1 Isoform 1 of Ryanodine receptor 1 98 113 59 ASCC3L1 Isoform 1 of U5 small nuclear ribonucleoprotein 200 kDa helicase 17 14 3

GTPBP4 Nucleolar GTP-binding protein 1 3 6 6 SFRS10 Isoform 1 of Splicing factor, arginine/serine-rich 10 28 32 0 PRPF8 Pre-mRNA-processing-splicing factor 8 20 16 0 MYCBP2 MYC binding protein 2 21 10 0 DHX30 Isoform 1 of Putative ATP-dependent RNA helicase DHX30 15 10 0

GYS1 GYS1 protein 14 4 0 RPS9 40S ribosomal protein S9 8 2 0 GEMIN4 Component of gems 4 6 7 0 MRPS22 Mitochondrial 28S ribosomal protein S22 5 2 0 RPS4X 40S ribosomal protein S4, X isoform 5 6 0 RPS24 Isoform 1 of 40S ribosomal protein S24 4 3 0 PTCD3 Isoform 1 of Pentatricopeptide repeat-containing protein 3, mitochondrial precursor 4 3 0

RPS16 40S ribosomal protein S16 4 2 0 MRPS31 28S ribosomal protein S31, mitochondrial precursor 4 2 0 PRPF6 Pre-mRNA-processing factor 6 3 2 0 EDC4 Isoform 1 of Enhancer of mRNA-decapping protein 4 3 2 0 SMN2 Isoform SMN of Survival motor neuron protein 3 1 0 MRPS10 Mitochondrial 28S ribosomal protein S10 3 1 0 RPS6 40S ribosomal protein S6 2 4 0 ATP5B ATP synthase subunit beta, mitochondrial precursor 2 3 0 RPL11 Isoform 1 of 60S ribosomal protein L11 2 3 0 PTPLAD1 Protein tyrosine phosphatase-like protein PTPLAD1 2 2 0

MRPS25 Mitochondrial 28S ribosomal protein S25 2 2 0 WDR26 Isoform 1 of WD repeat-containing protein 26 2 2 0 RSBN1 round spermatid basic protein 1 2 2 0 YTHDC1 Isoform 1 of YTH domain-containing protein 1 2 2 0 MRPS9 mitochondrial ribosomal protein S9 2 2 0 RPS13 40S ribosomal protein S13 1 2 0 RPS26 OTTHUMP00000018641 1 2 0 PHKB Isoform 4 of Phosphorylase b kinase regulatory beta 1 1 0

HERC1 guanine nucleotide exchange factor p532 2 0 1 Table 1 - 33 Potential RyR1 Interactors Found in >1 MS Run This table identifies the repeatedly detected 33 potential RyR1 interactors from figure 9 (yellow), ranked according to the number of purifications detected from (out of 3) and average intensity. Also provided is their corresponding total spectral counts detected during MS analysis of each purification. Highlighted in red are the only two proteins that were not functionally classified as ribosomal, mitochondrial, spliceosomal, histone and heat shock associated proteins. Phosphorylase b kinase was subsequently further investigated as a potential RyR1 interactor.

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Protein Fold Increase RYR1 Isoform 1 of Ryanodine receptor 1 90 CCT6A T-complex protein 1 subunit zeta 31 TCP1 T-complex protein 1 subunit alpha 28 LMAN1 Protein ERGIC-53 precursor 26.25 CCT5 T-complex protein 1 subunit epsilon 25.5 SFRS10 Isoform 1 of Splicing factor, arginine/serine-rich 10 20 CAD CAD protein 19 RPS15 40S ribosomal protein S15 16.6 VIM Vimentin 15.75 KRT14 Keratin, type I cytoskeletal 14 14.5 PRPF8 Pre-mRNA-processing-splicing factor 8 12 ASCC3L1 Isoform 1 of U5 small nuclear ribonucleoprotein 200 kDa helicase 11.33 CCT2 T-complex protein 1 subunit beta 10.8 MYCBP2 MYC binding protein 2 10.33 USP9X ubiquitin specific protease 9, X-linked isoform 4 10 RPS2 40S ribosomal protein S2 10 hCG_21078 hypothetical protein LOC389435 9.5 DHX30 Isoform 1 of Putative ATP-dependent RNA helicase DHX30 8.33 ATXN2L Isoform 1 of Ataxin-2-like protein 8 SF3B1 Splicing factor 3B subunit 1 7.25 CCT8 59 kDa protein 6.11 GYS1 GYS1 protein 6 EFTUD2 116 kDa U5 small nuclear ribonucleoprotein component 6 LUC7L2 Isoform 1 of Putative RNA-binding protein Luc7-like 2 6 PPP1CA protein phosphatase 1, catalytic subunit, alpha isoform 3 6 AKR7A2 Aflatoxin B1 aldehyde reductase member 2 6 RPS18;LOC100130553 40S ribosomal protein S18 5 GTPBP4 Nucleolar GTP-binding protein 1 5 FKBP1A Peptidyl-prolyl cis-trans isomerase 4.67 SF3B2 splicing factor 3B subunit 2 4.5 ZCCHC8 Isoform 1 of Zinc finger CCHC domain-containing protein 8 4.5 GEMIN4 Component of gems 4 4.33 CHERP Isoform 1 of Calcium homeostasis endoplasmic reticulum protein 4.33 TUBA1C Tubulin alpha-1C chain 4.25 Table 2 – Enrichment of Proteins in Cyto-RyR1 Purifications in Transfected HEK293 Cells LC-MS spectral counts were averaged for each of the three RyR1 runs and for each of the three control runs to attain the average number of spectral counts. For each protein the fold increase of each protein was calculated by dividing the average spectral count of the RyR1 runs by the average spectral count of the control. Here, is listed all proteins with a fold increase greater than 4.0. Highlighted in red are the only three proteins that were not functionally classified as structural, ribosomal, mitochondrial, spliceosomal, histone and heat shock associated proteins. TCP1, ERGIC-53 and CHERP were subsequently further investigated as a potential RyR1 interactor. In green, is FKPB1A, a known RyR1 interactor.

