Lecture 6 Flashcards
what is protein degradation
key part of protein folding quality control
essential regulatory and homeostatic mechanism
what is a major route of degradation
Ubiquitin mediated degradation by proteasome in the cytosol
also in ER - kicked out and degraded in cytosol
what is ub
ubiquitin
small 8kda, 76aa
can be covalently linked to lysine side chains of other proteins and to itself
what marks a protein for degradation
poly-ub chain= ptm, attach full other protein to another protein = covalent
attach one ub initially and then continue ubiquination to make chain
what needs to be maintained in the cell
balance between making and degrading
want to have good proteome = control components and proteins at a certain time and how they are folded
what is ups
Ubiquitin proteasome system
what does ubquitination do
enzymes attach chains of ub to substrate proteins
what selects the substrates of ups
E3 ub ligases
what are poly ub chains recognized by
receptors on proteasomes
what is the proteasome
large protein complex that unfolds and degrades substrates
describe E1
dozen of genes encode
in cytosol = need to be activated by E1
E1 has ub and transfers to E2
describe E2
conjugating enzyme
transfers ub to subtrate
50 diff genes encoding
describe E3
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
what is E1
E1 activating enzyme attaches Ub to itself in a chemically reactive state, on a Cys side chain (thioester bond)
what does E2 do
Conjugating enzyme
transfers ub to its own cys
what does E3 do
ligase
selects substrate to be modified
triggers ub transfer from E2 to lys side chain on substrate
what adds more ub to lys
E2-E3 adds more ub to lys on previous ub = makes poly ub chain
describe ubiquitin system - gen
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
describe K - ubiquitination
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
describe ubiquitination of lysine - structure
N terminus and another N terminus with C terminus in middle
N = protein target
2N & 1C terminus
what can ub c terminus be linked to
ub c-term can be linked to lys 63, 28 or 11 of another ub
linked to protein or another ub
what does E3 do - for ubiquitination
first attaches ub on substrate then extends poly ub chain
which lysines are ubiquitinated for degradation
long lys48 poly ub chains targeted for protein degradation by proteasome
mono-ub ot lys63 poly ub = not regonized but signal other things
describe diversity of E3 ligases
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
how many genes do most proteasome subunits have
1
since one type of proteasome only
why do cells have many genes coding for E3 and not proteasome
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
are all proteins degraded at same rate
NOO diff rates
but continually degraded
what controls degradation of substrate
Selectivity of E3 ligase
NOT BY proteasome
describe examples of protein degradation - why it would be degraded
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
describe quality control degradation of a misfolded protein
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
describe chip mechanism
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
describe constitutive degradation on native proteins - gen
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
which aa’s apply to N end rule
if 2nd aa = arg, lys, his, phe, trp, tyr, leu, Ile, = E3 ligases recognize the properties of the side chains and sends for degradation
does N end rule degrade proteins fast
degrades proteins rapidly = whether or not folded
other proteins are longer lived unless degraded by other means
which aas affected by N end rule
basic or large hydrophobic aas
describe N end rule modifications - gen
some n terminal residues are enzymatically modified to be recognized by N end rule
4 more if undergo modifcations
describe N end rule modifications - specific
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
describe N end rule pathway
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
describe degradation of a native protein in response to a signal: regulated degradation-SCF E3 = STEP 1
E3 ub ligase complex = Skp1/cullin/F-box
describe degradation of a native protein in response to a signal: regulated degradation-SCF E3 = STEP 2
scaffold (cullin) binds E2 that is ubiquitinated and substrate binding (F-box) protein
describe degradation of a native protein in response to a signal: regulated degradation-SCF E3 = STEP 3
F-box protein binds phosphorylated substrate
describe degradation of a native protein in response to a signal: regulated degradation-SCF E3 = STEP 4
substrate presented to E2 for ubiquitination
since in close proximity
describe degradation regulated by phosphorylation
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)
describe proteasome
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
what is proteasome responsible for
Responsible for general protein degradation in the cytosol and nucleus, and from the endoplasmic reticulum
name and describe 3 functions of proteasome cap
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
describe DUBs
de ubiquitinating
~90 genes
ub can be removed = when made misatke
what are UBPs
ub binding proteins
~300 genes
brings to proteasome - like garbage truck and breaks down and releases Ub to be recycled in cytosol
what modifications can lysine undergo
Acetylated
methylated
ubiquitinated
can only lysine be ubiquitinated
NOOO
cysteine too
describe chip mechanism - balance
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
function of proteasome center cylinder
20s middle
cuts polypeptide up
