L34 Post-translational regulation and Autophagy Flashcards
Protein folding
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
Hsp
Molecular chaperones
- rapidly induced by mild heat shock
- conserved in bacteria , plant, animals
Hsp70
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
Hsp60
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.
Ubiquitin proteasome system (UPS)
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
Post-translational mechanism
see onenote
Ubiquitination of proteins
Ubiquitin added to lysine residues in protein
Poly-ubiquitination of Lys48 targets proteins to proteasome
Poly-ubiquitinated proteins degraded by 26S proteasome
Ubiquitin ligases act on target proteins
see onenote
Diversity of E3 ubiquitin ligases
see onenote
Cullin-RING-ligase (CRL) type
Non-CRL type
E3 ligases interact with E2 and protein substrate
The unfolded protein response
see onenote diagram
Misfolded proteins in ER targeted to proteasome
Ubiquitinated by E3 ligase as they are exported from ER
The unfolded protein response and translational repression - PERK
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
All proteins are degraded eventually
regulated protein degradation occurs by UPS
Skp, Cullin, F-box (SCF) family of ubiquitin ligases
F-box proteins
- Define specificity
- CRL type ligase
- Important role in post-translation control in plants
Phytohormones
are signalling metabolites in diverse pathways
Genetic search for phytohormone receptors
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
F-box proteins
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
An f-box protein regulates Fe homeostasis in humans
- FBXL5
- IRP2
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
Autophagy
means to recycle cell components
pathways are conserved in fungi, plants, animals
directs material to lysosome
lysosome = acidic compartment containing enzymes for degradation
Genetic screen for autophagy mutants
see onenote slides
Autophagosomes - bodies accumulating in the vacuole
Ohsumi isolated 15 ATG mutants
Yeast ATG genes
conserved in animals
there are multiple autophagic pathways
Mitophagy - clearing of entire mitochondria
Initiation of formation of autophagosomes
see onenote
Initiation involves mTOR signalling
Ubiquitin-like conjugation system, involved in formation of phagosome
mTOR complex (TORC1)
see onenote
regulates autophagy
active TORC1 phosphorylates Atg13 to inhibit autophagy
under starvation => mTORC1 is inactive, Atg13 promotes autophage
Atg - ubiquitin-like conjugation system in elongation
see onenote
Atg12 and Atg8/LC3 are ubiquitin-like proteins
PE is a membrane phospholipid => required to form autophagosomes
Mitophagy an early onset Parkinson’s
- PINK1/PARKIN
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
