The Extracellular Matrix November 19, 2015 Jeff Miner, Ph.D. Renal Division 7717 Wohl Clinic...

The Extracellular MatrixNovember 19, 2015

Jeff Miner, Ph.D.Renal Division

7717 Wohl Clinic362-8235

[email protected]

All multicellular animals have ECM.

Why do all multicellular animals have ECM?

• Acts as structural support to maintain cell organization and integrity (endothelial tubes of the cardiovascular system; mucosal lining of gut; skeletal muscle fiber integrity)

• Compartmentalizes tissues (pancreas: islets vs. exocrine component; skin: epidermis vs. dermis)

• Provides hardness to bone and teeth (collagen fibrils become mineralized/calcified)

• Presents information to adjacent cells:• Inherent signals (e.g., RGD motif in fibronectin)• Bound signals (BMP7, TGFβ, FGF, SHH, etc.)

• Serves as a highway for cell migration during development (neural crest migration), in normal tissue maintenance (intestinal mucosa), and in injury or disease (wound healing and cancer)

Types of ECMs

• Basement membrane (basal lamina)• Epithelia, endothelia, muscle, fat, nerves

• Elastic fibers• Skin, lung, large blood vessels

• Stromal or interstitial matrix• Bone, tooth, and cartilage• Tendon and ligament

Generalizations• Most ECM proteins are large, modular,

multidomain glycosylated or glycanated proteins• Some domains recur in different ECM proteins

• Fibronectin type III repeats• Immunoglobulin repeats• EGF-like repeats• Laminin Globular (LG) domain• others• Exon shuffling the likely mechanism

Perlecan

The Major Basement Membrane Proteins

Perlecan

LM-511 α1α1α2

Major BM Proteins

• Can individually polymerize to form a network• Laminin• Collagen IV

• Linkage, regulatory, other functions• Perlecan, Nidogen, Agrin

• Glucoseaminoglycan (GAG) side chains• - impart negative charge to the BM

• In general, basement membranes appear very similar to each other by EM.

• But all are not alike!• There is a wealth of molecular and functional

heterogeneity among basement membranes, due primarily to isoform variations of basement membrane components.

Basement Membranes

• Cell proliferation, differentiation, and migration• Cell polarization and organization, as well as

maintenance of tissue structure• Separation of epithelia from the underlying

stroma/mesenchyme/interstitium, which contains a non-basement membrane matrix

• Kidney glomerular filtration (barrier between the bloodstream and the urinary space)

Basement Membranes are Involved in a Multitude of Biological Processes

LamininHeterotrimers are composed of

one a , one b, and one c chain.

• Major glycoprotein of basement membranes—it’s required!

• Chains are evolutionarily related.

• 15 heterotrimers described to date.

• Alpha chains are unique• contain a C-terminal laminin

globular “LG” domain, ~100 kDaLM-521

Laminin Trimers Polymerize

• Laminin chains assemble into trimers in the ER and are secreted as trimers into the extracellular space.

• Full-sized laminin trimers can self-polymerize into a macromolecular network through short arm-short arm interactions.

• The a chain LG domain on the long arm is left free for interactions with cellular receptors.

Receptor-mediated Assembly

Involves LG domains and receptors on the surface of cells.Results in laminin polymerization and signal transduction.

Sulfated Proteoglycans

• Have protein cores with large glycosaminoglycan (GAG) side chains (from 1 to >100) attached to serines

• Some PGs contain heparan sulfate• Perlecan, Agrin, Collagen XVIII

(endostatin)

• Others contain chondroitin, keratan or dermatan sulfate

• GAG chains are responsible for most of the biological properties of proteoglycans and provide negative charge to basement membranes

• Hydrated• Enriched in cartilage (lubrication)

Proteases Release Anti-Cancer PeptidesCleavage of Matrix proteins to peptides

MMP = Matrix MetalloproteinaseMT-MMP = Membrane-Tethered MMP

From Zent and Pozzi, 2005

Laminin cleavages

The Collagens

• The most ubiquitous structural protein. A triple helical protein containing peptide chains with repeating Gly-Xaa-Yaa (usually Pro) triplets.