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Figure 11 - Heat-map of Proteins Found in >1 MS Run and Proteins Whose Average Spectral Count in RyR1 Runs had a Four-Fold Increase over Controls This heat-map represents the proteins listed in table 1, consolidated with proteins whose average spectral count in RyR1 runs saw a four-fold increase over control runs. The fold-enrichment analysis generated an additional 21 proteins, however, six of those proteins were found in the original analysis (listed in table 1), resulting in a consolidated list of 48 proteins.

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Protein Function TCP1 T-complex protein 1 subunit alpha Molecular chaperone LMAN1 Protein ERGIC-53 precursor Glycoprotein receptor

MYCBP2 MYC binding protein 2 E3 ubiquitin-protein ligase FKBP1A Peptidyl-prolyl cis-trans isomerase Cis-trans isomerase GYS1 Protein Glycogen synthase CHERP Isoform 1 of Calcium homeostasis endoplasmic reticulum protein

Knockdown inhibits cytosolic Ca2+ rise

USP9X ubiquitin specific protease 9, X-linked isoform 4 Ubiquitin protease Phosphorylase kinase beta Phosphorylase, ∆ = Calmodulin

Table 3 - Final List of Candidate Proteins Identified in Cyto-RyR1 Purifications in Transfected HEK293 Cells After consolidation of the proteins listed in tables 1 and 2, represented in a heat-map in figure 11, 48 proteins remained. Here in table 3, are the eight proteins that were not functionally classified as structural, ribosomal, mitochondrial, spliceosomal, histone and heat shock associated proteins. From this eight, one was a known RyR1 interactor (FKBP1A, in green) and four others (in red) met one of the following criteria: The protein is effected by, or causes effect on, cytosolic calcium levels (CHERP, PHKB), the protein is located in, or near, the SR membrane (CHERP, ERGIC-53), or the protein is a known chaperone, as it is unknown how RyR1 is organized into the SR (TCP, FHL1). These four proteins went on for interaction verification via immunoblot.

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A

B

Figure 12 - ELISA and MS Analysis of SEC Fractions with FL-RyR1 Proteins A) HEK293 cells were transfected with FL-RyR1 and were subsequently lysed and treated with solubilization buffer. This was applied to the SEC column. ELISA showing the 32 SEC fractions probed against RyR1. The Sepharose-300 column was pumped at 0.75 mL/min and the void volume of the column was 8 mL as determined by dextran blue. RyR1 soluble construct elutes in fractions 7-10, consistent with the literature that RyR1 elutes in the void volume. B) After SEC, ELISA was performed as previously described. Fractions containing visualized RyR1 (7-10) were then combined and subjected to tryptic digest before LC-MS analysis. Control fractions containing no RyR1 (23-26) were also subjected to LC-MS analysis. Shown are the % coverage (%); # unique peptides (# unique); molecular weight (Mol. Size); name of protein identified (Accession and Description) for the RyR1 peptides.

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A

B

Figure 13 – Immunoblot and MS Analysis of Elutant Fractions of Complexiolyte Buffer Solubilized RyR1 A) Immunoblot showing Ni-NTA eluted fractions of Complexiolyte solubilized C2C12 FL-RyR1 stable cells. 4 mL of each buffer was used to solubilize 2 plates of cells. First lane shows crude homogenate of C2C12-FL cell line. B) An aliquot of the elutants observed in the western above, was digested with trypsin and run on LC-MS. Shown are the % coverage (%); # unique peptides (# unique); molecular weight (Mol. Size); name of protein identified (Accession and Description) for the RyR1 peptides.

