Mrs. Ofelia Solano Saludar

Department of Natural Sciences University of St. La Salle

PROTEIN STRUCTURE, SORTING AND TARGETING

Overview of protein structure and function.

(a) The linear sequence of amino acids (10 structure)

folds into helices or sheets (20 structure)

which pack into a globular or fibrous domain (30

structure). Some individual

proteins self-associate into complexes (40

structure). (b) Proteins display

functions that arise from specific binding interactions and conformational

changes in the structure of a properly folded

protein.

Sometimes the primary sequence of amino acids is sufficient to spontaneously direct the folding of proteins into their proper shape.

However, often newly-made proteins require the help of molecular chaperones to attain their final shape.

Members of the heatshock protein family (Hsp70 and Hsp60) briefly bind to and stabilize hydrophobic regions of proteins (especially rich in Trp, Phe, Leu) allowing proper folding instead of aggregation with other immature proteins.

Heat-denatured proteins can be renatured through the activity of molecular chaperones and heatshock proteins are made during times of stress.

A number of diseases, including Alzheimer's disease, may be considered to be protein-folding diseases.

Prion diseases, such as "mad cow" disease, may "self-propagate" based upon a misfolded protein that can, in turn, misfold other versions of the same protein.

Proteins have to be folded into the proper three dimensional conformation to work

properly

Amyloid Fibers-involved in Alzheimer’s

Protein amyloid fibers are often found to have a β-pleated sheet structure regardless of their

sequence, leading some to believe that it is the molecule's misfolding that leads to aggregation.

Enzymes act on the APP (Amyloid Precursor Protein) and cut it into fragments of protein, one of which is

called beta-amyloid and is crucial in the formation of senile plaques in Alzheimer.

Chaperone- and chaperonin-mediated protein folding. (a) Many proteins fold into their proper 3-D structures with the assistance of Hsp70-like proteins (top). These

chaperones transiently bind to a nascent polypeptide as it emerges from a ribosome. Proper folding of other proteins (bottom) depends on chaperonins such as the

prokaryotic GroEL, a hollow, barrel-shaped complex of 14 identical 60,000-MW subunits arranged in two stacked rings. One end of GroEL is transiently blocked by the co-

chaperonin GroES, an assembly of 10,000-MW subunits. (b) In the absence of ATP or presence of ADP, GroEL exists in a “tight” conformational state that binds partly folded

or misfolded proteins. Binding of ATP shifts GroEL to a more open, “relaxed” state, which releases the folded protein.

ER protein folding The ER membrane-

bound chaperone protein calnexin, or a resident chaperone calreticulin binds to incompletely folded proteins, trapping

the protein in the ER. Glucosyl transferase determines whether the protein is folded properly or not: if the protein is

still incompletely folded, the enzyme renews the protein's affinity for calnexin & retains it in the ER. The cycle repeats

until the protein has folded completely.

The export and degradation of misfolded ER proteins. Misfolded soluble proteins in the ER

lumen or membrane proteins are translocated back into the cytosol, where they are deglycosylated, ubiquitylated, and degraded in proteasomes. Misfolded proteins are exported through the same type of translocator that mediated their import; accessory proteins allow it to operate in the export

direction.

Ubiquitin proteolytic pathway(a) Enzyme E1 is activated by

attachment of an ubiquitin (Ub) molecule (1) and then transfers

this Ub molecule to E2 (2). Ubiquitin ligase (E3) transfers the bound Ub molecule on E2

to the side-chain-NH2 of a lysine residue in a target

protein (3).Ub molecules are added to the target protein by repeating steps 1–3 , forming a polyubiquitin chain that directs

the tagged protein to a proteasome (4). Within this

complex, the protein is cleaved into small peptide fragments

(5). (b) Computer-generated image reveals that a proteasome has a cylindrical structure with a

cap at each end of a core region. Proteolysis of ubiquitin-tagged proteins occurs along

the inner wall of the core.

After the amino chain is made, many proteins undergo posttranslational processing (including removal of stretches of amino acids). 1. In prokaryotes, the N-formyl

group is always removed in the mature protein and often the methionine and, sometimes, a number of N-terminal amino acids are cleaved away from the final protein product.

Example: Proinsulin is converted to the active hormone by the enzymatic removal of a long internal section of polypeptide.

