L34 Post-translational regulation and Autophagy

Question 1

Protein folding

Answer 1

see onenote slides

Hydrophobic regions move towards centre of protein

Protein folding begins during translation - some proteins can fold by the time synthesis is complete BUT most protein do not fold correctly without help

Some simple proteins don’t need any help with folding as long as the environment is optimum, it will self-fold

Larger, complex proteins need help folding

Question 2

Hsp

Answer 2

Molecular chaperones

• rapidly induced by mild heat shock
• conserved in bacteria , plant, animals

Question 3

Hsp70

Answer 3

see onenote

binds to hydrophobic region of unfolded proteins

rapid cycles of ATP hydrolysis induce conformational changes and disassociation/association of Hsp70 to assist folding

Question 4

Hsp60

Answer 4

see onenote

Provides an isolated environment for protein folding

Unsuccessfully folded protein will be hydrophobic on the outside drawn in by the hydrophobic Hsp60, correctly folded protein will be hydrophilic on the outside

Sequesters the incorrectly folded protein away to hopefully sort itself out

Hsp are constitutive as you always need their help to fold protein. More Hsp proteins produced under high temperatures as it is harder to fold protein correctly when the temperature is not optimal.

Question 5

Ubiquitin proteasome system (UPS)

Answer 5

see onenote

26S proteasome

proteins targeted by proteasome for degradation and recycling

Proteasome

• Identify proteins that have been tagged for degradation by ubiquitin
• Peptide passed through barrel which has protease activity to cleave polypeptide into aa or smaller polypeptides to be re-utilised and turned into new proteins

Question 6

Post-translational mechanism

Answer 6

see onenote

Question 7

Ubiquitination of proteins

Answer 7

Ubiquitin added to lysine residues in protein

Poly-ubiquitination of Lys48 targets proteins to proteasome

Poly-ubiquitinated proteins degraded by 26S proteasome

Question 8

Ubiquitin ligases act on target proteins

Answer 8

see onenote

Question 9

Diversity of E3 ubiquitin ligases

Answer 9

see onenote

Cullin-RING-ligase (CRL) type

Non-CRL type

E3 ligases interact with E2 and protein substrate

Question 10

The unfolded protein response

Answer 10

see onenote diagram

Misfolded proteins in ER targeted to proteasome

Ubiquitinated by E3 ligase as they are exported from ER

Question 11

The unfolded protein response and translational repression - PERK

Answer 11

see onenote

PERK is a e1F2-alpha kinase, global repressor of translational initiation

PERK detects unfolded proteins in ER

phosphorylation of e1F2-alpha leads to derepression of ATF4 translation due to uORFs

Question 12

All proteins are degraded eventually

Answer 12

regulated protein degradation occurs by UPS

Question 13

Skp, Cullin, F-box (SCF) family of ubiquitin ligases

Answer 13

F-box proteins

• Define specificity
• CRL type ligase
• Important role in post-translation control in plants

Question 14

Phytohormones

Answer 14

are signalling metabolites in diverse pathways

Question 15

Genetic search for phytohormone receptors

Answer 15

see onenote

phytohormone-insensitive mutants in F-box proteins

Receptor for signalling metabolites

If there is a mutation in protein that acts as a receptor to the metabolite, should be insensitive to that metabolite

Question 16

F-box proteins

Answer 16

see onenote slides

are receptors for phytohormones

F-box protein creates substrate specificity

TIR1, COI1, SLY1 are part of an SCF ubiquitin ligase

low levels of hormone => transcriptional repressors inhibit hormone-response genes

hormones bind to F-box protein to recruit repressor proteins

degradation of repressor proteins by UPS activates transcription

Question 17

An f-box protein regulates Fe homeostasis in humans

• FBXL5
• IRP2

Answer 17

see onenote slides

IRP2 degraded by proteasome under high Fe conditions

IRP2 has no aconitase activity, instead it is degraded under high Fe

FBXL5 is an Fe-binding F-box protein

• when bound by Fe, it forms the SCF complex to degrade IRP2
• when not boud by Fe, FBXL5 is ubiquitinated and degraded
• IRP2 binds to IRE to regulate Ferritin and Transferrin receptor

Question 18

Autophagy

Answer 18

means to recycle cell components

pathways are conserved in fungi, plants, animals

directs material to lysosome

lysosome = acidic compartment containing enzymes for degradation

Question 19

Genetic screen for autophagy mutants

Answer 19

see onenote slides

Autophagosomes - bodies accumulating in the vacuole

Ohsumi isolated 15 ATG mutants

Question 20

Yeast ATG genes

Answer 20

conserved in animals

there are multiple autophagic pathways

Mitophagy - clearing of entire mitochondria

Question 21

Initiation of formation of autophagosomes

Answer 21

see onenote

Initiation involves mTOR signalling
Ubiquitin-like conjugation system, involved in formation of phagosome

Question 22

mTOR complex (TORC1)

Answer 22

see onenote

regulates autophagy

active TORC1 phosphorylates Atg13 to inhibit autophagy

under starvation => mTORC1 is inactive, Atg13 promotes autophage

Question 23

Atg - ubiquitin-like conjugation system in elongation

Answer 23

see onenote

Atg12 and Atg8/LC3 are ubiquitin-like proteins

PE is a membrane phospholipid => required to form autophagosomes

Question 24

Mitophagy an early onset Parkinson’s

- PINK1/PARKIN

Answer 24

see onenote slides

damaged mitochondrial must be cleared from cell

increase damage, reduced clearance => parkinson’s

mutations in PINK1/PARKIN cause early onset Parkinson’s

PINK1 degraded by proteasome in healthy cells

• PINK1 retained on damaged mitochondria, recruiting PARKIN
• PARKIN is an E3 ligase that ubiquitinates mitocondrial proteins
• specific (non-lys48) modifications recruit ATG proteins