LEC32: Protein Quality Control in the Cell

Question 1

how is aggregation of newly-made proteins prevented in a crowded cellular environment?

Answer 1

chaperones prevent protein aggregation

Question 2

how do molecular chaperones promote polypeptide folding?

Answer 2

Bind to, stabilize otherwise unstable conformers of a protein and facilitate its folding

        Hsp70, Hsp60, Hsp90 families bind/relase polypeptides

e.g. = Hsp60, chaperonin- barrel-shaped, takes protein into its inner core, allows it to fold there where it is sequestered and protected

Question 3

what is mechanism by which misfolded proteins are targeted for degredation?

Answer 3

Proteasome/ubiquitin pathway

Ubiquitin tags proteins for degredation; works via E1, E2, E3; brings protein to proteasome for degredation

Question 4

what are processes of protein quality control?

Answer 4

1) protein folding
2) protein degredation

Question 5

how do proteins fold? how do we know this?

Answer 5

_by a self assembly process _

Anfinsen took RNase, treated w/ reducing agent that unfolded it via breaking disulfide bondes and w/ a **chaotrope **

saw that purified protein in dialysis bag w/o reagents of reducing agent and chaotrope refolded and activity was restored

indicates that proteins self assemble

Question 6

what is a chaotrope?

Answer 6

agent that disrupts hydrophobic interactions

Question 7

what are phases of polypeptide chain folding?

Answer 7

1) burst phase: 0-5 ms; hydrophobic collapse, formation of secondary structures

2) intermediate phase: 5-100 ms; secondary structures form molten globule intermediate; loose; high state of equilibirum

3) rate-limiting step: 100 ms-several minutes; global repacking of hydrophobic side chains and association of domains that were folded independently in intermediate stages; lowest free energy state so stops trying to fold itself

Question 8

why doesn’t protein folding happen spontaneously in cells?

Answer 8

b/c conditions are not ideal:

1) we do not exist at 4 degrees celsius, we are 37 degrees
2) concentration of proteins he used were on order of ng/ml, and protein concentration in humans is 100s of mg/ml

Question 9

what would happen if did Anfinsen’s experiment in human cell conditions? why?

Answer 9

aggregation would occur: when proteins **unspecifically interacts w/ its neighbor(s) **

occurs via **hydrophobic a.a. of other proteins that it normally isn’t interacting with **

occurs b/c hydrophobc Van Der Waals interactions get stronger when heated; so leads to aggregation

Question 10

what are molecular chaperones? what do they do?

Answer 10

proteins that promote protein folding and prevent aggregation in vivo by stabilizing an otherwise unstable conformer of another protein

may help with:

1) folding
2) oligomeric assembly
3) transport to a particular subcellular compartment
4) controlled switching between active, inactive conformations

Question 11

what do chaperones do to the folding reaction?

Answer 11

they **increase the yield, but not the rate **

thus a thermodynamic, not kinetic, effect

Question 12

what are the major families of molecular chaperones?

how do they work?

Answer 12

Hsp70

Hsp60 (chaperonin)

Hsp90

bind got and release polypeptides in a manner dependent on ATP binding, hydrolysis and ntd exchange

Question 13

do molecular chaperones work alone?

Answer 13

no; function in association w/ many co-chaperone proteins that regulate reaction cycle of polypeptide binding and release by chapersone

regulated cycles promote polypeptide folding

Question 14

what is the structure of bacterial chaperonin?

Answer 14

homo-oligomer of 14 subunits, each 60 kDa,

arranged in **2 stacked rings each of 7 subunits **

each ring = like a donut w/ a 7-fold axis and a chamber

smaller lid structure of 7 subunits on top of the barrels

Question 15

how does chaperonin act on misfolded proteins?