The two remaining chains continue to be covalently connected by disulfide bonds connecting cysteine residues in insulin.

2. Recently discovered, the process of protein splicing (analagous to RNA splicing) removes inteins and splices the exteins together to make a mature protein.

Free and bound populations of ribosomes are active participants in protein synthesis.

Free ribosomes are suspended in the cytosol and synthesize proteins that reside in the cytosol.

Bound ribosomes are attached to the cytosolic side of the endoplasmic reticulum.

They synthesize proteins of the endomembrane system as well as proteins secreted from the cell.

Secretory proteins are released entirely into the cisternal space, but membrane proteins remain partially embedded in the ER membrane.

While bound and free ribosomes are identical in structure, their location depends on the signal peptidase of proteins that they are synthesizing.

PROTEIN TARGETING AND SORTING

Overview of major protein-sorting pathways in

eukaryotic cells.

In cotranslational import, proteins to be targeted to the ER initially have an N-terminal peptide, the ER signal sequence, translated by a cytosolic ribosome.

The ER signal sequence is bound by a signal-recognition particle (SRP), a ribonucleoprotein complex composed of 6 peptides and a 300 nucleotide RNA molecule.

The SRP binds to the SRP receptor to dock the ribosome on the ER membrane. When the SRP receptor binds GTP, the nascent polypeptide enters the pore.

The SRP is released with hydrolysis of the GTP. The growing polypeptide translocates through a hydrophilic pore created by one or

more membrane proteins called the translocon. The ribosome fits tightly across the cytoplasmic side of the pore and the ER-lumen

side is somehow closed off until the polypeptide is about 70 amino acids long. When the polypepide is complete, the signal peptidase cleave the signal to release

the protein into the ER lumen while retaining the signal peptide, for a time, in the membrane.

Afterwards the ribosome is released and the pore closes completely.

Integral membrane proteins are inserted into the ER membrane as they are made, rather than into the

lumen.

Major topological classes of integral membrane proteins synthesized on the rough ER. The hydrophobic segments of the protein chain form helices embedded in the membrane bilayer; the regions outside the membrane are hydrophilic and fold into various conformations. All type IV proteins have multiple transmembrane

helices. The type IV topology depicted here corresponds to that of G protein–coupled receptors: seven helices, the N-terminus on the

exoplasmic side of the membrane, and the C-terminus on the cytosolic side. Other type IV proteins may have a different number of helices and

various orientations of the N-terminus and C-terminus.

Other kinds of signal peptides are used to target polypeptides to mitochondria, chloroplasts, the nucleus, and other organelles that are not part of the endomembrane system.

In these cases, translation is completed in the cytosol before the polypeptide is imported into the organelle.

Each of these polypeptides has a “postal” code that ensures its delivery to the correct cellular location.

In principle, a signal could be required for either retention in, or exit from a compartment.

Posttranslational import allows some polypeptides to enter organelles after protein synthesis. Like cotranslational

import into the ER, posttranslational import into a mitochondrion (and chloroplast) involves a signal sequence (called a transit sequence), a membrane receptor, pore-

forming membrane proteins, and a peptidase.

In the mitochondrion, the membrane receptor recognizes the signal sequence directly without the intervention of a cytosolic SRP.

Furthermore, chaperone proteins play several crucial roles in the mitochondrial process: o Chaperones keep the polypeptide partially

unfolded after synthesis in the cytosol so that binding of the transit sequence and translocation can occur.

o Chaperones drive the translocation itself by binding to and releasing from the polypeptide within the matrix, an ATP-requiring process

o Chaperones often help the polypeptide fold into its final conformation.

Protein import into the mitochondrial matrix. Precursor proteins synthesized on cytosolic ribosomes are maintained in an

unfolded or partially folded state by bound chaperones, such as Hsc70

(1). After a precursor protein binds to an import receptor near a site of

contact with the inner membrane (2), it is transferred into the general

import pore (3). The translocating protein then moves through this

channel and an adjacent channel in the inner membrane (4-5). Note that translocation occurs at rare “contact sites” at which the inner and outer

membranes appear to touch. Binding of the translocating protein by the

matrix chaperone Hsc70 and subsequent ATP hydrolysis by Hsc70 helps drive import into the matrix.

