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ANAT 212 > Lecture 6 > Flashcards

Lecture 6 Flashcards

1
Q

what is protein degradation

A

key part of protein folding quality control
essential regulatory and homeostatic mechanism

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2
Q

what is a major route of degradation

A

Ubiquitin mediated degradation by proteasome in the cytosol
also in ER - kicked out and degraded in cytosol

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3
Q

what is ub

A

ubiquitin
small 8kda, 76aa
can be covalently linked to lysine side chains of other proteins and to itself

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4
Q

what marks a protein for degradation

A

poly-ub chain= ptm, attach full other protein to another protein = covalent
attach one ub initially and then continue ubiquination to make chain

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5
Q

what needs to be maintained in the cell

A

balance between making and degrading
want to have good proteome = control components and proteins at a certain time and how they are folded

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6
Q

what is ups

A

Ubiquitin proteasome system

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7
Q

what does ubquitination do

A

enzymes attach chains of ub to substrate proteins

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8
Q

what selects the substrates of ups

A

E3 ub ligases

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9
Q

what are poly ub chains recognized by

A

receptors on proteasomes

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10
Q

what is the proteasome

A

large protein complex that unfolds and degrades substrates

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11
Q

describe E1

A

dozen of genes encode
in cytosol = need to be activated by E1
E1 has ub and transfers to E2

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12
Q

describe E2

A

conjugating enzyme
transfers ub to subtrate
50 diff genes encoding

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13
Q

describe E3

A

ub ligase
needs to be recognized by E3
scaffold protein
one domain recognizes E2 bound to ub and one recognizes substrate
only when E3 has the 2 components = Ub transferred
PTM leads to altered function
only one Ub
E2 and E3 keep going = poly ub then targeted

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14
Q

what is E1

A

E1 activating enzyme attaches Ub to itself in a chemically reactive state, on a Cys side chain (thioester bond)

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15
Q

what does E2 do

A

Conjugating enzyme
transfers ub to its own cys

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16
Q

what does E3 do

A

ligase
selects substrate to be modified
triggers ub transfer from E2 to lys side chain on substrate

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17
Q

what adds more ub to lys

A

E2-E3 adds more ub to lys on previous ub = makes poly ub chain

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18
Q

describe ubiquitin system - gen

A

binds to cysteine = thioester bond
E2 = also cys and thioester bond
scaffold protein = E3
another domain of E3 binds E2
DIRECT path = E2 transfers ub directly to substrate, RING E3 family
INDIRECT path = HECT E3 family, not as abundant, E2 transfers Ub to E3 (cys bond), and then transfers ub to substrate
Result = poly ub tail

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19
Q

describe K - ubiquitination

A

ub c terminus carboxyl covalently linked to side chain amine
lysine receives ub
isopeptide bond - not part of backbone
substrate can have multiple ub sites - many but not all lysines, depends on accessibility

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20
Q

describe ubiquitination of lysine - structure

A

N terminus and another N terminus with C terminus in middle
N = protein target
2N & 1C terminus

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21
Q

what can ub c terminus be linked to

A

ub c-term can be linked to lys 63, 28 or 11 of another ub
linked to protein or another ub

22
Q

what does E3 do - for ubiquitination

A

first attaches ub on substrate then extends poly ub chain

23
Q

which lysines are ubiquitinated for degradation

A

long lys48 poly ub chains targeted for protein degradation by proteasome
mono-ub ot lys63 poly ub = not regonized but signal other things

24
Q

describe diversity of E3 ligases

A

in humans = more than 600 genes code for E3
only 50 for E2 and dozen for E1
many for E3 since provides specificity = which proteins must be degraded
E1 = no specificity

25
Q

how many genes do most proteasome subunits have

A

1
since one type of proteasome only

26
Q

why do cells have many genes coding for E3 and not proteasome

A

cells express diff E3 enzymes for each degradation situation = which all use same proteasome
more effective than expressing hundred of diff proteases with diff specificities

27
Q

are all proteins degraded at same rate

A

NOO diff rates
but continually degraded

28
Q

what controls degradation of substrate

A

Selectivity of E3 ligase
NOT BY proteasome

29
Q

describe examples of protein degradation - why it would be degraded

A

quality control degradation of misfolded protein
Constitutive degradation of a native protein to control its level = folded but short half life, like beta actin, big turnover
degradation of native protein in response to signal = phosphorylation and cyclins
each case = has diff E3 lugs recognition mechanism

