LEC32: Protein Quality Control in the Cell Flashcards Preview

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Flashcards in LEC32: Protein Quality Control in the Cell Deck (46):
1

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

chaperones prevent protein aggregation 

 

2

how do molecular chaperones promote polypeptide folding?

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 

3

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

Proteasome/ubiquitin pathway

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

4

what are processes of protein quality control?

1) protein folding

2) protein degredation

5

how do proteins fold? how do we know this?

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

6

what is a chaotrope?

agent that disrupts hydrophobic interactions 

7

what are phases of polypeptide chain folding?

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

8

why doesn't protein folding happen spontaneously in cells?

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

9

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

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

10

what are molecular chaperones? what do they do?

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

11

what do chaperones do to the folding reaction?

they increase the yield, but not the rate 

thus a thermodynamic, not kinetic, effect

12

what are the major families of molecular chaperones?

how do they work?

Hsp70 

Hsp60 (chaperonin)

Hsp90

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

13

do molecular chaperones work alone?

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

14

what is the structure of bacterial chaperonin?

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

15

how does chaperonin act on misfolded proteins?

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

16

what controls chaperone gene txn?

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

17

what does Hsf respond to?

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

18

what does this demonstrate?

 

Q image thumb

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

19

what are methods of protein degredation?

1) proteasome 

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

20

what is the proteasome, what is its structure?

large gated protease  

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

 

 

 

21

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

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 

 

 

22

what is the structure of the 19S regulatory cap? 

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

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

23

what are the activities of eukaryotic proteasomes?

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

24

what special functions have been described in mammalian proteasomes?

1) cleaving after branched chain amino acids 

2) cleaving between small neutral amino acids

25

size of peptides resulting from proteasome activity?

7-9 amino acids

26

when are proteasomes therapeutic targets?

example?

for chemotherapy 

eg: velcade is v. useful for multiple myeloma

27

what is ubiquitin? its use?

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

28

how do successive ubiquitins become attached to each other?

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

29

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

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

 

30

what is CHIP? what does it do?

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 

31

how does Ub chain grow?

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

32

what is this?

Q image thumb

ubiquitylation visualized by gel electrophoresis 

multiple ubiquitins added to chain successively

33

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

form aggresomesaggregates of misfolded proteins bound together via hydrophobic interactions or ordered assemblies of amyloid fibers

occurs if overwhelm the ubiquitin/proteasome pathway 

 

34

how are aggresomes cleared?

via autophagy

35

what is healthy/normal function of aggregates in cells?

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

 

36

describe this

Q image thumb

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

37

what are prions?

proteins that form toxic amyloids that're transmissible 

 

38

what are amyloids?

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

39

what is genetic characterization of Alzheimer's disease?

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

40

what is genetic association of Parkinson's disease?

formation of aggregates called Lewy Bodies

= aggregates enriched in alpha-synuclein 

also involves loss of dopaminergic neurons in substantia nigra

41

what  causes poly-glutamine?

what does this cause?

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

42

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

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)

43

what is it about poly-Q that causes disease?

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

44

what are prions?

how are they transmitted?

what do they cause?

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

45

what is the purpose of protein quality control?

cellular process that integrates folding, degredation of proteins 

46

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

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

 

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