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Figure 14 – MS Identification of Mouse-D9-Cobalt Purification Products (Left) Heat-map representations of proteins identified by mass spectrometry in all tandem affinity purified samples from HEK-293 cells. Color intensities depict total spectral counts (as a function of log10). (Right) Original D9 MS datasets were filtered to remove proteins common to either negative or tag control purifications. Heat-map represents proteins detected only in D9 purifications. Proteins were arranged based on frequency of detection and intensity.

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Protein Fold Increase Ryanodine receptor 1 52.4 Hbb-b1 Hemoglobin subunit beta-1 7.33 Hbb Beta-2-globin 5 Fhl1 four and a half LIM domains 1 isoform 2 4 Table 4 - Enrichment of Proteins in D9-RyR1 Purifications Incubated with Mouse Skeletal Muscle Lysate LC-MS spectral counts were averaged for each of the three D9 runs and for each of the three control runs to attain the average number of spectral counts. For each protein the fold increase of each protein was calculated by dividing the average spectral count of the D9 runs by the average spectral count of the control. Here, proteins with a fold increase of 2 or greater are listed. Highlighted in red is FHL1, the only protein that is considered a potential RyR1 interactor based on functional relevance.

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Gene Name

Figure 15 - FHL1 Tandem Affinity Purification LC-MS Detection Data Experiments performed by Thiru Shathasivam MSc. A TAP-affinity tagged FHL1-SBP-CBP construct was transfected into HEK293 cells before a two-step purification, using firstly the streptavidin binding peptide (SBP) followed by calmodulin binding peptide (CBP).Runs 3 and 4 (containing RyR1) were from on-bead tryptic digested samples. This heat-map produced from filtered results, none of these listed proteins were found in control runs. Arrow, represents the bait RyR.

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Figure 16 - Preliminary Confirmation of RyR1 interacting candidates in HEK293 cells Top: Immunoblot analysis of over-expressed 6x-His tagged-RyR1 transfected HEK293 cell lysates and untransfected controls after sucrose density gradient purification and Ni-NTA purification. Fractions analyzed were the cell lysate, the flowthrough, or unbound fraction, which is lysate after incubation with Ni-NTA resin, the final wash and the elution. DHPR, a known interactor of RyR1 were used as positive controls. Bottom: RyR1 was pulled down by TAP purification of FHL1, performed by Thiru Shathasivam MSc.

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A

B C

Figure 17 - Subcellular Co-localization of CHERP and RYR1 in Skeletal Muscle A) Immunofluorescent analysis of subcellular distribution of endogenous CHERP and RYR1 in rat soleus skeletal muscle. CHERP was detected labeled with Alexa 488 (green) (first column), whilst RyR1 was alternatively labeled with Alexa 633 (red) (middle column). Regions of overlap are represented by yellow in the third column. The bottom right panel illustrates the co-localization at a higher magnification. B) Immunofluorescent analysis of subcellular distribution of endogenous CHERP and RYR1 in rat soleus skeletal muscle. RyR1 was detected labeled green (Alexa 488) and CHERP red (Alexa 633). Images were analyzed for 3D colocalization and 22.21% of the voxels within the region of interest seen above were determined to be colocalized. C) Magnification of image shown in B.

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A

B C

Figure 18 – Subcellular Co-localization of FHL1 and RYR1 in Skeletal Muscle A) Immunofluorescent analysis of subcellular distribution of endogenous FHL1 and RYR1 in rat soleus skeletal muscle. FHL1 was detected labeled with Alexa 488 (green, first column), whilst RYR1 was alternatively labeled with Alexa 633, (red, middle column). Regions of overlap are represented by yellow in the third column. The bottom right panel illustrates the co-localization at a higher magnification. B) Immunofluorescent analysis of subcellular distribution of endogenous FHL1 and RYR1 in rat soleus skeletal muscle. FHL1 was detected labeled green (Alexa 488) and RYR1 red (Alexa 633). Images were analyzed for 3D colocalization, but there was no colocalization (0%) determined by the algorithm. C) Magnification of image shown in B.

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A

B

Anti-CHERP Anti-FHL1

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Figure 19 – 340/380 Ratio Calcium Transients of RyR1 Expressing HEK293 Cells A) HEK293 were transfected on glass cover slips with RyR1 containing cDNA, followed by transfection 24 h later with either blank lipofectamine (+RyR1), or siRNAs knocking down either CHERP (+RyR1 – CHERP), FHL1 (+RyR1 – FHL1), or scramble siRNA (+RyR1 – Scramble) which knocks down no protein. 24 h after transfection with siRNAs, calcium transient 380/340 analysis was performed. Cells were incubated with 1 uM fura-2 for 1 h followed by a media change with no fura-2 for 10 minutes. Cells were then loaded into a cover slip holding unit where media was added to, and read at 340 and 380 nm every three seconds, over a period of 60 seconds, with addition of 5 mM caffeine at 30 seconds. All values were normalized by dividing by the baseline value. B) Western blot analysis showing CHERP and FHL1 protein levels in cells transfected first with RyR1, and then with empty lipofectamine (untransfected), CHERP, or FHL1 siRNAs. There were three siRNAs against CHERP and FHL1, they were tested individually and in tandem and it was found that tandem administration of the siRNA molecules yielded the highest knockdown of protein levels.

