Professor Ugalde lecture 6-7 Flashcards
What is the key part of protein folding quality control?
Ubiquitination bc it has an important regulatory/homestatic mechanism
What is the major route of ubiquitination, what is it done by?
ubiqutin mediated degradation is done by proteasoms in the cytosol
Where can ubiquitin be covalently linked?
it can be covalently linked to lysine side chains of other proteins and to itself since it is a small 8kDa protein
what does the poly-Ub chain mark?
it marks a protein for degradation
What enzymes add length to the Ub tail?
What receptor detects the chain?
E2/E3 get together and add UB to Lys on the previous UB the tail
-receptors on proteasomes recognize the chain
What enzyme determines specificity in the ubiquitin proteasome system?
E3 Ub ligases select the substrates to determine what protein to degrade
What does the proteasome do in the UPS?
-it degrades/unfolds the substrates, and cuts the UB tail to recycle the amino acids (like a garbage recycling truck)
What is the first enzyme to bind UB in the UPS, and where does UB end up after that in E2/E3?
E1 binds ubiquitin to itself,then transfers it to E2 enzyme, which transfers it to E3 or the protein that is going to be degraded
What is the role of DUB (deubiquinated)?
can remove the UB if the protein is found to be useful and it does not want to get rid of it
Where does the E1 enzyme bind the UB
it binds on a cys side chain forming a thioester bond, and attaches a Ub to itself
Where does the E2 enzyme bind the UB?
it binds Ub to its own Cys, after being transfered from E1
What type of transfer does E3 trigger?
E3 triggers UB transfer from E2 to Lys side chain on substate
what bond does the UB c-terminus covalent link form?
How many Ub sites can a substrate have?
it forms an isopeptide bond and it binds to side chain amine of lysine
-it can have many sites
what lysines can the UB c-terminus be linked to?
Lys 63, 48 or 11 of another Ub
What lysine form can be recognized for degradation and which ones cannot?
Lys 48 poly-Ub chains target protein for degradation by proteasomes
-mono Ub or lys 63 Ploy Ub are not recognized but signal other things
How many E3 enzyme genes are there and what does this diversity signify? can the same proteasome be used?
more than 600 E3 enzymes
-bc of diversity cells express diff E3 enzyme for various degradation situations (which all use the same proteasome since genes have only have 1)
What enzyme controls the substrate rate of degradation?
E3 ligase selectivity controls the degradation
What are 3 examples of E3 ligase selectivity degradation control?
- quality control degradation of a misfolded protein
- constitutive degradation of a native protein to control its level
- degradation of a native protein in response to a signal
What co-chaperone regulates mislfolded protein degradation, and what do the TPR domain and E3 ligase domain bind?
CHIP co chaperone
-TPR domain binds HSC70 and HSP90
-E3 ligase domain binds E2
What forms a complete E3 ligase complex?
What is selectively Ubiquintinated?
Chaperone, CHIP, and E2 from the E3 ligase complex
Chaperone-bound substrate is selectively UB
Why are interactions with the chaperones transient (fast binding and release)
this is bc if the substrates ar bound by chaperones for a long time they will form a complex with chip and be UB, so the short time allows them to escape being UB.
what are all proteins translated with?
with N-terminal Methionine (AUG start codon)
Why does a different residue become the N-terminus in proteins?
many proteins are processed by clevage within their sequences, so a diff residue is present at N-terminus
What does the N-End rule do and what happens when N-terminal residues are bound by N-end rule E3 ligases?
-N -end rule degrades the proteins rapidly, whether or not they are folded
-N-terminal residue bound by N-end rule E3 ligases Ub the protein
What amino acids side chains are recognized by E3 ligases?
Arg, Lys, His, Phe, Trp, Tyr, Leu, Ile (basic or large hydrophobic amino acids)
How are n-end modifications done on acidic amino acids vs amindes?
Acidic- for aspartic acid and glutamic acid–>Arginine is added to N-terminus
Amides-for asparagine (Asn) and glutamine (Gln)—> side chains converted to Aspartic acid (Asp), and Glutamic acid by removal of amine, then Arginine is added to N-terminus
What is the N-end rule pathway for Asparagine (N) vs Glutamine (Q)?
if n-terminal N, convert to aspartic acid (D)
if n terminal Q, convert to glutamic acid (E)
what is N-end rule if N-terminal is D (aspartic acid) or glutamic acid (E)
-add N-terminal arginine (R) by arginine transferase
What is N-end rule for Arginine (R), lysine (k), histidine (H), phenylalanine (F), tryptophan (W), tyrosine (Y), Leucine (L), isoleucine (Ile)?
ubiquintinate the protein
what is the E3 UB ligase complex composed of in the degradation of the native state protein (SCF complex)?
- its composed of Skp1, cullin (scaffold protein), f-box
What does the scaffold protein bind to
it binds E2 and substrate binding (f-box) protein
what does F-box protein bind to?
binds to the phosphorlyated substrate
Where is the fully binded substrate presented in the degradation of native protein?
presented to E2 for UB
what is used as a signal for degradation, what signal prevents degradation?
phosphorlyation by kinase is used as a signal for degradation
dephosphorlyation prevents degradation
what phospholipid can act as a signaling molecule?
phosphophatidyl-inositol (PI) is not abundant but can be phoshphorylated and act as a signaling molecule