• The triple helix forms through the association of three related polypeptides (α-chains) forming a coiled coil, with the side chain of every third residue directed towards the center of the superhelix. Steric constraints dictate that the center of the helix be occupied only by Glycine residues.

• Many Proline and Lysine residues are enzymatically converted to hydroxyproline and hydroxylysine.

• ~28 distinct collagen types; each is assigned a Roman numeral that generally delineates the chronological order in which the collagens were isolated/characterized.

Collagen IV Network

Trimers (aka protomers) associate with each other, four at the N-terminus and two at the C-terminus (hexamer), to form a chicken wire-like network that provides strength and flexibility to the basement membrane.

Fibrillar Collagens (I, II, III, V)• Connective tissue proteins that

provide tensile strength• Triple helix, composed of three a

chains• Glycine at every third position (Gly-

X-Y)• High proline content

• Hydroxylation required for proper folding and secretion

• Found in bone, skin, tendons, cartilage, arteries

Biosynthesis of Fibril-forming Collagens

Adapted from: Keilty, Hopkinson, Grant. In: Connective Tissue and Its Inheritable Disorders, Wiley-Liss, 1993.

Prolyl hydroxylasesLysyl hydroxylaseGlycosyltransferases

Procollagen N- and C- proteinasesLysyl oxidase

Collagen Crosslinking

• Once formed, collagen fibrils are greatly strengthened by covalent crosslinks that form between the constituent collagen molecules.

• The first step in crosslink formation is the deamination by the enzyme lysyl oxidase of specific lysine and hydroxylysine side chains to form reactive aldehyde groups.

• The aldehydes then form covalent bonds with each other or with other lysine or hydroxylysine residues.

• If crosslinking is inhibited (Lysyl hydroxylase mutations; vitamin C deficiency), collagenous tissues become fragile, and structures such as skin, tendons, and blood vessels tend to tear. There are also many bone manifestations of under-crosslinked collagen.

• Hydroxylation of specific lysines governs the nature of the cross-link formed, which affects the biomechanical properties of the tissue. Collagen is especially highly crosslinked in the Achilles tendon, where tensile strength is crucial.

Collagen Crosslinking

Scurvy

• Liver spots on skin, spongy gums, bleeding from mucous membranes, immobility, depression

• Caused by Vitamin C deficiency• Ascorbate is required for prolyl

hydroxylase and lysyl hydroxylase activities

• Acquired disease of fibrillar collagen

Illustration from Man-of-War by Stephen Biesty (Dorling-Kindersley, NY, 1993)

Bone is Composed of Mineralized Type I Collagen Fibrils

Mineral is Dahllite,similar to hydroxyapatite(contains calcium, phosphate, carbonate)

Bone is 70% mineral and 30% protein, mostly collagen

Different Types of Mutations in Collagen I aChain Genes Cause Different Disease Severities

Gene location mutation SyndromeCOL1A1 17q22 Null alleles OI type I

Partial deletions; C-terminal substitutions

OI type II

N-terminal substitutions OI types I, III or IV

Deletion of exon 6 EDS type VII

COL1A2 7q22.1 Splice mutations; exon deletions OI type I

C-terminal mutations OI type II, IV

N-terminal substitutions OI type III

Deletion of exon 6 EDS type VII

Osteogenesis Imperfecta(brittle bone disease)

Clinical: Ranges in severity from mild to perinatal lethal

bone fragility, short stature, bone deformities, teeth abnormalities, gray-blue sclerae, hearing loss

Biochemical: Reduced and/or abnormal Type I collagen

Molecular Genetics: Mutations in either Type I collagen gene, COL1A1 or COL1A2, resulting in haploinsufficiency or disruption of the triple helical domain (dominant negative: glycine substitutions most common)

COL1 Haploinsufficiency (Dominant)

Byers P. Connective Tissue and Its Inheritable Disorders 1993, pp317-50.

(α1)2α2

Dominant Negative COL1 Mutations

Byers P. Connective Tissue and Its Inheritable Disorders 1993, pp317-50.

