Lecture 15- Protein degradation by the ubiquitin-proteasome system

Question 1

Ubiquitin basic structure

Answer 1

beta sheet around an alpha helix, flexible c-terminal tail- allows recognition by a lot of proteins

Question 2

why is protein degradation importany

Answer 2

preventing cytotoxicity (removing damaged or aggregating proteins), fine-tuning the levels of a protein within a cell

Question 3

where is Ub attached

Answer 3

lysine side chains, c-terminus attached to the gamma-amine by an isopeptide linkage

Question 4

enzymes involved in Ubation- steps

Answer 4

Ub attaches to E1- ATP dependent
E1 transfers Ub to a Cys on E2
E2 collaborates with E3, catalyses isopeptide bond formation

Question 5

variations in each enzyme type

Answer 5

2 E1s, 40ish E2s, varying numbers of E3s- 500 human 1200 A. thaliana

Question 6

what is the ubiquitin code

Answer 6

repetitive Ubation of a protein, which can have distinct consequences for a cell

Question 7

how many lysines in Ub

Answer 7

7

Question 8

examples of different Ub chain topologies and their function

Answer 8

K63 (on 63rd lysine)- non-proteolytic functions
K48- proteolysis

Question 9

what does PolyUb do

Answer 9

targets the protein towards the 26s proteasome, a ‘degron’

Question 10

what is the 26S proteasome

Answer 10

major eukaryotic proteasome, responsible for protein degradation in the cytosol and nucleus

Question 11

2 functional parts of the proteasome

Answer 11

barrel-like 20S core particle, contains active sites
19S regulatory particle, acts as a cap at one/both ends

Question 12

what is the benefit of having separate subunits

Answer 12

allows for compartmentalisation, and high levels of control

Question 13

rough structure of 20S core

Answer 13

heptameric rings, beta subunits are proteolytically active and cut at different points into peptides of average length 7-8aas

Question 14

how is access to the chamber regulated

Answer 14

opening and closing of axial pores via changes in the N terminal tails of the alpha subunits

Question 15

regulatory subunit composition

Answer 15

Rpn (non-ATPase) and Rpt (triple A ATPase) subunits

Question 16

how do substrates move into the core through the RP

Answer 16

loops projecting from ATPases push the substrate through and unfold it

Question 17

examples of Rpn subunits and their roles

Answer 17

Rpn10- Ub receptor, provides a ‘platform’ to load peptides
Rpn 1- scaffold protein
6 subunits forming a ‘lid’, acts as a stabilising anchor

Question 18

how many ‘stages’ can the proteasome exist in

Answer 18

4- where s1 has a closed core gate, and s4 has an open gate (subunits are sligned)- s2 and 3 are intermediates

Question 19

what changes the state

Answer 19

substrate binding can lead to conformational shifts

Question 20

prerequisites for proper folding into the proteasome

Answer 20

polyubiquitin tagging and a disordered region

Question 21

what is ERAD

Answer 21

ER associated protein degradation

Question 22

4 main steps of ERAD

Answer 22

recognition
dislocation and ubiquitination
targeting
proteosomal degradation

Question 23

example of ERAD pathways

Answer 23

ERAD-L (at the Lumen)
ERAD-M (at the Membrane)
-named based on location of the degron

Question 24

function of Cdc48

Answer 24

retrotranslocation motor- pulls proteins out of the ER to deliver them to the 26SP

Question 25

what are ubiquitin like proteins and what are some of their functions

Answer 25

proteins related in structure and sequence to Ub, can be used in proteolysis as well as autophagy, immune response etc

Question 26

what is SUMO

Answer 26

a ubiquitin like protein- SUMOylation of a protein is non-proteolytic and can interfere with ubiquitination and alter protein-protein interactions or protein localisation