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MCB - Molecular Bio > Post Translation > Flashcards

Post Translation Flashcards

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Biology 101 flashcards
1
Q

where does most protein synthesis start?

A

on free cytosolic ribosomes not on ER

apart from mitochondria and plastid (chloroplast is one) translation

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2
Q

polysomes

A

multiple ribosomes can bind on same mRNA and make diff length proteins

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3
Q

macromolecular crowding

A

crowded state of cytoplasm in eukaryotic

conc. of substrates is high so fast reactions and drives cellular biochemistry, aggregation of proteins

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4
Q

nascent proteins

A

not fully born
non-native aggregation prone conformation (aggregation because crowded env.)
extruded in close proximity

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5
Q

newly synthesised proteins are…

A

non-functional
unfolded/misfolded proteins have exposed hydrophobic residues so protease-sensitive and prone to aggregation
when folded, the sites are tucked away so stable and protease resistant (with help of chaperones)

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6
Q

chaperones

A

favour correct folding for cytosolic proteins

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7
Q

chaperone mechanisms

A
  1. hydrophobic patches on unfolded proteins are recognised by Hsp40 co-chaperone which shields them so keeps protein soluble
  2. Hsp40 transfers protein to Hsc70 chaperone which is ATP-bound in open conformation, but stimulates ATPas activity so ATP to ADP
  3. ADP-bound Hsc70 is closed and shields hydrophobic patches so prevent aggregation
    solubility allows hydrophilic parts to fold and find final conformation
  4. release by NEF binding Hsc70 forming client complex so nucleotide exchange (removes ADP) so ATP to nucleotide binding site of Hsc70 and Hsc70 opens so releases substrate in partly folded shape
    (diff co-chaperones release at diff locations (BAG1/2, HASPBP1, CHIP, chips come in bags remember)
  5. multiple fates can occur now: released and find final conformation, or pass to other chaperone, or transport to organelle, or to proteasome for degradation
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8
Q

Hsp40

A

heat shock protein 40

co-chaperone

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9
Q

Hsc70

A

heat shock cognate protein 70

chaperone

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10
Q

study to see what is needed for solubility of protein

A

heat protein and separate aggregated (P) and soluble (S) by centrifugation

most protein is insoluble without chaperones
adding Hsp40 increases solubility and Hsc70 greatly increases and together even more - recover 50% activity

adding ATP is max solubility because system can regenerate

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11
Q

NEF

A

nucleotide exchange factor

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12
Q

Hsc70 co-chaperones pass protein onto other chaperones like…. or they…..

A

Hsp90 accepts partly folded and assembles multimeric complexes

pass onto chaperonins

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13
Q

chaperonins (definition and mechanism)

A

a class of chaperones that assist in folding of (largely) newly synthesized proteins with the help of ATP, i.e. all chaperonins can be referred to as chaperones, however, all chaperones need not be chaperonins

2 cages of 7, protein enters upper cage and finished in lower cage, shuts and lower released, forces conformation
very ATP expensive

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14
Q

how is a protein’s fate decided (role of each co-chaperone)

A

there is no absolute control over fate but is competition of co-chaperones for protein which depends on conc. of Hsc70 and Hsp90

if HOP get there first then transfer protein from Hsc70 to Hsp90
BAG-1 sends to proteasome
BAG-2 favours folding
HIP maintains Hsc70:client complex so competes with NEF

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15
Q

proteasome structure

A

central core of 4 stacked rings (7a top and bottom and 2 7b in the middle) forms 20S core

3 proteolytic activities (lecture 8 page 2) inside barrel encoded by b

19S regulatory particle cap on 1 or both ends
so together is 26S or 30S

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16
Q

proteasome targetting

A

polyubiquitylation - ubiquitin (Ub) covalent addition to protein, chain of 4 Ub means degradation signal

E1 (9 of it): ubiquitin-activating enzyme activates Ub because H removed from SH when Ub added covalently

E2 (30): ubiquitin-conjugating enzyme chosen by E1 transfers Ub from E1 to E2 (on SH)

