Lecture 6

Question 1

what is protein degradation

Answer 1

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

Question 2

what is a major route of degradation

Answer 2

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

Question 3

what is ub

Answer 3

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

Question 4

what marks a protein for degradation

Answer 4

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

Question 5

what needs to be maintained in the cell

Answer 5

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

Question 6

what is ups

Answer 6

Ubiquitin proteasome system

Question 7

what does ubquitination do

Answer 7

enzymes attach chains of ub to substrate proteins

Question 8

what selects the substrates of ups

Answer 8

E3 ub ligases

Question 9

what are poly ub chains recognized by

Answer 9

receptors on proteasomes

Question 10

what is the proteasome

Answer 10

large protein complex that unfolds and degrades substrates

Question 11

describe E1

Answer 11

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

Question 12

describe E2

Answer 12

conjugating enzyme
transfers ub to subtrate
50 diff genes encoding

Question 13

describe E3

Answer 13

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

Question 14

what is E1

Answer 14

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

Question 15

what does E2 do

Answer 15

Conjugating enzyme
transfers ub to its own cys

Question 16

what does E3 do

Answer 16

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

Question 17

what adds more ub to lys

Answer 17

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

Question 18

describe ubiquitin system - gen

Answer 18

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

Question 19

describe K - ubiquitination

Answer 19

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

Question 20

describe ubiquitination of lysine - structure

Answer 20

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

Question 21

what can ub c terminus be linked to

Answer 21

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

Question 22

what does E3 do - for ubiquitination

Answer 22

first attaches ub on substrate then extends poly ub chain

Question 23

which lysines are ubiquitinated for degradation

Answer 23

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

Question 24

describe diversity of E3 ligases

Answer 24

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

Question 25

how many genes do most proteasome subunits have

Answer 25

1 since one type of proteasome only

Question 26

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

Answer 26

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

Question 27

are all proteins degraded at same rate

Answer 27

NOO diff rates but continually degraded

Question 28

what controls degradation of substrate

Answer 28

Selectivity of E3 ligase NOT BY proteasome

Question 29

describe examples of protein degradation - why it would be degraded

Answer 29

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

Question 30

describe quality control degradation of a misfolded protein

Answer 30

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

Question 31

describe chip mechanism

Answer 31

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

Question 32

describe constitutive degradation on native proteins - gen

Answer 32

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

Question 33

which aa's apply to N end rule

Answer 33

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

Question 34

does N end rule degrade proteins fast

Answer 34

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

Question 35

which aas affected by N end rule

Answer 35

basic or large hydrophobic aas

Question 36

describe N end rule modifications - gen

Answer 36

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

Question 37

describe N end rule modifications - specific

Answer 37

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

Question 38

describe N end rule pathway

Answer 38

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

Question 39

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

Answer 39

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

Question 40

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

Answer 40

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

Question 41

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

Answer 41

F-box protein binds phosphorylated substrate

Question 42

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

Answer 42

substrate presented to E2 for ubiquitination since in close proximity

Question 43

describe degradation regulated by phosphorylation

Answer 43

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)

Question 44

describe proteasome

Answer 44

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

Question 45

what is proteasome responsible for

Answer 45

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

Question 46

name and describe 3 functions of proteasome cap

Answer 46

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

Question 47

describe DUBs

Answer 47

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

Question 48

what are UBPs

Answer 48

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

Question 49

what modifications can lysine undergo

Answer 49

Acetylated methylated ubiquitinated

Question 50

can only lysine be ubiquitinated

Answer 50

NOOO cysteine too

Question 51

describe chip mechanism - balance

Answer 51

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

Question 52

function of proteasome center cylinder

Answer 52

20s middle cuts polypeptide up