Gly subst. in COL4A2*

*Gly subst. in COL4A1

½ of the trimers are abnormal

¾ of the trimers are abnormal

Elastin and Elastic Fibers Exhibit Rubber-Like Properties

• Physiological importance lies in the unique elastomeric properties of elastin. Found in tissues in which reversible extensibility or deformability are crucial, such as the major arterial vessels (esp. aorta), the lung and the skin.

• Elastin is characterized by a high index of hydrophobicity (90% of all the amino acid residues are nonpolar). One-third of the amino acid residues are glycine with a preponderance of the nonpolar amino acids Ala, Val, Leu, and Ile. As in collagen, one-ninth of the residues are proline (but with very little hydroxylation).

• Early in development, the elastic fibers consists of microfibrils, which define fiber location and morphology. Over time, tropoelastin accumulates within the bed of microfibrils.

Elastic Fiber Biogenesis

• Elastic fibers are very complex, difficult to repair structures

• There are two morphologically distinguishable components

• Microfibrils• Elastin

• Assembly follows a well-defined sequence of events:

1. Assembly of microfibrils2. Association of tropoelastin aggregates with

microfibrils3. Crosslinking of tropoelastins with each other

by lysyl oxidase to form polymers

Shifren and Mecham, 2006

Copyright ©2004 American Physiological Society

Ramirez, F. et al. Physiol. Genomics 19: 151-154 2004;doi:10.1152/physiolgenomics.00092.2004

Major steps underlying the assembly of microfibrils and elastic fibers

Crosslinking

Microfibril Components: ~30

• Fibrillin--three forms• Microfibril-associated glycoproteins

(MAGPs)--two forms• Latent TGFb Binding Proteins (LTBPs)--

four forms• Proteoglycans, MFAPs, Fibulins,

Emilins, Collagens, Decorin, et al.

Fibrillins

• Large glycoproteins (~350 kDa) whose primary structures are dominated by Ca++ binding EGF domains (cbEGF) that, in the presence

of Ca2+, adopt a rodlike structure

• Limited intracellular assembly may occur, but microfibril assembly initiates at the cell surface after secretion, perhaps with the help of cellular receptors

• Members of the fibrillin superfamily• Maintain TGFb in the inactive state by forming the

“large latent complex”• TGF b – secreted signaling protein

• Promotes the expression of ECM

Latent TGFb Binding Proteins

Marfan Syndrome

• Caused by dominant Fibrillin-1 (FBN1) mutations

• Haploinsufficiency is the culprit

• Deficiency of elastin-associated microfibrils

• Syndrome seems to result from increased TGFb signaling, because there are not enough microfibrils present to bind TGFβ (and its associated proteins) to keep it inactive.

Cell-Matrix InteractionsNovember 24, 2015

Jeff Miner, Ph.D.

Renal Division

7717 Wohl Clinic

362-8235

[email protected]

Twitter: @JeffMinerPhD

Fibronectin

• A glycoprotein associated with many extracellular matrices and present in plasma/serum

• Alternative splicing generates many isoforms that heterodimerize covalently via S-S bonding

• Fibroblasts make it, assemble it, stick to it, and respond to it

• FN harbors the “RGD” motif (in domain III-10) that serves as a ligand for various integrins, especially a5b1

• Fn-/- mouse embryos die at E8.5 due to defects in the vasculature and in heart development

Mao and Schwarzbauer, Matrix Biol. 2005

Fibronectin and Branching Morphogenesis

Sakai et al., Nature 2003

Fibronectin and Branching Morphogenesis

Inhibiting FN expression with siRNA reduces branching

Adding FN promotes branching

Sakai et al., Nature 2003

Integrins Direct FN Fibril Formation

Mao and Schwarzbauer, Matrix Biol. 2005

Secreted compact soluble FN binds integrin

FN binding induces reorganization of actin and signaling

Cell contractility leads to changes in FN conformation, exposing FN interaction domains and allowing fibril formation

Integrins

• Large family of transmembrane receptors for extracellular matrix and cell surface proteins.