Once the uptake-targeting sequence is removed by a matrix protease and

Hsc70 is released from the newly imported protein (6), it folds into the mature, active conformation within

the matrix (7). Folding of some proteins depends on matrix

chaperonins.

Pathways for transporting proteins from the cytosol to the inner mitochondrial membrane.

In all three pathways, proteins cross the outer membrane via the Tom40 general import pore.

Proteins delivered by pathways A and B contain an N-terminal matrix-targeting sequence that is recognized by the Tom20/22 import receptor in the outer membrane.

Although both these pathways use the Tim23/17 inner-membrane channel, they differ in that the entire precursor protein enters the matrix and then is redirected to the inner membrane in pathway B. Matrix Hsc70 plays a role similar its role in the import of soluble matrix proteins.

Proteins delivered by pathway C contain internal sequences that are recognized by the Tom70 import receptor.

A different inner-membrane translocation channel (Tim22/54) is used in this pathway.

Two intermembrane proteins (Tim9 and Tim10) facilitate transfer between the outer and inner channels.

Two pathways for transporting proteins from

the cytosol to the mitochondrial

intermembrane space. Pathway A, the major one for

delivery to the inter-membrane space, is similar to pathway A

for delivery to the inner membrane.

The major difference is that the internal targeting sequence in proteins such as cytochrome b2 destined for the intermembrane space is recognized by an innermembrane protease, which cleaves the protein on the inter-membrane-space side of the membrane. The released protein then folds and binds to its heme cofactor within the intermembrane space. Pathway B involves direct delivery to the intermembrane space through the Tom40 general import pore in the outer membrane.

Two of the four pathways for transporting proteins from the

cytosol to the thylakoidlumen. In these pathways, unfolded

precursors are delivered to the stroma via the same outer-membrane proteins that

import stromal-localized proteins. Cleavage of the N-terminal stromal-import sequence

by a stromal protease then reveals the thylakoid-targeting sequence. At this point

the two pathways diverge. In the SRP dependent pathway (left), plastocyanin and

similar proteins are kept unfolded in the stromal space by a set of chaperones and,

directed by the thylakoid targeting sequence, bind to proteins that are closely related to the bacterial SRP, SRP receptor,

and SecY translocon, which mediate movement into the lumen. After the

thylakoid-targeting sequence is removed in the thylakoid lumen by a

separate endoprotease, the protein foldsinto its mature conformation. In the pH

dependent pathway (right), metal-bindingproteins fold in the stroma, and complexredox cofactors are added. Two arginineresidues (RR) at the N-terminus of thethylakoid-targeting sequence and a pH

gradient across the inner membrane arerequired for transport of the folded proteininto the thylakoid lumen. The translocon in

the thylakoid membrane is composed ofat least four proteins related to proteins in

the bacterial inner membrane.

(1) Catalase and most other peroxisomal matrix proteins contain a C-terminal PTS1 uptake-targeting sequence (red) that binds to the cytosolic receptor Pex5. (2) Pex5 with the bound matrix protein interacts with the Pex14 receptor located on the peroxisome membrane. (3) The matrix protein–Pex5 complex is then transferred to a set of membrane proteins (Pex10, Pex12, and Pex2) that are necessary for translocation into the peroxisomal matrix by an unknown mechanism. (4) At some point, either during translocation or in the lumen, Pex5 dissociates from the matrix protein and returns to the cytosol, a process that involves the Pex2/10/12 complex and additional membrane and cytosolic proteins. Note that folded proteins can be imported into peroxisomes and that the targeting sequence is not removed in the matrix.

Import of peroxisomal matrix proteins directed by PTS1

targeting sequence.

Mutations can affect protein structure and

function Mutations are changes in the genetic

material of a cell or virus. MUTATION AND DNA REPAIR MECHANISMS.pptx

These include large-scale mutations in which long segments of DNA are affected (for example, translocations, duplications, and inversions).

A chemical change in just one base pair of a gene causes a spontaneous or point mutation.

If these occur in gametes or cells producing gametes, they may be transmitted to future generations.

For example, sickle-cell disease is caused by a mutation of a single base pair in the gene that codes for one of the polypeptides of hemoglobin.

A change in a single nucleotide from T to A in the DNA template leads to an abnormal protein.

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