Autophagy (Martens) Flashcards
Explain differences between the three main types of autophagies.
Chaperone mediated:
- unfolded proteins
1) Upon unfolding, protein exposes KFERQ seq. -> recognized by Hsp70
2) Translocation to lysosome
3) Hsp90 (LAMP-2A) binding
4) Hsp70 dissociation
5) Further protein unfolding
6) Protein translocation into lumen
7) LAMP-2A dissasembly and degradation (not constitutively in the membrane)
Microautophagy
- similar to endocytosis
- ESCRT involvement
- lysosome form extension to grab cytosolic molecules
Macroautophagy
- non-selective (starvation induces) or selective
- double isolation membrane formation
- de-novo
- ESCRT involvement
State all generalized steps of macroautophagy.
1) Cargo recognition by receptor
2) Scaffold protein interaction
3) Atg9 vesicle formation and binding via scaffold to cargo
4) PI3K -> PI3P, important for binding of many Atg proteins
5) Lipid transfer (or membrane protein rearrangement?)
6) E3-like ligation by Atg12 system
7) Atg8 lipidation (outgrowth of autophagosome) and de-lipidation
Name autophagy functions.
- recycling of nutrients in starvation
- protein aggregation removal and neurodegeneration prevention
- recycling of damaged organels and cancer prevention
- pathogen removal
Why is autophagy tumor-supressive before developing cancer but is tumor-promoting when already developed?
Before cancer development:
Damaged mitochondria cause ROS -> removed by autophagy
After developing cancer:
Damaged mitochondria cause ROS -> cell is rescued by autophagy and does not enter apoptosis
Explain how the starvation-induced autophagy is activated.
Presence of AA (nutrients) -> active mTOR -| inactive Atg1 and thus inactive autophagy
Absence of AA (nutrients) -| active mTOR -> active Atg1 and thus active autophagy
Where is the Atg9 vesicle formed? From which organel arises? Why?
Omegasome-intermediate formation: ER proximity, in the cytoplasm (no direct contact). It has similar densities.
Where (in the cell) do the autophagy proteins and Atg9 vesicle with bound cargo translocate after being formed? What intermediates do they form?
Closer to vacuole (yeast) or lysosome (eukaryots). They form pre-autosomal structures (PASs) before forming autophagosome.
Briefly explain how do autophagosomes fuse with lysosomes.
Autophagosome: vSNARE Stx17
+ SNAP29 addaptor
Lysosome: tSNARE VAMP8
Shortly explain cargo recognition and degradation in Cvt pathway.
Cvt: cytoplasm-to-vacuole:
- typical cargo: prApe1 (aminopeptidase) needs to be degraded
1) prApe1 is colocalized and recognized by Atg19
2) Atg19s bind to Atg8s
3) Inner and outer membrane degraded
4) prApe1 -> mApe1 (cleaved)
What is the role of Atg19?
Conjugating cargo with autophagy machinery.
Domains:
-ZF, UBA: ubi-binding
-LIR motif: Atg8/LC3 binding, protein on the membrane
-FIR motif: binds FIP200
-PB1: oligomerization
Explain cargo recognition in aggrephagy *in detail**. In which disease is the receptor mutated? What domain is mutated?
Cargo receptor: p62
-PB1: oligomerization dom.
-LIR motif: LC3 interacting region, LC3 = Atg8
-UBA dom.: ubi/cargo binding
Equilibrium between UPR and aggrephagy (misfolded protein -> UPR and proteasome, poly- and mono-ubi block aggrephagy) is maintained.
1) NBR1 binds aggregate via UBA dom.
2) NBR1 colocs with p62 via PB1 dom., p62s condensate
3) NBR1 recruits TAX1BP1
4) TAX1BP1 recruits FIP200
5) FIP200 + adaptors bind Atg19 which binds Atg8-isolation membrane
Diseases: ALS, FTD, mutated UBA dom.
Explain E3-like ligation by ATG12 system in detail. What is its purpouse? Name an important Atg homolog in humans.
Purpouse: conjugation of phosphatidylethanolamine (PE) with Atg8
Note for self: cargo recognition or lipidation or both????
1) Atg7 (E1) conjugation with Atg8 (ATP -> AMP)
2) Binding of Atg3 to Atg7-Atg8
3) Transfer of Atg8 to Atg3
4) Binding of Atg12-5-16L1 to Atg7-Atg3-Atg8
5) Transfer of Atg8 to PE
Atg8 = LC3
Explain mitophagy.
Physiological state:
1) (Usually 2) PINK1 proteins are integrated into TIM/TOM compex, passing both membranes
2) PINK1 is cleaved by PARL in IMM and by MPP in lumen
3) PINK1 is translocated into cytosol
4) Ubi by UBR1-2-4 complex (E3 ligase) and degradation
Pathophysiology state: mitochondrial stress and mitophagy
1) PINK1 translocation into both membranes is incomplete and PINK1 is only retained in the OMM -> no cleavage and sequestering
2) Transphosphorylation of PINK1 -> phosphorylation of Parkin and poly-ubi substrates on OMM
3) Parkin further phosphorylates ubi chains on substrates
Substrates:
- Optoneurin (OPTN)
- TAX1BP1 (also in aggrephagy)
- NDP52
ZF dom: ubi-binding
dimerization dom.
autophagy machinery interacting dom.
Explain mitophagy induced cGAS/STING.
Mitochondrial stress -> membrane burst -> ROS and mitDNA leakage
1) cGAS is activated and produces cGAMP (cyclic AMP-GMP)
2) Recognition by STING receptor on ER
3) Activation of TBK1 and IKK proteins
4) NF-kappaB TF pathway activation via TF
Explain the role of deubiquitinases (DUBs) in mitophagy.
DUB USP8: deubiquitinates Parkin -> Parkin can phosphorylate ubi substrates -> mitophagy takes place
DUB UPS8 not present: Parkin inacive -> DUB USP15-30 deubiquitinate ubi substrates -> mitophagy inhibited