• Consist of an a and a b subunit, both with a single-pass transmembrane domain.

• 16 different a chains and 8 different b chains associate to form 22 distinct heterodimers.

• Cytoplasmic tails of both a and b chains mediate cell signaling events in response to ligand binding.

Integrins

• Some integrins bind to a specific site on matrix proteins, such as Arg-Gly-Asp (RGD), which is found in fibronectin, vitronectin, tenascin, et al.

• Ligand binding absolutely requires divalent cation** (Mg++ or Ca++)

• As mechanotransducers, integrins link the extracellular matrix to the force generating actin-myosin cytoskeleton. This both allows the cell to influence the nature of the extracellular matrix, and allows the ECM to influence cellular architecture and behavior.

Integrins Need to be Activated

• Integrin adhesiveness can be dynamically regulated through a process termed inside-out signaling.

• Ligand binding transduces signals from the cellular environment to the interior of the cell through outside-in signaling.

• Protein structure analyses have provided insights into the mechanisms whereby integrins become activated to bind ligand and how ligand binding translates to changes in intracellular signaling.

Adair and Yeager, Meth. Enzymol. 2007

Model for Integrin Activation

• Involves a switchblade-like motion when the headpiece extends

• Downward movement of the a7-helix leads to b subunit hybrid domain swing out, separation of the knees, and opening of the headpiece for high affinity ligand binding

• Activation can occur by PKC stimulation, GPCR activation, or binding of proteins such as talin to the b subunit tail.

• A delicate equilibrium among the different conformation states exists.

Anoikis (Greek for Homelessness)

• Apoptosis induced by inadequate or inappropriate cell/matrix interactions.

• Resistance to anoikis can lead to metastasis of epithelium-derived cancer cells (carcinomas).

Receptors for the Basement Membrane

• Cells are thought to recognize the basement membrane through receptors that interact with specific basement membrane components, primarily with laminin.

• Integrins• Dystroglycan

• Binding of receptors to the basement membrane can result in signal transduction and alterations in cell behavior.

Laminin-Binding Integrins

• a3b1, a6b1, a7b1, and a6b4

• They are found on the surface of many epithelial (a3 and a6), endothelial (a3, α6), and muscle (a7) cells.

• They bind primarily to laminin α chains and demonstrate some specificity.

• Their activities are modulated by members of the tetraspanin family of 4-pass transmembrane proteins

• CD9, CD81, CD151

Tetraspanin

Hemidesmosome Assembly vs. Disassembly

• The binding of integrin a6b4 to plectin plays a central role in HD assembly. Disrupting the association between these two proteins, through serine/threonine phosphorylation of the b4 cytoplasmic domain (perhaps by PKC and PKA), is a critical event in the disassembly of HDs.

• De-phosphorylation of residues distal to the plectin binding domain leads to unfolding of the tail, exposing the binding site for plectin.

• EGF signaling can lead to phosphorylation of integrin b4 and HD disassembly.

Discoidin Domain Receptors (DDRs)Bind fibrillar and BM collagens

Members of the transmembrane RTK family.

Two distinct family members: DDR1 and DDR2

DDR1: epithelial cells in lung, kidney, colon, and brain

DDR2: mesenchymal cells including fibroblasts, myofibroblasts, smooth muscle, and skeletal muscle

The N-terminal DDR discoidin domains are homologous to discoidin I, a secreted protein from the slime mold Dictyostelium discoideum

DDR1 binds to all known collagens, whereas DDR2 binds to fibrillar collagens

• Slow activation process vs. other RTKs

• Receptors exist as dimers even before ligand stimulation.

• Collagen stimulation induces rapid aggregation and internalization of the receptor

Mechanism of Activation

Dystroglycan

• Highly glycosylated

• Dystroglycan is involved in and perhaps necessary for laminin polymerization at the surface of some cells

• Laminin polymerization initiates basement membrane formation (certain cell types).

