34 – Function & Regulation of Autophagy Flashcards
What is autophagy?
self-degradative process found in all eukaryotic cells
Role of autophagy in normal cells function & in stress response
- Clearance of cytoplasm
- Nutrient recycling
Clearance of cytoplasm
Removal of aged ribosomes, abnormal protein aggregates, long lived proteins, aged supramolecular structure, invasive viruses
Turnover of excessive/defective/harmful organelles
-ER, mitochondria, peroxisomes, nucleus
Nutrient recycling
In response to new cell development need, starvation & other stress, it provides:
-Amino acids for protein synthesis
-Energy source essential for new cell development need, survival under starvation & other stressed
Level of autophagy can be …
basal or induced
All lives are short lived but relentless
Recycling = essential for life, important ability for survival
-Different between organisms & machine
Cell=balanced state between synthesis & degradation:
-Replacement of most proteins every 3 months
* New central dogma?
General process in autophagy
- Induction & nucleation
-Phagophore
-Omegasome - Expansion/Maturation
-Phagophore - Autophagosome fuse with lysosome = autolysosome
- Degradation of what was inside autophagosome& recycling
Molecular regulation of autophagy:
Ohsumi’s screen for autophagy mutants (apg(atg)) in yeast:
differene with WT and mutant
- Treat ~38000 yeast cells with EMS mutagen
-Single point mutation - 1st screen by plate assay of loss of viability in SD(-N) medium (nitrogen starvation)
-Media lacking N (induce autophagy)= which cells can’t survive = 2700 defective - 2nd screen for lack of autophagic body accumulation in vacuole by light microscopy of yeast cells in SD(-N) medium
=99 defective - Complementation analysis by adding WT gene back to 99 mutant cell lines
=To see the defect is in atg or not - After 2 screen, identified 14 Apg/atg (autophagy) mutant isolated
In WT: accumulation of autophagic bodies inside vacuole
Mutant: lacks autophagic bodies inside vacuole
Empty vacuole= autophagy carrying mutant from cytosol to vacuole (defective)
Atg 1 function & how
Atg 1 = kinase induces autophagy
-Key autophagy initiator
once starved (stress) = dephosphorylation of Atg13 = Atg 1- Atg13 complex = tethered ER domain: Omegosome
to grow phagophores
Lipidated Atg8 (LC3-II) attaches onto growing phagophores to grow them
-Lipidation needs Atg5 complex
–phagophore grow around Defective ER, mitochondria
Atg8/LC3 on growing phagophores also serves as
cargo ligand that recruits cargoes
Mitophagy visualized by GFP-LC3
Hepatocytes expressing GFP-LC3 loaded with TMRM - Dye marks active mitochondria
Cells then placed in nutrient deprivation condition
After 120mins, GFP-LC3 appeared around mitochondria
In 5 mins, mitochondrion fully surrounded by GFP-LC3
who supplies membrane for phagophore expansion?
Atg9 tagged vesicles & Atg18 + Atg2 channel = supplies membrane for phagophore expansion
-Atg18+Atg2: channel between ER & phagophore
-Have lipids required to grow phagophore
Membranes must be supplied to growing sites
How Atg18 tethered onto ER?
Rab18 GTP promote autophagy by tethering ATG18a to ER in response to nutrient starvation
What regulates fusion autophagosomes with lysosome?
Atg8/LC3 needs to be released from autophagosome
-so that the vesicle can fuse with target membrane
SNAREs: SNAP29 & other
Rab proteins: Rab7
Tethering factors: Atg14
-Downstream effector of Rab protein
Autophagy in development & pathogenesis:
Starvation adaptation
what happened with the mice experiment?
Autophagy transiently induced immediately following birth
Autophagy monitored by accumulation of GFP-LC3 in heart muscle cells of embryonic & post-natal mice
-Increased LC3 seen post-natally till~24 hours
Reason: Embryo nourished by placenta
-Upon birth, neonates suddenly face starvation
–Mice can take nutrient from mild immediately after birth
-Autophagy activated immediate after birth to self-nourish
Atg5 knockout mice
die neonatally
Require Atg5 for Atg8 for autophagy
Atg5 mutant appear normal at birth, but die within 24 hours
Sars-CoV-2 & autophagy
Autophagy in viral infection
-Elimination of invasive viruses
-GFP-LC3
Arms race between Sars-CoV-2 & autophagy
Endocytosis of Sars-CoV-2 with ACE 2
Makes early endosome - fusion = late endosome
Usually degraded in lysosome, BUT
-Can release protein to break/inhibit fusion with lysosome
Phagophore - autophagosome has virus inside - autophagosome no fused with lysosome due to ORF7a
Smooth walled vesicle with new virus inside to release it.