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P = 0.0127

P = 0.0320

0

5

10

15

20

25

30

35

40

+ RyR1 + RyR1 - CHERP + RyR1 - FHL1 + RyR - Scramble

Perc

enta

ge in

crea

se a

bove

bas

elin

e of

340

/380

nm

***

Figure 20 – Percentage Increase in 340/380 nm Ratio of RyR1 Expressing HEK293 Cells HEK293 were transfected on glass cover slips with RyR1 containing cDNA, followed by transfection 24 h later with either blank lipofectamine (+RyR1), or siRNAs knocking down either CHERP (+RyR1 – CHERP), FHL1 (+RyR1 – FHL1), or scramble siRNA (+RyR1 – Scramble) which knocks down no protein. 24 h after transfection with siRNAs, calcium transient 380/340 analysis was performed. Cells were incubated with 1 uM fura-2 for 1 h followed by a media change with no fura-2 for 10 minutes. Cells were then loaded into a cover slip holding unit where media was added to, and read at 340 and 380 nm every three seconds, over a period of 60 seconds, with addition of 5 mM caffeine at 30 seconds. The percentage increase of the 340/380 nm ratio over baseline values after the addition of 5 mM caffeine was calculated. Each condition was repeated two times with at least 10 cells measured per repeat.

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Chapter Four: Discussion I. Protein Complex Isolations

To identify novel RyR1 protein interactions, metal affinity chromatography was

applied in HEK-293 cell systems. This particular method was chosen for its efficiency,

potential for high-throughput applications, and capacity for purifying protein complexes

from in vivo systems. Divalent metal affinity purifications have been performed

efficiently from various organisms. However, successful application to higher eukaryotic

organisms, particularly mammalian cells, has continued to face several limitations.

Requirement of large cell mass, overall low yield of bait and interacting proteins, and

endogenous protein competition for interactors are some of the common issues faced.

(Porath et al. 1975; Hubert et al. 1980; Giannone et al. 2007)

For the purification of RyR1 containing protein complexes from mammalian cells,

HEK-293 cells proved to be the most practically applicable. Although RyR1 is expressed

predominantly in skeletal muscle tissue, the purification of a 2 MDa ion channel required

a cell type with relatively less of a cytoskeletal network, in order to adequately solubilize

the RyR1 proteins. Purifications were attempted in C2C12 mouse myotubes, however,

isolating RyR1 proteins was unsuccessful, suggesting that the highly complex contractile

muscle network didn’t allow for the solubilization of RyR1 proteins. Furthermore, due to

its relatively high transfection efficiency, HEK-293 cells also reduced the amount of

starting cells necessary when compared to C2C12 myoblasts, almost by one half.

However, due to the T4 promoter driven plasmid transcription, concerns of over-

expression consequences persisted. Unfortunately, multiple attempts to generate stably

expressing cells to resolve this issue were unsuccessful.

Considering the purification itself, the recovery and concentration of bait protein

from the cell lysate was satisfactory in a limited number of runs. However, the recovery

of the 6xHIS tagged RyR1 bait proteins bound to the Ni-NTA resin proved to be difficult.

In order to retrieve a decent proportion of the bound protein that was adequately pure,

multiple lengthy washes and a single elution were necessary. This issue resulted in

75

purification complications, hence the exploration of alternative purification methods,

particularly size exclusion chromatography, isolation of RyR1 proteins using the

Complexiolyte buffer system, and the generation and purification of small N-terminal

RyR1 truncations. Each method proved to possess particular benefits and disadvantages.

For instance, size exclusion chromatography showed a decent purification by ELISA

analysis of the column void volume, but transferring from the biochemistry to the

proteomic analysis was difficult, yielding low spectral counts for RyR1. The

Complexiolyte buffer system also showed good purification of RyR1 by western blot, but

the in-gel digest and the TCA precipitation of the original samples led to poor recovery of

RyR1 peptides. In comparison, passing a muscle lysate over bound D9-RyR1 fragment

worked well in achieving a high spectral counts in subsequent MS analyses.

The D9-RyR1 work was successful compared to the other methods for a number

of reasons. Firstly, producing the D9 fragment in bacteria prior to its binding to

mammalian muscle proteins is easier. Bacteria, grown in suspension, grow much faster

than HEK293 cells which are grown in monolayers. In addition, purification and

subsequent binding of mouse muscle lysates required similar, and delicate, biochemical

conditions throughout the entire experimental procedure. This allowed for a smoother

transition to the MS analysis. Finally, working with a smaller, more manageable bait

protein illustrated how difficult it was to express, and then attempt to purify a 2 MDa

transmembrane ion channel.