• Dystroglycan KO embryonic stem cells cannot assemble soluble laminin at the cell surface

Dystroglycan Function Requires Extensive Glycosylation

• DG isolated from certain muscular dystrophy patients or mice does not bind a DG antibody with an epitope dependent on glycosylation

• This DG also shows reduced binding to laminin

• Six glycosylation enzymes are mutated in human muscular dystrophies (called “dystroglycanopathies”)

• The protein core of DG has little receptor function on its own; glycosylation is critical!

• MD is a disease characterized by defective muscle cell/matrix interactions.

Martin, P. T. Glycobiology 2003 13:55R-66R

Indirect Promoters of Muscle Pathology in Muscular Dystrophy

Heydemann et al., J. Clin. Invest. 2009

A polymorphism/mutation in LTBP4 impacts disease in a mouse model of muscular dystrophy.

Genetic modifiers of disease (different backgrounds)

Polymorphisms in human LTBP4 impacts disease in patients with Duchenne’s muscular dystrophy.

Basement Membrane Proteins Regulate Mammary Cell Gene Expression:

Streuli et al,J. Cell Biol. 1991

What is the Active EHS Matrix Component?Which Receptors Recognize It?

• Dystroglycan and integrins cooperate to organize laminin, transduce the information from the ECM, induce cell polarization, and activate expression of milk proteins.

Weir et al., J. Cell Sci. 2006

Intracellular Protein Degradation

Chris Weihl MD/[email protected] of Neurology

Consequence of impaired protein degradation

• Protein aggregates• Ubiquitinated inclusions• Vacuolation (impairments in autophagy)• Damaged organelles• Secondary impairment in other cellular processes• Cell Death

• Underlying pathogenesis of degenerative disorders (neurodegeneration, muscle degeneration, liver degeneration, lung disease, aging)

Protein Degradation (regulated process)

Turnover of protein is NOT constant

Half lives of proteins vary from minutes to infinity

“Normal” proteins – 100-200 hrs

Short-lived proteinsregulatory proteins

enzymes that catalyze committed stepstranscription factors

Long-lived proteinsSpecial cases (structural proteins, crystallins)

Protein Degradation

Example: Lactic Acid DehydrogenaseTissue Half-lifeHeart 1.6 daysMuscle 31 daysLiver 16 days

• May depend on tissue distribution

• Protein degradation is a regulated processExample: Acetyl CoA carboxylase

Nutritional state Half-lifeFed 48 hoursFasted 18 hours

Protein Degradation Ubiquitin/Proteasome Pathway

80-90%Most intracellular proteins

• Lysosomal / Autophagosomal / Endosomal processes

10-20% Extracellular proteins

Cell organellesSome intracellular proteins

UBIQUITIN

K

G

Small peptide that is a “TAG” 76 amino acids C-terminal glycine - isopeptide

bond with the e-amino group of lysine residues on the substrate

Attached as monoubiquitin or polyubiquitin chains

Ubiquitination of proteins is a FOUR-step process

First, Ubiquitin is activated by forming a link to “enzyme 1” (E1 ubiquitin ligase).

Then, ubiquitin is transferred to one of several types of “enzyme 2” (E2).

Then, “enzyme 3” (E3) catalizes the transfer of ubiquitin from E2 to a Lys e-amino group of the “condemned” protein. Where specificity occurs.

Lastly, molecules of Ubiquitin are commonly conjugated to the protein to be degraded by E3s & E4s (chain extension)

AMP

The UPS is enormous!

The genes of the UPS constitutes ~5% of the genome

• E1’s- 1-2 activating enzymes

• E2’s- 10-20 conjugating enzymes

• E3’s- 500-800 ubiquitin ligase- drives specificity

• DUBs- 100 ubiquitin specific proteases- regulators of pathway

The UPS is enormous!