E3 (100s): ubiquitin ligase, E2-Ub associates with E3 and binds target protein to transfer Ub to it

each E is specific to the next E and to a protein

19S cap of proteasome recognises Ub

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17
Q

monoubiquitylation vs poly

A

mono sends to lysosome while poly to proteasome

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18
Q

proteasome destruction

A

protein binds 19SRP (19S regulatory particle cap)
RP uses ATP to unfold protein and feeds through 20S core where it’s degraded and comes out other end as small peptides
deubiquitylases (DUBs) recycles Ub

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19
Q

other function of proteasome

A

fail-safe mechanism and can re-fold proteins

RPT5 subunit in cap checks if protein worth saving and acts as chaperone so refold and recover activity

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20
Q

UPS fail?

A

ubiquitin-proteasome system
proteins that need to be destroyed accumulate and aggregate
cell cycle proteins not degraded so cell proliferation and cancer

on the other hand, overactive proteasome causes autoimmune diseases

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21
Q

other protein modification (other than Ub)

A 
proteolytic cleavage
lipids for membrane targeting
phosphorylation
ADP ribosylation
methylation
22
Q

proteolytic cleavage

A

inactive protein form is cleaved and subunits are rearranged so activated
e.g. so digestive enzymes don’t eat us

23
Q

lipids for membrane targeting (Rab)

A

Rabs regulate membrane trafficking
Rab-GDP is cytosolic and nucleotide exchange causes GDI (GDP dissociation inhibitor) to dissociate so no longer mask prenyl group so Rab-GTP enters membrane

24
Q

phosphorylation

A

add phosphates by CAK and Wee1 and remove by Cdc25 so alter activity of cell cycle
activated by single phosphate but inactive with more

25
Q

ADP ribosylation

A

add ADP-ribose residues for cell signalling/DNA repair/apoptosis

Cholera toxin is an ADP-ribosylase so interferes with signalling

26
Q

methylation

A

on arginine or lysine residues

e.g. histones epigenetics to repress/activate gene expression

27
Q

co-translational targeting

A

target while translated

nascent protein has N-terminal signal which directs to ER then other secretory pathways

28
Q

N-terminal signal

A

5’ first few AAs (15-30) fused to RNA encoding protein

no ER signal peptide is the same but there are 3 key similarities: +ve N-terminus (Arg or Lys), long hydrophobic stretch of AAs, then cleavage site after small AA (serine/cysteine/glycine)

29
Q

entry into the ER

A
  1. binds SRP (signal recognition particle) receptors in cytosol
  2. SRP/SP to alpha subunit of SRP receptor in ER membrane
  3. SRP receptor recruits closed translocon and forms channel
  4. translocon opens so SP enters as loop/nook shape, and SRP recycled
  5. signal peptidase removes SP so released to ER membrane and now mature protein without SP
    cleaves SP so broken and extracted from membrane so SPs don’t build up
  6. translation terminates and protein is released to ER lumen to fold
30
Q

what happens in the ER lumen?

A

membrane-bound polysomes are formed during secretion so proteins in ER are formed in crowded conditions and need chaperones

proteins may become N-glycosylated or disulfide-bonded
if not folded properly then fail check by chaperones and ejected from ER for degradation (retro-translocation)

31
Q

ER chaperones equivalent to cytosol chaperones?

but also ER specific chaperones?

A

BiP in ER does same thing as Hsp/Hsc70
GRP94 in ER does further folding like Hsp90

PDI and ERp57 do disulfide bonding
CRT and CNX do N-glycan

32
Q

N-glycosylation definition

A

covalent addition of oligosaccharide tree of sugars (core N-glycan) from lipid carrier to target protein by OST

33
Q

OST

A

oligosaccharide transferase
in membrane with active site in ER lumen
recognise protein motifs and add N-glycan

34
Q

core n-glycans

A

2 N-acetyl glucosamines with branch tree of 9 mannose and on largest branch are terminal 3 glucose residues

is soluble

more detail in lecture 9 page 2

35
Q

N-glycosylation functions

A

flags for folding and ER quality control
increase protein solubility
influence folding rates and final protein conformation

36
Q

how does n-glycosylation flag protein for folding and quality control?