Regardless, a collection of 33 potential RyR1 protein interactions was identified

using metal affinity chromatography from HEK-293 cells. Individual analysis of these

proteins revealed that many were involved in biological processes which were not

directly related to RyR1 and calcium regulation, thus they were removed from contention

as potential interacting candidates. These proteins fell into categories such as metabolic

pathway proteins, cytoskeletal, ribosomal, spliceosomal, and heat-shock proteins. There

were a select few however, that were associated with calcium homeostasis, and muscle

processes, allowing us to efficiently pair down the list to a handful of proteins for further

analysis.

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In addition to this, although our stringent data filtering criteria removed many

known RyR1 interactors from final protein lists, there were known interactors identified

in both the RyR1 and D9 MS runs. FKBP1A was identified in numerous runs, and

FKBP1A spectral counts were an average of 4.67 enriched over controls. One DHPR

spectra was identified in one RyR1 run, but because it was only identified in a single run,

it did not meet the initial filtering criteria. Recently identified RyR1 interactor dysferlin

was identified in one RyR1 run. In the D9-RyR1 purifications, which had significantly

fewer proteins identified overall, well-characterized RyR1 interactor calsequestrin was

identified in two of three runs, while being found in no controls. These identifications

serve as positive controls for the expression and purification protocols.

Thus, the metal affinity chromatography method was moderately successful in

identifying potential interactors for a membrane embedded ion channel, which could be

mapped to a broad range of cellular processes. And while a variety of techniques are

available for identifying novel protein interactions, many of which were observed with

the cyto-RyR1, D9-RyR1, SEC, specialized buffers employed in thus study, each

methodology presents with its own set of pros and cons.

II. The Biochemistry-Mass Spectrometry Interface

A. Post-purification identification of RyR1 peptides via LC-

MS

Performing protein purifications of RyR1, a bait protein with complex

transmembrane sequences and a high molecular weight, led to numerous challenges in the

transfer between biochemically isolating the protein and preparing said proteins for MS

analysis. Finding a purification technique that was effective, yet did not employ materials

that interfered with MS analysis was a large task. The difficulty lied in the fact that the

biochemical purification of such a naturally insoluble and large protein required high

concentrations of detergents to remove it from the SR membrane. These detergents were

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necessary for the solubilization of the bait and were subsequently difficult to remove

before MS analysis. The detergents had a range of interfering effects; their non-volatile

nature meant they became more concentrated when the sample size was reduced for

loading onto the MS column, thus generating interfering spectra, and most likely causing

ion suppression. In addition, purifying the protein using Ni-NTA required significant

concentrations of imidazole throughout the binding and washing stages of the protocol

(as the volatile acetic acid could be used for elution). Imidazole, which is critical to the

purification process because it prevents non-specific binding to the Ni-NTA resin,

generates interfering spectra under MS analysis, and is almost impossible to completely

remove from a sample.

There were extensive efforts made throughout the experimental protocol to

determine the most effective method of detecting RyR1 proteins and potential interacting

partners by MS analysis. The recovery of 6xHIS tagged RyR1 bait bound to the Ni-NTA

resin proved to be difficult. In order to retrieve a decent proportion of the bound protein,

multiple lengthy elutions were necessary. This issue also resulted in complications to the

ideal tryptic digestion method for subsequent MS analysis, as we tried in-solution, in-gel,

and post-TCA precipitation digestions. Each method proved to possess particular benefits

and disadvantages. For instance, in-solution digestion identified the greatest number of

bait proteins, however, there was significant contamination by chemicals critical to the

purification process. In an attempt to remove these contaminating agents, SDS-PAGE

was employed on a 5-20% polyacrylamide gradient in order to remove detergents, salts

and imidazole. In-gel digestions identified very few bait peptides and a limited number of

overall proteins. Even after visualizing RyR1 by immunoblot in a duplicate lane and

excising an identical band, recovery of peptides from resolved SDS-PAGE gels proved to

be ineffective. Digestion after TCA precipitated samples was the most ineffective method

for identifying RyR1 peptides. After SEC, TCA precipitation was employed to

concentrate the high volume sample to a more manageable volume. The TCA pellet was

resolubilized in 8M urea (later diluted within the working range for trypsin), however,

resolubilization was not effective and lead to no recovery of bait proteins.

B. Identification of non-specific binding proteins

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Of the 856 combined total proteins identified in both the Ni-NTA RyR1 and

cobalt D9 purifications, only a handful had potential interest as possible RyR1

interactors. In particular, there were many ribosomal and heat-shock proteins that

managed to co-purify with the affinity tagged RyR1. Ribosomal proteins can co-purify

with bait proteins that are incompletely translated, but this only can occur in systems

expressing N-terminal affinity tags. Since our RyR1 was 6xHIS tagged at the C-terminal

end, this was not a possible explanation. Detergents used in the purification process may

have solubilized various ribosomal and heat shock proteins, thus, preventing them from

being removed by centrifugation. Washing the beads throughout the purification process

does not remove all contaminants, and thus contaminant proteins can make it through to

MS identification.

