Autophagy

Question 1

What are the functions of autophagy/lysosomal pathways?

Answer 1

Recycling of nutrients
Downregulation of signalling pathways
Cellular remodelling
Killing of intracellular pathogens
Homeostasis
Removing damaged components

Question 2

What is the UPS?

Answer 2

Ubiquitin-Proteasome system: Proteins are tagged with ubiquitin and recognised by the UPS. Major turnover pathway for short-lived proteins, doesn’t require a lysosome. Some bacteria have a similar system called pupylation.

Question 3

What is macroautophagy?

Answer 3

Lysosomal, bulk digestion pathway using autophagosomes. Can digest entire organelles, the molecules released support metabolism.

Question 4

What is microautophagy?

Answer 4

Lysosomal, engulfment of cytoplasmic material, lower volume than macroautophagy.

Question 5

What is CMA?

Answer 5

Chaperone-mediated autophagy. Proteins with a CMA-targeting motif are recognised by chaperones which deliver them to the lysosome for degradation. Similar to Microautophagy but much more specific.

Question 6

Tell me about nutrient recycling

Answer 6

Autophagy is rapidly upregulated under starvation conditions, causes non-selective bulk-degradation of the cytosol.
Autophagy deficient mice die during neonatal starvation, likewise, autophagy-deficient cells die during starvation conditions. Cancer cells in solid tumours require autophagy until the vascular system develops.

Question 7

Tell me about cellular remodelling

Answer 7

Autophagy is the only mechanism that can degrade entire organelles. In erythropoiesis, the nucleus, mitochondria and ER are removed. Sperm-derived mitochondria are removed after eggs are fertilised.

Question 8

Tell me about the removal of damaged components

Answer 8

Overtime, cellular components accumulate damage, can be due to oxidative stress and misfolding. Particularly relevant to ageing and neurodegenerative diseases; as you age your autophagic flux capacity decreases. Long-lived cells and muscle cells are more susceptible.

Question 9

Tell me about the role of autophagy in killing intracellular pathogens/viruses

Answer 9

Many pathogens have evolved to escape or survive in lysosomes & escape into the cytoplasm. This includes Mycobacterium and some Salmonella strains.

Question 10

What can we use autophagy for?

Answer 10

Inhibit it to starve cancer cells or reduce the cellular uptake of pathogens.
Upregulate it to reduce ageing, reduce muscle dystrophy, reduce neurodegeneration, prevent cancers developing, kill more pathogens/viruses.

Question 11

How many proteins are involved in autophagy, and what initiates it?

Answer 11

There are 50-60 proteins involved (atg genes). VLK1 complex initiates the pathway (it’s a kinase), and it’s controlled by mTORC1 & AMPK. Initiation leads to autophagosome formation & elongation until it closes and fuses with a lysosome.

Question 12

What is the role of autophagy in neurodegenerative diseases?

Answer 12

Autophagy is a house keeping function ensuring that misfolded or damaged proteins are degraded.
Neurons are particularly sensitive to misfolded proteins due to their long lifespan and transport of proteins over long distances.
Dysfunctional autophagy can lead to accumulation of ubiquitinated aggregates & increased apoptosis. Neurons cannot reproduce.

Question 13

What causes Huntington’s disease?

Answer 13

Polyglutamine expansion in the huntingtin protein. When Q<18, individual is healthy, when Q>35, huntingtin protein is disease causing. PolyQ huntingtin protein is misfolded and aggregates where it is ubiquitinated, usually it would be removed by proteasomal degradation but it can form aggresomes which have to be removed via autophagy.

Question 14

Why is polyQ huntingtin protein toxic?

Answer 14

Can lead to loss of normal huntingtin protein function.
Interacting proteins are sequestered & degraded
Can form toxic oligomers and aggresomes.
Can damage the proteasome/proteasomal system
Sequester adaptors required for other damaged proteins

Question 15

Tell me about the molecular basis of Parkinson’s disease

Answer 15

Affects 1-2/1000 people in the UK. Involves the loss of inhibitory dopaminergic neurons. α-synuclein is rarely mutated itself.
α-synuclein is usually degraded via CMA, the A53T mutation blocks the CMA.

Question 16

Tell me about the genetic basis of Parkinson’s disease

Answer 16

Complex genetics, only 5-10% of cases are familial. Involves mutations of α-synuclein, PINK1/PARKIN proteins which regulate mitophagy.
In sporadic early-onset cases:
5-10% mutations are loss of function mutations in PINK1
10-15% mutations are loss of function mutations in PARKIN

Question 17

Tell me about mitophagy in Parkinson’s disease

Answer 17

Damaged mitochondria are the main source of ROS in a cell and undergo mitophagy (mitochondrial autophagy) to prevent ROS production.
PINK1 is a kinase on the surface of mitochondria.
PARKIN1 is an E3 ubiquitin ligase (mutated in 50% of autosomal recessive cases).

Accumulation of damaged mitochondria results in a feedfoward loop which spirals out of control.

Question 18

What is the proteinopathy of Alzheimer’s?

Answer 18

β-Amyloid plaques

Question 19

Tell me about autophagy in cancer

Answer 19

Cancer is caused by an accumulation of DNA damage, some of this damage comes from toxic substances within the cell. Autophagy can remove toxic substances to prevent oxidative stress, DNA damage and tumorigenesis (tumour suppressive).
Monoallelic deletion of Beclin2 (atg6) gene is involved in 40-75% of ovarian, breast and prostate carcinomas.

Autophagy is upregulated in hypoxic, nutrient poor tumour regions. This allows cancer cells to survive until vasculation occurs. Blocking autophagy can lead to an increase in necrosis/apoptosis, stopping or slowing tumour growth when there’s low vasculation.

Question 20

How does autophagy inhibit apoptosis?

Answer 20

Beclin1 is key in autophagosome formation & mitophagy. When activated, it sequesters Bcl2 (B Cell Lymphoma 2), a protein on mitochondria which prevents mitophagy.
Beclin1 activation is key in allowing damaged mitochondria to be degraded preventing ROS production and eventual cell apoptosis. This mechanism also drives tumour survival and chemotherapy resistance.

Question 21

List how autophagy is anti and prooncogenic

Answer 21

Anti: ensures cell homeostasis, removes damages proteins/components, reduces ROS/genotoxicity, reduces inflamamtion
Pro: Promotes the survival of tumours, prevents cell apoptosis, confers chemotherapy resistance

Question 22

What are the strategies for autophagy therapies?

Answer 22

Inhibit autophagy to block survival to metabolic stress and increase apoptosis during chemotherapy
Promote autophagy to remove damage & prevent tumorigenesis