The genes of the UPS constitutes ~5% of the genome

E1’s- 1-2 activating enzymes E2’s- 10-20 conjugating enzymes E3’s- 500-800 ubiquitin ligase- drives specificity DUBs- 100 ubiquitin specific proteases- regulators of pathway

De-ubquitinases

PROTEASOME COMPONENTS

20S Proteasome(Catalytic core, ATP independent)

19S Particle (cap, recognizes ubiquitin tag, deubiqutinase (Usp14), ATP dependent (AAA ATPase, unfolds the protein)

26S Proteasome

MURF/Atrogin – E3 ligaseConfer specificity for myosin

Knockout of Atrogin (E3) Rescues atrophy

Dynamic regulation:Proteasome inhibition increases deubiquitinase activity Increased expression of deubiquitinase impairs protein degradation

Decrease steady-state levels of aggregate prone proteins in the absence of DUB Usp14 (pharmacologic inhibitors are coming online)

Lee, BH et alNature 467:179-842010

Autophagy – lysosomal degradation

• Lysosomal degradation of proteins and organelles• Occurs via three routes

• Macroautophagy• Microautophagy (direct uptake of cellular debris via the

lysosome)• Chaperone mediated autophagy (selective import of

substrates via Hsc70 and Lamp2a)

Double layer membrane

Direct invagination of cytosolic components

Direct insertion of proteins into lysosome

Macroautophagy

Autophagosome

Induction(Stress, starvation, etc)

mTOR

BeclinATG7

SequestrationPhagophoreATG5-ATG12-ATG16L1

Nucleation

Lysosome

Autolysosome

Degradation

FOXO3

Trafficking Fusion

“Autophagic Flux”

& Cargo loading

• Degeneration of CNS tissue• Hepatomegaly in Liver; Komatsu et al 2005

• Atrophy and weakness of skeletal muscle; Masiero et al 2009

• Pathologic similarities• Ubiquitinated inclusions• Aberrant mitochondria• Oxidatively damaged protein

Tissue specific requirements of autophagy

Complete loss of ATG5 leads to lethality

Basal Autophagy

• Autophagy has a “housekeeping” role in the maintenance of cellular homeostasis

• Autophagy is responsible for the clearance of ubiquitinated proteins

Selective Autophagy

• Aggregaphagy– p62/SQSTM1, Nbr1• Mitophagy – Parkin, Nix• Reticulophagy – endoplasmic reticulum• Ribophagy – translating ribosomes• Xenophagy – e.g. Salmonella via optineurin• Lipophagy – autophagy mediated lipolysis

• Performed by an expanding group of ubiquitin adaptors

LC3 on the autophagosome membraneVia receptor, pull autophagic cargo into the growing autophagosome

Ubiquitin adapter proteins UBA and LIR domains

p62 as an autophagic tool• p62 associates with ubiquitinated proteins and LC3• p62 is an autophagic substrate

• Used to monitor autophagic degradration• Autophagosome and its contents get degraded

Lysosomal inhibitionProteasome inhibition

LC3 as an autophagic tool

LC3-I (18kD)

(Soluble) LC3-II (16kD)

GFP-LC3

starved

Why do autophagosomes accumulate?

• Upregulation of functional autophagosomes

• Decrease in autophagosome degradation or “autophagic flux”• Phagophore closure• Autophagosome-lysosome fusion• Absence of functional lysosomes

Rapamycin as an inducer of autophagy

Immunosuppressant used to treat transplant rejection Inhibits the mTOR pathway mTOR integrates extrinsic growth signals and cellular

nutrient status and energy state Active mTOR

Protein synthesis and cell growth Inactive mTOR (rapamycin, mito damage, starvation)

Inhibition of protein synthesis and increased autophagic degradation of protein

TITLE PAGE INTRODUCTION THE PROCESS SIGNALING PATHWAYS BCL-2 PROTEINS PORE FORMING STRUCTURE

INITIATIN

HISTORICAL LANDMARKS AND NONLINEAR DEVELOPMENTAL PROGRAM

THE CELL BIOLOGY OF

APOPTOSIS

TO LIVE IS TO DIE – METALLICA(2007)

Paul H. SchlesingerDepartment of Cell Biology and Physiology

Office McDonnell 401Washington University Medical School

[email protected]

December 9, 2014

SCHLESINGER

(WUMS)APOPTOSIS DECEMBER 9,

201477 / 37

mailto:[email protected]

