Autophagy

Question 1

What is autophagy?

Answer 1

Autophagy the process by which material is delivered to the lysosomes.

Question 2

What are the three main types of autophagy?

Answer 2

Microautophagy
Macroautophagy
Chaperone mediated autophagy

Question 3

In which type of autophagy are heat shock proteins involved, and how are proteins targeted for this pathway?

Answer 3

Chaperone mediated autophagy involves hsp70 and hsp90. Only proteins that contain a specific targeting sequence (KFERQ) can be targeted to the lysosome via this method.

Question 4

What is an autophagosome?

Answer 4

A double membraned vesicle that contains the cellular contents that are targeted for destruction in the lysosome. The double membraned autophagosome fuses with the lysosome and the contents of the autophagosome are degraded.

Question 5

What separates protein degradation via autophagosomes and lysosomes from degradation via proteasomes?

Answer 5

On average, shorter-lived proteins are degraded by proteasomes, longer-lived proteins are degraded in the lysosome. Autosomal/lysosomal degradation is the only known method of degrading organelles. Mitochondria are degraded via lysosomes, as are lysosomes themselves.

Question 6

Is autophagy regulated or unregulated?

Answer 6

It is both regulated and unregulated. Autophagosomes can be directed to take specific items to lysosomes, or they can indiscriminately envelop proteins and degrade them all. The process that determines regulation/unregulation is unknown.

Question 7

What is the process of autophagy-mediated degradation of extracellular material?

Answer 7

Extracellular material is encapsulated in an endosome that binds with an autophagosome, creating an amphisome. The amphisome then binds with a lysosome to create an autolysosome in which all contents are degraded.

Question 8

What effect does autophagy have on aging?

Answer 8

Increased autophagy increases life span of worms and other model organisms. Calorie restriction diets likely extend life in multicellular organisms by increasing rates of autophagy. mTOR inhibitors have been shown to increase average life span as well, likely through increased autophagy.

Question 9

What are the effects of chemotherapeutic agents on autophagy?

Answer 9

All tested chemotherapeutic drugs affected autophagy rates. 3/4 increased autophagy, while 1/4 inhibited it. Current studies are looking at the effects of deliberate manipulation (increase or decrease) of autophagy to affect cancer outcomes.

Question 10

What induces autophagy?

Answer 10

Autophagy is induced during times of nutrient stress. Double knock-out mice develop fine in utero but die at birth due to sudden lack of nutrients. Birth is the most severe time of nutrient stress experienced by mammals (constant nutrition to zero nutrition between feedings).

Question 11

Why does examining fixed tissue samples (EM imaging) present problems with determining the rate of autophagy?

Answer 11

Fixed tissue samples only show the number of autophagosomes present at one time. Increased numbers of autophagosomes may represent an increase in their rate of creation or a decrease in their rate of degradation. Thus, the same fixed state may represent an increase or a decrease in autophagy and they cannot be distinguished.

Question 12

How are autophagy rates assessed in living cells?

Answer 12

GFP tagged LC3 protein. LC3 is incorporated into autophagosomal membranes and can be visually tracked.

Question 13

What are the 6 or 7 steps of the autophagosome cycle?

Answer 13

1 Induction (nutritional stress, rapamycin, chemo drugs, etc)
2 Vesicle nucleation (Phagophore)
3 Vesicle expansion (Omegasome - shaped like omega)
4 Cargo targetting (via LC3II and p62)
5 Vesicle closure
6 Vesicle fusion with endosome (Amphisome, if it occurs)
7 Vesicle fusion with lysosome (Autolysosome)

Question 14

Why are Atg genes of interest to pharmaceutical companies, and what complicates their use?

Answer 14

Atg genes code for proteins that control the autophagy process, and could be drug targets for affecting autophagy. However, all of the Atg products function in multiple areas and drugs would have varied effects/side effects.

Question 15

How are ubiquitinated proteins recognized by autophagosomes?

Answer 15

The protein p62 has a region (UBA) that recognizes ubiquitin and can attach to poly-ubiquitinated proteins. p62 also has a region that binds to LC3, which localizes to autophagosome membranes, so the p62 also attaches to LC3 and draws the poly-ubiquitinated protein into the autophagosome.

Question 16

What is the role of the protein “parkin”?

Answer 16

Parkin plays a role in mediating the attachment of p62 to LC3 when it is bound to ubiquitinated mitochondria. Parkin is often mutated in familial cases of Parkinson’s disease, thus implicating the action of autophagy in the pathology of the disease.

Question 17

Why is the mTOR pathway so important in autophagy?

Answer 17

Many of the processes that lead to autophagy converge on mTOR.

Question 18

How does Beclin-1 illustrate the complexity of control mechanisms in autophagy?

Answer 18

Under normal conditions, Beclin-1 induces autophagy. When an apoptotic process is initiated, Beclin-1 is cleaved by caspase-8 or caspase-7 and formed Beclin-1c, which inhibits autophagy.

Question 19

What proteins does Beclin-1 interact with and what is the mechanism that prompts it to induce autophagy?

Answer 19

Beclin-1 also interacts with Bcl2/Bcl-XL which are apoptosis regulators. A third protein, BH3 can compete with Beclin-1 for binding with Bcl2/Bcl-XL, freeing Beclin-1 to initiate autophagy.

Question 20

How does the BH3 mimetic ABT 737 function, and what effect does it produce?

Answer 20

ABT 737 is the first drug developed that does not interrupt an enzyme, but effects protein-protein interactions. It interacts with Bcl2/Bcl-XL at the surface of the mitochondria to induce apoptosis. It also interacts with Bcl2/Bcl-XL where it binds to Beclin-1, freeing Beclin-1 to initiate autophagy. The cancer cells die, but the whether they die due to apoptosis or autophagy is unclear.

Question 21

How does rapamycin interact with autophagy?

Answer 21

Rapamycin inhibits mTOR, which inhibits Atg proteins. In the absence of autophagic proteins (in knockout models), rapamycin does not prevent cell death. In the presence of autophagic proteins, rapamycin promotes autophagy and cell survival is enhanced.

Question 22

One example of how the inhibition of autophagy may result in increased effectiveness of chemotherapeutics.

Answer 22

Administration of CQ (an anti-malarial drug that inhibits autophagy) improved the effectiveness of vemurafenib in a 13 year old ganglioglioma patient.

Question 23

How might rapamycin be therapeutic for neurodegenerative diseases?

Answer 23

Rapamycin induces autophagy. Increased rates of autophagy may result in improved clearing of aggregate-prone proteins, such as poly-glutamine tracts in Huntington’s disease or other aggregate prone proteins such as Tau in Alzheimers.