Lecture 13 - Autophagy

Question 1

Define Autophagy

Answer 1

Process of Self-digestion using action of enzymes originating in the same cell

Question 2

State the three main types of Autophagy

Answer 2

1. Chaperone-Mediated Autophagy (CMA)
2. Microautophagy
3. Macroautophagy (Autophagy) - involves the sequestration and transport of protein aggregates/organelles to the lysosome in double membraned structures

Question 3

Why does Autophagy Occur?

(2 Reasons)

Answer 3

1. Cell Starvation - non-essential components are recycled to provide energy and amino acids for synthesis of essential proteins
2. Removal of Protein Aggregates, Damage Organelles, Invading bacteria

Question 4

What Substrates may undergo autophagy?

(4 Points)

Answer 4

1. Organelles - Nucleus, Mitochondria, Ribosomes
   * Ribophagy - pathway involved in coordination of protein synthesis and degradation
2. Components - Aggregate-prone proteins, Lipids, RNA
3. Microbes

Question 5

Autophagy is a selective process. What is the principle signal for Autophagy of a substrate?

Answer 5

K63-linked Polyubiquitination

Question 6

Describe the mechanism of substrate recruitment into a developing autophagosome

(3 Points)

Answer 6

• Protein Aggregate/Organelle surface protein is ubiquitinated with K63-linked chain
• Adaptor Proteins (Autophagy Receptor) - recognises both K63 Linked chain and LC3-II on surface of autophagosome
• Substrate is recruited into developing autophagosome

Question 7

(i) What is the ATG8 Family?
(ii) What are their Structures?

Answer 7

(i) Consists of Six Different Proteins with different functions (e.g., LC3s - involved in Autophagy)

(ii) Ubiquitin-like 3D/Tertiary Structure

Question 8

(i) Compare LC3-I Lipidation to Ubiquitylation

(ii) Why is LC-I Lipidated

(3 Points)

Answer 8

(i):
* Both utilise Three different enzymes with similar functions (E1, E2, E3)
* Both require ATP

(ii) LC-I is lipidated with PE to LC-II to allow it to associated with autophagosome membrane

Question 9

What Binding domains do Autophagy Receptors require to carry out their function?

(3 Points)

Answer 9

1. UBD (e.g., UBA, UBZ)
2. LIR or LC3 Interacting Regions
   * Very Simple (6aa), often containing WXXL consensus (very degenerate)

Question 10

How do Autophagy Receptors achieve high affinity interactions with polyubiquitin chains?

Answer 10

• Rarely have tandem UBDs, instead oligomerising through different oligomerisation domains (e.g. PB1), or via coiled-coil

Question 11

Describe how Ubiquitin signalling can be amplified in response to Mitochondrial Damage

Answer 11

• Mitochondrial damage leads to activation of membrane associated kinase PINK1
• PINK1 phosphorylates Parkin (E3 Ligase), inducing conformational change that activates it
• PINK1 also phosphorylates Ubiquitin monomers on Ser65
• Parkin utilises phosphoubiquitin to form K63-linked chains on mitochondria that favour autophagy receptor binding, and disfavour DUB activity

Question 12

What is TBK1? How does it facilitate Autophagy?

Answer 12

• Kinase recruited by interaction with autophagy receptor, and activated by receptor oligomerisation
• Once activated it phosphorylates the autophagy receptors UBD and LIR domains, facilitating binding and recruitment of cargo into autophagosomes