There are a number of shortcomings inherent to immobilized metal affinity

chromatography. Firstly, proteins of interest containing affinity tags need to be easily

solubilized, and fully soluble throughout the entire purification process. This makes the

purification of all transmembrane proteins, as well as those closely associated with

structural components very difficult.

This was illustrated in this study as the purification of cytosolic RyR1 proved to

be quite difficult throughout the research program, under a plethora of different

solubilization conditions. A minimal number of cytosolic RyR1 proteins bound to the Ni-

NTA resin provide additional opportunity for the binding of non-specific proteins. This

was also highlighted by the fact that the D9 RyR1 N-terminal fragment, demonstrated to

be highly soluble was purified with a much higher efficiency, with a vast reduction in the

number of total proteins identified, meaning there was markedly reduced non-specific

binding.

The issue of non-specific binding to the affinity resin is the main shortcoming

associated with metal affinity chromatography. As previously described, all untransfected

control runs performed throughout our research program involved the incubation of

immobilized metal ion resin into untransfected cellular lysates, obviously containing no

affinity tags. Yet, in each of these runs, after repeated washes and elution, hundreds of

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proteins were still identified by MS. Most immobilized metal ion resin is made of

agarose, which is a polysaccharide, a polymer of galactose molecules. Polysaccharides,

although not a strong candidate to abundantly bind cellular proteins, will inherently do so.

Finally, there are many contaminant proteins commonly found in proteomics

experiments that are present either by accident or through unavoidable contamination of

protein samples. These proteins include common laboratory proteins (i.e. albumin),

proteins added by accident through dust or physical contact (i.e. keratins) and proteins

used as molecular weight or mass spectrometry quantization standards. These proteins are

listed in the contaminant repository of adventitious proteins (cRAP)

(http://www.thegpm.org/crap/index.html).

III. Interactions with RyR1

A. CHERP

Calcium homeostasis endoplasmic reticulum protein, or CHERP, is a novel

protein that was first described in 2000 after the development of a monoclonal antibody

raised against IP3-receptor-rich dense-tubular-system membranes of platelets (West et al.

2002). This antibody inhibited Ca2+ release by IP3 receptor from internal membrane

vesicles derived from platelets, cerebellum, smooth muscle, sea urchin eggs and from

HEL cells, a human leukemic cell line with megakaryoblastic features. The antibody did

not react with the IP3-receptor and the protein with which it did, later characterized as

CHERP, and was localized to the ER and found to play a key role in the regulation of

intracellular Ca2+ mobilization. Like RyR1, CHERP is an integral membrane protein

spanning the cytosol to the lumen of the SR.

Further, more in depth, functional work was later performed in Jurkat cells.

Transfection of Jurkat cells with a lac-regulated mammalian expression vector containing

CHERP antisense cDNA caused a knockdown of CHERP and impaired the rise of

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cytoplasmic Ca2+ levels caused by phytohaemagglutinin (PHA) and thrombin (O'Rourke

et al. 2003). A 50% fall of CHERP decreased the PHA-induced rise of the cytoplasmic

free Ca2+ concentration, but calcium influx was unaffected. Furthermore, cell

proliferation was slowed. And while these findings provided further evidence that

CHERP is an important component of ER calcium mobilization, and that its loss impairs

Ca2+-dependent biochemical pathways and progression through the cell cycle, no

mechanism of these effects was ever offered, despite the finding that calcium influx was

not affected.

Jurkat cells also express RyR1 according to a 2001 study elucidating the

expression of RyR isoforms in immune cells (Hosoi et al. 2001). It was found that the

RyR-stimulating agent 4-chloro-m-cresol induced Ca2+ release and thereby confirmed

functional expression of the RyR in Jurkat cell lines expressing RyR1 mRNA. Since

calcium influx was unaffected, and because Jurkat cells express RyR1, it is plausible that

the CHERP suppression in the O’Rourke et al. study was responsible for lower cytosolic

calcium levels because of reduced RyR1 activity.

In the present study, CHERP suppression affected cytosolic calcium levels in a

similar way compared to the previously discussed study. Here, siRNA knockdown of

CHERP resulted in a reduction of total calcium release from the ER by 56%. Based on

the four-fold enrichment of CHERP spectra in MS-analyzed RyR1 purifications, and the

CHERP-RyR1 co-localization results, which saw subcellular localization of both CHERP

and RyR1 to the SR in skeletal muscle with 22.21% co-localized voxels when analyzed

by 3D software, it is plausible to suggest that CHERP directly interacts with RyR1 and

affects calcium release.

This correlation is not trivial however. Cytosolic calcium levels are determined by

numerous factors including calcium uptake by SERCA, extracellular calcium release by

the PMCA and the NCX, as well as calcium uptake and release by the mitochondria. The

properties of the calcium release channels and pumps associated with calcium

homeostasis within the cell indicate that RyR is the only calcium mover capable of

generating the magnitude of calcium release observed in the calcium transient results.