TITLE PAGE INTRODUCTION THE PROCESS SIGNALING PATHWAYS BCL-2 PROTEINS PORE FORMING STRUCTURE INITIATIN

MOTIVATIONS FOR THE STUDY OF CELL DEATH

DRAMATIC, UNIVERSAL, INTEGRATED

Apoptosis is characteristic of plants and metazoans≡animals

Allows for non-linear development – e.g. temporary and scaffold type structures

Immense change in membrane structure during apoptosis, but membrane integrity is maintained

Most cancers suppress apoptosis – different mutations

Many viruses suppress apoptosis

Apoptosis monitors the cell for stressPast a threshold – programmed cell death

SCHLESINGER


20142 / 37


CLASSIFICATION OF CELLULAR DEATH

HOW CELLS ACHIEVE MORTALITY

Apoptosis (Programed Cell Death)

Necrosis – cell loses control of environment, parts of it are genetically programmed (e.g. autophagy)

Autophagy – see Weihl lecture

Senescence – telomeric shortening, genotoxic damage

SCHLESINGER


20143 / 37


INITIATIN

CLASSIFICATION OF CELLULAR DEATH

Cellular death can

be Initiated by:

Stress

Death Receptors

DNA Damage

Cell Infection

SCHLESINGER


20144 / 37

Nuclear changes associated with Cell Death

Pyknosis – Nuclear Shrinkage

Karyorrhexis – Nuclear/Chromatin Fragmentation

Karyolysis – Nuclear/Chromatin Dissolution

Mitochondrial Permeability Transition

Apoptosis occurs at the Outer Mitochondrial Membrane

Inner mitochondrial membrane is impermeable to protons

Loss of this permeability barrier occurs through a series of events

Impairment in ATP production

Swelling of mitochondria and rupture of OMM -> release of CytC


MORPHOLOGICAL

APOPTOSIS: MORPHOLOGY

Morphological Progression

Retain Membrane Barriers

SCHLESINGER


20149 / 37


MORPHOLOGICAL


Apoptotic Cells Shrink

Intact Membranes — Volume Reduction — Membrane Channel Activity

SCHLESINGER


20149 / 37


MORPHOLOGICAL


Phagocytosis

phosphotidylserine as a signal for phagocytosis

SCHLESINGER


20149 / 37

APOPTOSISIn

itiat

ion

Exe

cutio

n

Stress

Genetic Clonal Selection

Development

Death Decision

Cytochrome c

Caspases

Nucleases

Membrane Packaging

Cytochrome c – released from IMM

APOPTOSISIn

itiat

ion

?E

xecu

tion

Stress

Genetic Clonal Selection

Development

Death Decision

Cytochrome c

Caspases

Nucleases

Membrane Packaging

ATP Dependence


CE,FLY,MOUSE

APOPTOSIS CHANGE ACROSS CHORDATA

Apotosis is different across species, but same basic rheostat mechanism:

SCHLESINGER


201412 / 37


CE,FLY,MOUSE

APOPTOSIS CHANGE ACROSS CHORDATA

Apotosis is different across species, but same basic rheostat mechanism:

Commonalities:

Proteins with Caspase activation and recruitment domains (CARD): Ced-4, , dark, Apaf-1

Caspase activation requires mitochondrial membranes and soluble proteins

The combined protein-protein and protein-membrane interactions are critical to the regulation of apoptosis

SCHLESINGER


201412 / 37


MODES OF APOPTOSIS

EXTRINSIC PATHWAY OF APOPTOSIS

Engage Cell Surface “Death” Receptor- Activates Caspase 8, initiator

caspase- Cleaves BID

- Causes Mito to release CytC

SCHLESINGER


201413 / 37


MODES OF APOPTOSIS

INTRINSIC PATHWAY OF APOPTOSIS Consensus Intracelluar Stress Sensing

Conformational change in Bcl2 family proteins (BAX, BAK)

BAX and BAK and/or Mito permeability transition cause CytC release (point of no return)

Caspase 9 activation (Intrinsic pathway is “Caspase independent”)

BID is an activator of BAX (Changes its conformation)

SCHLESINGER


201414 / 37


CASPASES

EXECUTION BY CASPASES

cysteine-aspartic-acid-proteases Regulatory and signalling proteins

SCHLESINGER


201415 / 37


INITIATIN

TUMOR SUPPRESSOR P53DIRECT GENETIC AND APOPTOSIS

CONTROL

p53 is a tumor suppressor found in the nucleus and cytosol.

