Lecture 13. Autophagy Flashcards
What is the role of autophagy (self-eating)?
Removal of protein aggregates, old and damaged organelles and invading microbes
Developmental remodelling
Providing amino acids, nucleotides, lipids and sugars under low nutrient conditions
What is the most well-characterised form of autophagy?
Macroautophagy
Overview of macroautophagy?
A phagophore captures cargo forming an autophagosome
Autophagosome fuses with a late endosome forming an amphisome
Amphisome fuses with a lysosome forming an autolysosome (can miss out amphisome step)
What is mircoautophagy?
Direct targeting into a lysosome
What is chaperone-mediated autophagy?
Entry via a membrane channel (Hsc70)
How does a protein that is directed into the ER end up in the lysosome?
Entry into ER as a preproenzyme, signal peptide cleavage to generate a proenzyme, N-glycosylation and folding but not activated
Transported into the trans-Golgi network (TGN) and still inactive and sorted to the late endosome (LE) where the pH 5.5 starts to activate proenzyme
LE fuses with lysosome, delivering the lysosomal protein to a hybrid endolysosome and the protein is fully activated through pH reduction (4.5)
Lysosome regenerated through tubulation and reformation of the endolysosome
How is the low lysosomal pH (~4.5-5) maintained?
Lysosomes maintain their pH gradients using proton-pumping V-type ATPases, which hydrolyse ATP to pump protons into the lysosome lumen
This generates a transmembrane voltage, so another ion must move to dissipate this so that net pumping can continue
The counterion may be either a cation (positive) moving out of the lysosome or an anion (negative) moving into the lysosome
There are multiple ion channels that move Ca²⁺ and Cl⁻ ions across the lysosomal membrane, maintaining low pH
What are the requirements for chaperone-mediated autophagy (CMA)?
Selective lysosomal degradation of proteins bearing Hsc70-binding KFERQ motifs, via a membrane channel formed from lysosome-associated membrane protein type 2A (LAMP-2A)
What is LAMP-2A?
Receptor and channel for CMA, dedicated channel for proteins with the KFERQ motif
What is the KFERQ motif?
Up to two positively charged residues (K, R)
Up to two hydrophobic residues (I, F, L, V)
A single negatively charged residue (E, D)
A single Q that can be at either the N- or C- terminus
K, E, F and R can be in any order
What are LAMP-2B and LAMP-2C required for?
LAMP-2B required for fusion of autophagosomes with late endosomes/lysosomes
LAMP-2C required for autophagy of nucleic acids
How many mammalian proteins contain a canonical KFERQ motif?
Approximately 40%
What are examples of post-translational modifications to non-KFERQ proteins that can make them become KFERQ?
A motif lacking a negative charge can be converted to an active motif by phosphorylation of S, T or Y
In some instances, Q is replaced by K: following acetylation, this K mimic a Q
What are KFERQ, driven CMA and microautophagy used for?
Disposal of a very wide range of subunits
What are examples of a substrates/proteins with a KFERQ motif?
α-synuclein, Ub, HSc70, RNase A
How does CMA work?
- Recognition of KFERQ motif by Hsc70 which is captured (by cochaperones) and delivered to LAMP-2A
- Mutlimerisation of LAMP-2A aided by Lys-Hsp90, substrate unfolding, translocation mediated by Lys-Hsc70 and hydrolysis
- Hsc70-mediated dismantling of the CMA translocation complex, transport to cholesterol-containing lipid domain, and degradation of LAMP-2A by cathespin A and a metalloproteinase in lipid microdomains
How is CMA down-regulated and what does this result in?
- mTORC2 (has kinase activity) complex activates Akt1 (important) by phosphorylation
- Activated Akt11 phosphorylates GFAP (glial fibrillary acidic protein)
- Phosphorylated GFAP (controls CMA) keeps LAMP-2A inactive
The result is reduced substrate docking
What stimulates mTORC2 activity?
Nutrient rich diets, high fat content diets and increasing age, therefore these factors have a role of inhibiting CMA
How is CMA up-regulated and what does this result in?
Inhibition of mTORC2 and Akt1 reduce phosphorylation of GFAP
Non-phosphorylated GFAP stabilises multimerised LAMP-2A
The result is increased substrate docking
What decreases mTORC2 activity?
Withdrawal of growth factors, Starvation, Oxidative stress, DNA damage, Hypoxia, Small molecules inhibitors all stimulate CMA
These also inhibit Akt1 which s required for stabilising trimeric genome
How is CMA linked to cancer cells?
CMA normally reduces malignant transformation, when CMA goes wrong:
Degrades: tumour suppressors and pro-apoptotic proteins
Protects against radiation and hypoxia
Favours growth and metastasis
How is CMA linked to neurodegeneration?
Healthy individuals contain the CMA substrates α-SYN, PARK7 and Tau
When CMA goes wrong it can lead to CMA dysfunction (mRNA maturation, LAMP2 promoter variants, accumulation of Tau) and CMA toxicity (mutant α-SYN, PTM α-SYN, and Tau varients)
What is the mechanism of microautophagy?
During microautophagy, autophagic cargoes are taken up directly by late endosomes and lysosomes
Some microautophagy substrates bear KFERQ motifs and are delivered by Hsc70 by direct binding of the chaperone to phosphatidylserine in the LE membrane
The autophagic cargos are then degraded in the endolysosomal or lysosomal lumen
What are digestive lysosomes?
Fusions of lysosomes with phagosomes (forming phagolysosomes)
Fusions of autosomes forming autolysosomes
