Lecture 13. Autophagy

Question 1

What is the role of autophagy (self-eating)?

Answer 1

Removal of protein aggregates, old and damaged organelles and invading microbes
Developmental remodelling
Providing amino acids, nucleotides, lipids and sugars under low nutrient conditions

Question 2

What is the most well-characterised form of autophagy?

Answer 2

Macroautophagy

Question 3

Overview of macroautophagy?

Answer 3

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)

Question 4

What is mircoautophagy?

Answer 4

Direct targeting into a lysosome

Question 5

What is chaperone-mediated autophagy?

Answer 5

Entry via a membrane channel (Hsc70)

Question 6

How does a protein that is directed into the ER end up in the lysosome?

Answer 6

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

Question 7

How is the low lysosomal pH (~4.5-5) maintained?

Answer 7

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

Question 8

What are the requirements for chaperone-mediated autophagy (CMA)?

Answer 8

Selective lysosomal degradation of proteins bearing Hsc70-binding KFERQ motifs, via a membrane channel formed from lysosome-associated membrane protein type 2A (LAMP-2A)

Question 9

What is LAMP-2A?

Answer 9

Receptor and channel for CMA, dedicated channel for proteins with the KFERQ motif

Question 10

What is the KFERQ motif?

Answer 10

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

Question 11

What are LAMP-2B and LAMP-2C required for?

Answer 11

LAMP-2B required for fusion of autophagosomes with late endosomes/lysosomes
LAMP-2C required for autophagy of nucleic acids

Question 12

How many mammalian proteins contain a canonical KFERQ motif?

Answer 12

Approximately 40%

Question 13

What are examples of post-translational modifications to non-KFERQ proteins that can make them become KFERQ?

Answer 13

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

Question 14

What are KFERQ, driven CMA and microautophagy used for?

Answer 14

Disposal of a very wide range of subunits

Question 15

What are examples of a substrates/proteins with a KFERQ motif?

Answer 15

α-synuclein, Ub, HSc70, RNase A

Question 16

How does CMA work?

Answer 16

1. Recognition of KFERQ motif by Hsc70 which is captured (by cochaperones) and delivered to LAMP-2A
2. Mutlimerisation of LAMP-2A aided by Lys-Hsp90, substrate unfolding, translocation mediated by Lys-Hsc70 and hydrolysis
3. 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

Question 17

How is CMA down-regulated and what does this result in?

Answer 17

1. mTORC2 (has kinase activity) complex activates Akt1 (important) by phosphorylation
2. Activated Akt11 phosphorylates GFAP (glial fibrillary acidic protein)
3. Phosphorylated GFAP (controls CMA) keeps LAMP-2A inactive
   The result is reduced substrate docking

Question 18

What stimulates mTORC2 activity?

Answer 18

Nutrient rich diets, high fat content diets and increasing age, therefore these factors have a role of inhibiting CMA

Question 19

How is CMA up-regulated and what does this result in?

Answer 19

Inhibition of mTORC2 and Akt1 reduce phosphorylation of GFAP
Non-phosphorylated GFAP stabilises multimerised LAMP-2A
The result is increased substrate docking

Question 20

What decreases mTORC2 activity?

Answer 20

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

Question 21

How is CMA linked to cancer cells?

Answer 21

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

Question 22

How is CMA linked to neurodegeneration?

Answer 22

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)

Question 23

What is the mechanism of microautophagy?

Answer 23

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

Question 24

What are digestive lysosomes?

Answer 24

Fusions of lysosomes with phagosomes (forming phagolysosomes)
Fusions of autosomes forming autolysosomes

Question 25

What are the four things to consider about how autophagosomes are formed (the order of events in autophagosome formation)?

Answer 25

Initiation: Formation of the phagopore
Nucleation
Growth of the phagopore
Closure of the phagopore

Question 26

What happens in the initiation stage of autophagosome formation?

Answer 26

Formation of the phagopore
Inhibition of mTORC complexes (mammalian/mechanistic target of rapamycin) due to starvation stimulating macroautophagy (from withdrawal of insulin, growth factors and nutrients) and unphosphorylates ULK1
Activation of ULK1 complexes which associate and capture Atg9 vesicles. ULK1 kinase phosphorylates Beclin which activates PI3K activity
Activation of PI3K complex, phosphorylation of membrane lipids

Question 27

What happens in the nucleation stage of autophagosome formation?

Answer 27

Recruitment of WIPI proteins to phosphorylated membrane lipids and bind to PI3P on the ATG9 vesicles
Recruitment of ATG16 complex (by WIPI proteins) and lipidation of LC3-I attaches to membrane lipids by ATG16 complex, becoming LC3-II
Recruitment of ATG2 (lipid transporter) which binds to the omegasome, anER-domain that might be the same as an ER-exit site

Question 28

What happens in the vesicle fusion stage of autophagosome formation?

Answer 28

A phagophore (isolation membrane, IM) is formed: captured by ATG13, connected via ATG2 to the omegasome, and bearing multiple molecular markers from multiple cellular locations; the phagophore is now expanded
Expansion takes place at ATG2 and ATG9

Question 29

What happens in the growth stage of autophagosome formation?

Answer 29

ATG9 vesicle fusion - ATG9 is a lipid ‘flippase’ so both inner and outer leaflets are expanded with ER-derived lipids
Transfer of membrane lipids from the omegasome generate new outer leaflet membrane lipids, resulting rapid expansion of the phagopore

Question 30

What happens in the closure stage of autophagosome formation?

Answer 30

Role of ESCRT (endosomal sorting complex required for transport) complexes
ESCRT-I bridges the gap, ESCRT-III subunits are recruited by FIB200 (a member of the ULK complex) to form a plug
The plug allows the membranes to seal and the ESCRT proteins are removed
Autophagosome now compete for lysosomal fusion

Question 31

How is LC3 process by proteolytic cleavage and lipidation?

Answer 31

Three isoforms of LC3 (LC3A, LC3B, LC3C) are processed by proteolytic cleavage agter a gly residue becoming LC3A-1, LC3B-1, LC3C-1
And on demand, a proportion of LC3-I is conjugated to phosphatidylethanolamine (PE) to generate LC3-II

Question 32

How do we know LC3 isoforms associate with different membranes?

Answer 32

Despite both being associated with autophagosomes/amphisomes, there is no co-localisation between LC3A-II and LC3B-II, implying they associate with different vesicles

Question 33

How does LC3-I in the cytosol catch cargo?

Answer 33

Ubiquitylated cargo can be captured by cargo receptors such as p62 which can be collected by cytosolic LC3-I, stimulating conjugation to membrane PE by ATG16 complex
Thus availability of cargo can stimulate selective autophagy (allowing response to cellular stress)

Question 34

How does LC3-II capture cargo receptors?

Answer 34

LC3-II captures cargo receptors that contain an LC3-interacting region (LIR) motif
The cargo receptor then recruits cargo e.g., the p62 cargo receptor binds ubiquitylated proteins
Other cargo receptors have different specificities