Genotoxic and oncogenic stress that stabilizes p53 which transcriptionally regulates cell-cycle arresting and apoptosis genes.

The cytosolic p53 induces apoptosis.

p53 has a BH3 interaction site – Bcl2 family member?SCHLESINGER


201418 / 37


HISTORY

BCL-2 PROTEINS

B-cell Lymphoma 2 Gene, BCL-2

The constitutitve expression of this protein resulted from a gene translocation in chromosomes 14 and 18 of B-cell follicular lymphomas

Loss of Programmed Cell Death

Genetic analysis in C. elegans suggested effector (EGL-1) and repressor (CED-9) functions

The biochemical basis of BCL-2 action was unknown

BCL-2 associated x-protein BAX

Proposed Neutralizatin Interaction

Homology and Interaction Now Defines a Family of ≈25 Proteins

SCHLESINGER


201420 / 37


HISTORY

BCL-2 PROTEINS

B-cell Lymphoma 2 Gene, BCL-2

Genetic analysis in C. elegans suggested effector (EGL-1) and repressor (CED-9) functions

The biochemical basis of BCL-2 action was unknown

BCL-2 associated x-protein BAX

Isolated by immunoprecipitation of BCL-2, sequenced and clonedSignificant, domain specifc homology with BCL-2



SCHLESINGER


201420 / 37


HISTORY

BCL-2 PROTEINS

B-cell Lymphoma 2 Gene, BCL-2Genetic analysis in C. elegans suggested effector (EGL-1) and repressor (CED-9) functionsThe biochemical basis of BCL-2 action was unknownBCL-2 associated x-protein BAX


BCL-2 overexpression prevents death

BAX overexpression sensitizes to pro-apoptotic stress

Interaction is central to regulation


SCHLESINGER


201420 / 37


INITIATIN

FOLD HOMOLOGY – BAX and Colicin

Structural homology to colicinsColicins – bacterial toxins that make pores in membranes

Homologus regions of colicins insert to form channels

The oligomerize in target membranes forming a large pore

The pore tranports a toxin protein

Molten globule structure

SCHLESINGER


2014


INITIATIN

THE ROLE OF MITOCHONDRIA IN APOPTOSIS

Cytochrome c movement into the cytoplasm results in apoptosis

Cytosolic cytochrome c activates the apoptosome, caspase 9, and effector caspases

During apoptosis Bax translocates to the mitochondria

Bax oligomerizes in the mitochondria – larger, more

stable tetramer form allows CytC to leave

SCHLESINGER


201427 / 37


PORE ACTIVATION

BAX ACTIVATION – occurs via BID, inhibited by Bcl-xl

Inactive Bax doesn’t bind to membranes

Bcl-xl (Bcl-x-long) inhibits the activation of Bax

Bcl-xl binds BID strongly bind to each other

Hypothesis – Bcl-xl changes the PM structure – which inhibits the bax

SCHLESINGER


201429 / 37

040

0080

00

0.3

0.6

0.9BCL cBID (nM)

XL

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50 100

50 50

No

rma

lize

d P

ore

Ac

tiv

ati

on

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BH3-INHIBITOR

SEQUESTRATION AND DIRECT ACTIVATION

FCS – Fluorescence Correlation Spectroscopy

Direct measurement of protein interactions at single molecule level with statistical significance.

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The Extracellular Matrix November 19, 2015 Jeff Miner, Ph.D. Renal Division 7717 Wohl Clinic...

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Transcript of The Extracellular Matrix November 19, 2015 Jeff Miner, Ph.D. Renal Division 7717 Wohl Clinic...