81

RyR and DHPR are the only calcium transporters that are channels, rather than a

pump. The RyR channel transports calcium ions at a rate of 1.566 x 107 ions per s-1 (Endo

2009), consistent with the typical ion channel rate in the order of magnitude of 107 ions

per s-1 (Gadsby et al. 2009). Calcium pumps, like the PMCA, NCX and the mitochondrial

calcium pumps transport calcium ions at a typical rate of 102 ions per s-1 (Gadsby et al.

2009). In addition to the fact that caffeine acts as a activator for only RyR in the group of

calcium transporters, and that RyR transports ions at five orders of magnitude faster than

other calcium pumps, the calcium transient amplitudes are a result of RyR calcium

release and that the knockdown of CHERP plays a role in this event.

Finally, it should it noted that while HEK293 cells have been shown to contain

RyR mRNA, and express minute numbers of endogenous RyR channels, in our calcium

transient work, there was no legible response to caffeine by wild-type HEK293 with

which we could generate a baseline value to subtract from all subsequent readings.

Because the amino acid sequence of CHERP described by O’Rourke et al.

contains two transmembrane domains at the N and C terminal ends of the protein

respectively, it can be suggested that CHERP interacts with RyR1 at the membrane. The

large loop between the transmembrane domains is predicted to be on the luminal side of

the ER membrane, and may cause effect of the luminal pore of RyR1, perhaps through

interaction with the various luminal loops of RyR1.

B. FHL1

FHL1 is a member of the four-and-a-half-LIM-only protein family. Family

members contain two highly conserved, tandemly arranged, zinc finger domains with

four highly conserved cysteines binding a zinc atom in each zinc finger. The LIM

domains have been proposed to function as modular protein-protein binding interfaces

upon which the coordinated assembly of multimeric protein complexes occurs. These

scaffold proteins are capable of interacting with other LIM domain containing proteins,

forming homo- or heterodimers. Furthermore, the presence of a LIM domain was

82

recently recognized as a potential hallmark of proteins associating with both the actin

cytoskeleton and transcriptional machinery.

It was previously shown here that there is clearly no direct interaction between

FHL1 and RyR1. However, considering the association between FHL1 and the

cytoskeleton, and the scaffolding properties of LIM proteins, it is plausible that FHL1

functions to indirectly anchor RyR1 to the underlying cytoskeleton.

Cytoskeletal proteins include an array of proteins which underlie and interact with

cell membrane elements. In mouse T-lymphoma cells, RyR was identified from internal

calcium storage vesicles, and an interaction was described with I-ankyrin (Bourguignon

et al. 1995). Ankyrin is a membrane-associated cytoskeletal protein which also

demonstrated a capacity to regulate internal calcium release during lymphocyte

activation. It also effectively prevented the binding and calcium release inhibitions by

ryanodine (Bourguignon et al. 1995). Additionally, in cultured neuronal cells, disruption

of the actin cytoskeleton resulted in diminished RyR-mediated calcium release.

Disassembly of the actin cytoskeleton was caused by treatment with cytochalasin D,

which inhibits actin polymerization (Bose et al. 2009). In our studies, suppression of

FHL1 resulted in a significant decrease in total calcium release in HEK293 cells

expressing RyR1. It is plausible that an absence of the association between FHL1 and the

cytoskeleton alters the organization of the RyR1 macromolecular complex, thus affecting

its ability for optimal calcium release.

Moreover, an interaction has previously been described between FHL1 and

another ion channel protein, KCNA5. Patch clamp experiments demonstrated a

functional role for FHL1, whereby KCNA5 activity was modified by increased K+

current density, altered channel gatings, and enhanced slow inactivation (Yang et al.

2008).

Similarly, FHL2 was also discovered to interact with a pore-forming K+ channel

subunit, human ether-a-go-go-related gene (HERG) (Lin et al. 2008). HERG contributes

to the rapidly activating delayed rectifier potassium current (IKr), which is vital for action

potential repolarization in myocardium. When co-expressed in cells, FHL2 significantly

83

amplified the HERG current amplitude and accelerated the deactivation rate of the tail

currents (Lin et al. 2008). FHL2 may also be involved in repolarization of cardiac cells

via its interaction with the β-subunit minK of voltage-gated K+ channels encoding the

delayed rectifier current IKs (Kupershmidt et al. 2002). Thus, FHL1 protein may also

mediate a structural connection between RYR1 and the cytoskeleton, in addition to

regulating electrophysiology.