30
Q

describe quality control degradation of a misfolded protein

A

regulated by chip cochaperone
tpr domain binds hsc70 or hsp90 (to misfolded substrate)
E3 ligases doman binds E2
other domain = E3 ligase Ubox, brings E2 that is ubiquitinated = easy to transfer now to misfoled substrate
chaperone, chip and E3 form complete E3 ligase complex
chaperone bound substrate is selectively ubiquinated

31
Q

describe chip mechanism

A

try to always save the proteins since energetically expensive to make them
CHIP interactions with chaperones are transient = relatively fast binding and release, if protein being difficult = chip binds

32
Q

describe constitutive degradation on native proteins - gen

A

all proteins translated with n terminal MET
many proteins processed by cleavage within their sequences = so diff residue becomes n terminus = no more met
certain n terminal residues are bound by N end rule E3 ligases which ubiquitinate proteins
= short lived proteins

33
Q

which aa’s apply to N end rule

A

if 2nd aa = arg, lys, his, phe, trp, tyr, leu, Ile, = E3 ligases recognize the properties of the side chains and sends for degradation

34
Q

does N end rule degrade proteins fast

A

degrades proteins rapidly = whether or not folded
other proteins are longer lived unless degraded by other means

35
Q

which aas affected by N end rule

A

basic or large hydrophobic aas

36
Q

describe N end rule modifications - gen

A

some n terminal residues are enzymatically modified to be recognized by N end rule
4 more if undergo modifcations

37
Q

describe N end rule modifications - specific

A

asp, glu (acidic) = arg added to n terminus
asn,gln (amides) = side chains converted to asp, glu by removal of amine then arg added
add arg = now peptide can be degraded

38
Q

describe N end rule pathway

A

Computational
if N terminal N, convert to D; If Q convert to E
if N terminal D or E = add N terminal R (arginine)
if N terminal R, K, H, F, W, Y, L, I = ubiquitinate protein
else = leave alone

39
Q

describe degradation of a native protein in response to a signal: regulated degradation-SCF E3 = STEP 1

A

E3 ub ligase complex = Skp1/cullin/F-box

40
Q

describe degradation of a native protein in response to a signal: regulated degradation-SCF E3 = STEP 2

A

scaffold (cullin) binds E2 that is ubiquitinated and substrate binding (F-box) protein

41
Q

describe degradation of a native protein in response to a signal: regulated degradation-SCF E3 = STEP 3

A

F-box protein binds phosphorylated substrate

42
Q

describe degradation of a native protein in response to a signal: regulated degradation-SCF E3 = STEP 4

A

substrate presented to E2 for ubiquitination
since in close proximity

43
Q

describe degradation regulated by phosphorylation

A

many f-box proteins recognize phosphorylated peptide sequences
phosphorylation by kinase used as signal for degradation
de phosphorylation then prevents degradation
SCF ligases degrade native, functional proteins to stop their function (cullin)

44
Q

describe proteasome

A

Large oligomeric complex with a central 20s core particle and two 19s regulatory particles (caps)
Core and 2 caps form complete 26s proteasome – around 2.5MDa

45
Q

what is proteasome responsible for

A

Responsible for general protein degradation in the cytosol and nucleus, and from the endoplasmic reticulum

46
Q

name and describe 3 functions of proteasome cap

A

19S cap
control proteins that get into ring to be degraded
recognizes poly ub in protein and brings to proteasome
cuts poly ub tail = ubs recycled in cytosol

47
Q

describe DUBs

A

de ubiquitinating
~90 genes
ub can be removed = when made misatke

48
Q

what are UBPs

A

ub binding proteins
~300 genes
brings to proteasome - like garbage truck and breaks down and releases Ub to be recycled in cytosol

49
Q

what modifications can lysine undergo

A

Acetylated
methylated
ubiquitinated

50
Q

can only lysine be ubiquitinated

A

NOOO
cysteine too

51
Q

describe chip mechanism - balance

A

balance between chaperone mediated folding and degradation
substrates bound by chaperones for long times are more likely to form complex with chip and be ubquitinated
Substrates bound for short times likely escape ubquitination
several rounds of folding *tpr domain = hsp 70 and 90

52
Q

function of proteasome center cylinder

A

20s middle
cuts polypeptide up

ANAT 212 (34 decks)

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