Answer 15

1) misfolded proteins bind to rim of barrel
2) proteins displaced into cavity of barrel by lid structure
3) protein folds in sequestered, protected environment of barrel chamber, which is lined w/ hydrophilic a.a. that promote folding
4) ATP hydrolysis **causes lid to dissociate, due to conformational changes in large subunits **

5) folded protein emerges into cytosol

Question 16

what controls chaperone gene txn?

Answer 16

Hsf, heat shock factor

txn factor

Hsf responds to presence of unfolded protein or heat shock or other type sof proteotoxic stress

stressed cells’ Hsf activates upon stress signal, binds to promoter element of Hsp70 gene, stimulates expression, sometimes hundred fold, and quickly

Question 17

what does Hsf respond to?

Answer 17

1) environmental stress: heat shock, a.a. analogues, heavy metals, inhibitors of energy metabolism

2) pathophysiological state: fever/inflammation, hypertrophy, oxidative injury, ischemia, infection, xenobiotics

3) non-stress: cell cycle, growth factors, development, differentiation

Question 18

what does this demonstrate?

Answer 18

Hsf expression is increased under conditions of stress, i.e. high temperature

Question 19

what are methods of protein degredation?

Answer 19

1) proteasome
2) target system that targets proteins in the proteasome, via covalent attachment of ubiquitin

Question 20

what is the proteasome, what is its structure?

Answer 20

large gated protease

central catalytic core (20S proteasome) and regulatory cap (19S) come together = 26S proteasome

Question 21

what are the functions of the 20S subunit of the proteasome?

Answer 21

catalytic particle

outer subunits = alpha, inner subunits = beta

access to inside is via tunnel formed by alpha subunit rings

catalytic activity occurs in beta subunits

Question 22

what is the structure of the 19S regulatory cap?

what are the functions of the 19S regulatory cap of the proteasome?

Answer 22

comprised of at least 15 subunits, 6 of which are AAA-ATPases that perform unfolding

1) binds ubiquitinated proteins
2) de-ubiquitinates proteins
3) unfolds substrates
4) presents substrate to catalytic particle (CP) of the proteasome

Question 23

what are the activities of eukaryotic proteasomes?

Answer 23

1) chymotrypsin-like: cleaves after hydrophobic amino acid chains
2) trypsin-like: cleaves after basic amino acids
3) peptidyl-glutamyl peptide hydrolyzing activity: cleaves after a.as

Question 24

what special functions have been described in mammalian proteasomes?

Answer 24

1) cleaving after branched chain amino acids
2) cleaving between small neutral amino acids

Question 25

size of peptides resulting from proteasome activity?

Answer 25

7-9 amino acids

Question 26

when are proteasomes therapeutic targets?

example?

Answer 26

for chemotherapy

eg: velcade is v. useful for multiple myeloma

Question 27

what is ubiquitin? its use?

Answer 27

76 a.a. protein that becomes covalently attached to polypeptides that’re substrates for degredation by the proteasome

has a G at the end

has a K at position 48 which conjugates via an isopeptide bond of its epsilon-amino group to the glycine of the next ubiquitin

Question 28

how do successive ubiquitins become attached to each other?

Answer 28

via action of enzymes E1, E2, E3:

E1: activates ubiqutin via ATP-dependent activation of C-terminal glycine

E2: ubiquitin-conjugating enzymes, accept ubiquitin from E1 & transfer it to protein substrate

E3: ubiquitin ligase, specifies substrate selection, brings it in close proximity to ubiquitin, catalyzes transfer to substrate

Question 29

describe process of E1, E2, E3 w/ ubiquitin –> protein substrate

Answer 29

1) E1 carries out ATP-dependent activation of C-temrinal glycine by first forming a ubiquitin-adenylate, then transfers activated ubiquitin to thiol site in E1

high energy thiol important for Ub tarnsfer to E2

2) E2 enzymes accept ubiquitin from E1, transfer it to protein substrate w/ help of E3 enzyme

Question 30

what is CHIP? what does it do?