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Chapter Five: Limitations

The current study applied metal affinity chromatography to identify RyR1

interactions from HEK-293 cells. Our initial aim, to purify full-length and cytosolic

domains of RyR1, in excess of 1.5 MDa, proved to be highly difficult as there were

persistent issues of protein expression and solubility throughout the research program. In

addition, considering RyR1 is predominantly expressed in skeletal and cardiac muscles,

interactions would ideally have been identified from muscle cells. Thus, we attempted to

repeat the experimental approach in C2C12 skeletal muscle cells, however, poor

transfection efficiencies and purification from the C2C12 cells inhibited further

purification studies. Furthermore, concerns regarding the transient over-expression of

RyR1 in cells could not be appeased. Multiple attempts at generating stably expressing

cell lines were unsuccessful.

Using HEK293 cells in the experimental program had various attributes and

limitations. Firstly, the absence of a highly structuralized cytoskeletal network, as seen in

C2C12 myotubes, may have allowed for more efficient solubilization of RyR1 proteins.

In C2C12 myotubes, the cytosolic domain is occupied by the muscle contractile

components, which may have bound to soluble RyR1 proteins, removing them from the

soluble fraction of the cellular lysate.

HEK293 cells however, express many RyR1 interactors at low levels, or not at all.

In addition, HEK293 over-expression systems, generate bait protein levels to that way

above physiological levels. This can result in the binding of potential interactors at

elevated concentrations of bait protein, however, these said interactions may not exist at

physiological levels.

Another limitation to the entire process was the loss of interacting proteins at

various stages. First, transient and weak interactions were inherently lost during the

purification process with multiple washes and varying elution efficiencies. Interacting

proteins were also lost during the tryptic digestion process and solid-phase extraction. In

addition, low abundant proteins are often overlooked during MS analysis, masked by the

85

presence of more abundantly present proteins. Without at least two unique peptides,

these proteins would have failed to make the minimum requirements for further

consideration.

One potential strategy for avoiding this limitation is the chemical cross-linking of

transient protein complexes. In this experimental program, a mass spectrometry

compatible chemical cross-linker, such as disuccinimidyl tartarate (DST) or

bis(sulfosuccinimidyl) suberate (BS3), could be applied after the initial lysis of the RyR1

over-expressing cells, allowing transient interactions to be immobilized in a RyR1 protein

complex prior to metal affinity chromatography purification and characterization.

With respect to MS identification, we experienced difficulty between the

biochemical purification of protein and the proteomic analysis of those proteins. In-

solution, on-bead, and in-gel digestion methods each presented various limitations. In

addition, during the purification process, buffer compatibility with the mass spectrometer

posed a continued limitation as imidazole and detergents necessary for RyR1 solubility

were non-compatible with MS analysis methods.

Considering the validation of direct protein interactions, an alternative method is

necessary to strengthen the association between CHERP and FHL1 with RYR1.

Colocalization verification via confocal microscopy is limited by its resolution. Co-IP is

also not plausible because RyR1 is a massive size, has solubility issues, and lacks a

commercial antibody that is capable of co-IPs. For this, forster resonance energy transfer

(FRET) is a method that could be utilized for better resolution and identification of a

direct interaction. With a resolution in the range of 10Å, it provides a 10-fold increase in

resolution over conventional fluorescence microscopy, elevating any results from

subcellular localization of each protein, to identifying actual protein-protein interactions.

86

Chapter Six: Future Directions

In the present study, 33 potential RyR1 potential interactions were identified, with

validation and functional relevance experiments performed for two potential interactions.

For generation of a larger interaction network including the identification of additional

candidates, the isolation and identification of RyR1 molecules via mass spectrometry

would need improvement. The issue of RyR1 expression and purification was exhausted,

and it was concluded that moving forward, smaller and soluble fragments of RyR1 that

lie in the MH/CCD domains should be pursued. We explored this with the D9-RyR1

fragment, and the potential for more easily isolatable constructs like it are the type of bait

candidates that should be pursued.

For CHERP, in which a potential novel interaction with RyR1 was established,

characterization of the interaction is warranted. Since RyR1 exists as a large

homotetramer with various domains that mediate different interactions, interaction

domain analysis may be required. In addition, the kinetics of binding affinities can also

be assessed. For instance, Biacore™ sensor chips can be prepared with immobilized

RyR1 domains and subjected to an analyte solution consisting of an interacting protein.

The automated Biacore™ system can then provide details regarding, for instance, the

interaction kinetics (i.e. association and dissociation), affinity constants, and

concentration dependencies.

Aside from the current interaction study, other studies involving RyR1 can be

performed. The long-term goal, that inspired this project, was to contribute to the

elucidation of how RyR1 is regulated at a molecular level. But finding RyR1 interacting

partners is only one of many necessary pieces of the puzzle. Moving forward, elucidating

the structure at a low resolution, as well as assigning function to the known and unknown

post-translational modifications of RyR1 will be essential to achieving the overall aim.

While high-resolution structure elucidation of the intact RyR1 channel is a long

way away, crystallization of smaller, more manageable fragments of RyR1 should be a

87

priority moving forward. In addition, the discovery of new, and characterization of

known post-translation modifications of RyR1 could be pursued.

88

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