A

removes 2 glucose so allows interaction with chaperones for folding in ER

final glucose and 1 mannose removed so ready to pass to Golgi

so can see how far down folding pathway by how much glucose and mannose it has

37
Q

how does n-glycosylation increase protein solubility

A

it adds large and hydrophilic sugars so prevents aggregation

38
Q

how does n-glycosylation affect folding rates and final conformation

A

N-glycan is bulky so contrains alpha-C backbone so it’s no longer flexible so affects shape and folding
e.g. determines Ab structure or enzyme rates

39
Q

disulfide bonding (definition, affect, conditions)

A

2 cysteines brought close during folding by PDI (protein disulfide isomerase) so covalent bond which contributes to stability or tertiary structure

only under oxidising conditions so in ER and not in reducing conditions of cytosol

40
Q

PDI

A

binds to unstable proteins
2 SH of cysteines interact to form a mixed disulfide heterodimer which breaks and shuffles (isomerisation) till stable conformation
breaks bond if in wrong place

41
Q

coordination of ER protein folding with other things

A

lecture 9 page 2 bottom

MHC Class 1 assembly
BiP maintains HC solubility till beta2 microglobulin binds and n-glycosylated
HC enter folding env. provided by calreticulin and ERP57 w/ chaperone shuffle and stabilise disulfide bonds
ERP57 bond to tapasin so folding env around HC and folds so groove for peptide fed through TAP transporter recruited by tapasin

42
Q

other secretory system modifications

A

O-glycosylation - sugars to oxygen of target protein
proteolytic cleavage to activate
addition of lipids for membrane targeting

43
Q

Alzheimer’s link to protein modifications

A

alpha or beta cut first then gamma
if beta cuts first then fragment aggregates and plaques
if alpha cuts first then don’t aggregate

44
Q

is mitochondrial targeting co-translational?

A

no

45
Q

origin of mitochondria

A

prokaryote Rickettsia obligate intracellular parasite

alpha-proteobacterium

46
Q

problems with import into mitochondria

A

2 membranes and crowded env. so aggregation

47
Q

leader peptide (LP) (definition and types for mitochondria)

A

N-terminal LPs for targeting from free ribosomes in cytosol to secretory pathway
longer than signal peptides

to the matrix: matrix-targeting sequence, matrix protease, alcohol dehydrogenase III

to inner membrane: matrix protease, IMS targeting, IMS protease, cytochrome b2

to outer membrane: matrix protease, OM localisation, P70, no cleavage so remain as membrane anchors

48
Q

LPs (leader peptides) for mitochondria

A

18-80 AAs long
no 2 the same
form amphipathic helices (hydrophobic and +ve part)
+ve N-terminus on 1 face and -ve on another

49
Q

mitochondrial (mt) targeting process

A
  1. precursors of mt proteins made in cytosol are kept soluble with Hsc70 and delivers to mt
  2. LP is recognised by import receptors of TOM complexes
    Hsc70 interact with receptors associated with TOM40 so it opens and proteins enter
  3. channel formed by Tim23 and Tim17 at rare contact sites between OM and IM (membranes)
    which lines up with TOM and channel forms to matrix
  4. Tim-mediated transport requires proton-motive force across IM so proteins move through channel, requires ATP
    Hsp70 bound to Tim44 uses ATP and drags 1 residue at a time
  5. protein released from mortalin (Hsp70) and matrix signal removed by matrix protease if in matrix
50
Q

TOM

A

translocase outer membrane

51
Q

targeting to other mitochondrial locations (not matrix)

A

p70 exits laterally from TOM so stays in membrane and matrix signal not cleaved

cytochrome oxidase exits laterally from Tim in IM, no cleavage of IM signal

cytochrome c1 enter matrix into IMS - IMS signal cleaved (have to go to matrix and cross IM to IM space)

52
Q

differences between cytosolic vs ER vs mt targeting

A

lecture 10 page 2

MCB - Molecular Bio (7 decks)

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