Answer 30

C-terminal Hsp interacting protein

protein w/ an E3 ligase domain & a chaperone binding domain

misfolded proteins bind chaperone binding domain of CHIP; this stimulates ubiquitin ligation

CHIP binds directly to Hsp70 and catalyzes ubiquitinylation of misfolded proteins

Question 31

how does Ub chain grow?

Answer 31

as several ubiquitins tagged on to substrate, higher number of ubiquitins tagged = growing affinity = chain grows, too

Question 32

what is this?

Answer 32

ubiquitylation visualized by gel electrophoresis

multiple ubiquitins added to chain successively

Question 33

what happens to misfolded proteins that cannot be destroyed quickly enough?

Answer 33

form aggresomes, **aggregates **of misfolded proteins bound together via hydrophobic interactions or ordered assemblies of amyloid fibers

occurs if overwhelm the ubiquitin/proteasome pathway

Question 34

how are aggresomes cleared?

Answer 34

via autophagy

Question 35

what is healthy/normal function of aggregates in cells?

Answer 35

aggresomes

large collections of misfodled aggregated proteins

are central meeting point for molecular chaperones & proteasome- so although aggregated, it’s ok

form in micro aggregate that are then transported along cytoskeleton to form a central protective aggresome near center of cell- way to path misfolded proteins and make sure they don’t do harm to the cell

usually get cleared by autophagy

Question 36

describe this

Answer 36

Model for aggresome formation

Small aggregates formed at the cell periphery are delivered along microtubule tracks (green lines) on retrograde motors (grey) to a juxtanuclear, pericentriolar location

A pair of centrioles is shown in blue next to the nuclear indentation

Question 37

what are prions?

Answer 37

proteins that form toxic amyloids that’re transmissible

Question 38

what are amyloids?

Answer 38

extremely stable aggregates of proteins that’re formed by stacked beta sheets

build up of amyloids in brain is toxic, causes death

leads to diseases like alzheimer’s

Question 39

what is genetic characterization of Alzheimer’s disease?

Answer 39

extracellular amyloids of a peptide called AB, A-beta40 and A-beta42

are 40 and 42 a.a. long peptides which are formed by clipping a protein called APP, alzheimer’s precursor protein

A.D. also caused by **intracellular deposits, or neurofibrillary tangles, of microtubule binding protein, Tau **

aggregating form of Tau is hyperphosphorylated

Question 40

what is genetic association of Parkinson’s disease?

Answer 40

formation of aggregates called Lewy Bodies

= aggregates enriched in alpha-synuclein

also involves loss of dopaminergic neurons in substantia nigra

Question 41

what causes poly-glutamine?

what does this cause?

Answer 41

some cases, the triplet encoding Q (CAG) expands during replication

if expands beyond ~36 Q residues then get diseases state

currently, know of 9 proteins w/ polyQ resulting in neurotoxicity

Question 42

what diseases are known to be caused by poly-Q strectches?

Answer 42

must be beyond ~36 Q residues to cause disease state

disease (protein)

Huntington’s (Huntingtin)

DRPLA (Atrophin 1)

SCA 1, 2, 3, 6, 7, 17 (spinocerebellar ataxias: Ataxin 1,2, 3,7,17 & TATA box binding protein)

Question 43

what is it about poly-Q that causes disease?

Answer 43

when >36 bp of polyQ, toxicity results b/c glutamines in nucleus of these neurons aggregate, this aggregation causes neurons to degenerate

Question 44

what are prions?

how are they transmitted?

what do they cause?

Answer 44

proteins that form transmissible amyloids that cause disease in humans & other mammals

transmission occurs by eating contaminated food, so they’re infectious

cause scrapie, CJD, KURU, BSE, fatal fetal insomnia

all known prion diseases attack the brain

Question 45

what is the purpose of protein quality control?

Answer 45

cellular process that integrates folding, degredation of proteins

Question 46

why are so many late-onset diseases assocaite w/ protein misfolding?

Answer 46

b/c quality control apparatus becomes less robust w/ age