Autophagy

Autophagy translates to

“self-eating”

Autophagy

evolutionarily conserved catabolic process in eukaryotic cells

Catabolic Process

breakdown molecules

Functions of Autophagy

• removal of misfolded or aggregated proteins

• clearance of damaged organelles

• elimination of intracellular pathogens

Where was autophagy first discovered?

a survival mechanism in yeast subjected to nutrient deprivation

How many autophagy-related (Atg) proteins have been identified and where?

>40 and mainly by genetic screens in S. cerevisiae

Atg proteins assemble into functional complexes to promote

autophagosome formation, trafficking, and fusion with lysosomes

3 major forms of autophagy

1. macroautophagy

2. micrautophagy

3. chaperone-mediated autophagy (CMA)

Macroautophagy

how organelles and proteins are broken down

Macroautophagy uses an autophagosome to deliver

cytoplasmic cargo to the lysosome

Microautophagy

cytoplasmic material is brought into lysosome

What happens during microautophagy?

invagination of the lysosomal membrane

Chaperone-Mediated Autophagy (CMA)

Hsc70/Hsp73-dependent; very selective

What happens during CMA?

• uses chaperone proteins that are recognized by lysosomal-associated membrane protein 2A (LAMP-2A)

• results in unfolding and degradation

What do all 3 forms of autophagy promote?

proteolytic degradation of cytosolic components at the lysosome

Microautophagy is mediated by

direct engulfment of the cytoplasmic cargo

For microautophagy, cytoplasmic material is trapped in the lysosome/vacuole by

membrane invagination

Mostly observe non-selective microautophagy in

mammalian cells

Steps of microautophagy

• membrane invagination and autophagic tubes formation

• vesicle formation

• vesicle expansion and scission

• vesicle degradation and recycling

  • cargo degraded by hydrolases

Chaperone-Mediated Autophagy Process

• chaperone-dependent selection of proteins that are targeted to lysosomes and directly translocated across the lysosome membrane for degradation

• CMA is selective and allows for direct shuttling of proteins across the lysosomal membrane without the requirement for the formation of additional vesicles

• Hsc70 targets cytosolic proteins to CMA via recognition of KFERQ

• lysosome-associated membrane protein type 2A (LAMP-2A) is the receptor for this pathway and recognizes chaperone-bound substrates

Steps of CMA (from figure)

1. recognition of KFERQ-motif bearing substrate proteins by hsc70/cochaperones in the cytosol

2. binding of substrate-chaperone complex to LAMPS-2a monomer

3. unfolding of the substrate

4. LAMP-2A multimerization, substrate translocation and subsequent degradation

5. disassembly of LAMP-2A multimer/translocon. LAMP-2A monomers are degraded in lipid microdomains

In macroautophagy, part of the cells are delivered to the lysosome in the membrane-bound vesicles for

degradation

Macroautophagy is induced in response to

different stessors

Where can these different stressors in response to macroautophagy be induced?

nutrient or growth factor deprivation, hypoxia, damaged proteins and organelles, genotoxic stress

Autophagy is initiated by the assembly of the core autophagy machinery proteins. Where is this located?

yeasts and mammals

Where is the ULK1 complex located in and what is it?

mammals and a starting protein

Macroautophagy Process

• formation of the PAS (phagophore assembly site) initiates autophagy and defines the site of autophagosome formation

• autophagy begins with the extension of the phagophore

• the phagophore engulfs the molecules and organelles to be eliminated, forming a double membrane vesicle called autophagosome

• autophagosomes are targeted to lysosomes and fusion occurs, forming an autolysosome. the sequestered material is degraded and released back into the cytosol

Phagophore

a specialized membrane derived from the endoplasmic reticulum (ER), the mitochondria, and the Golgi cisternae

Mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) are central in

macroautophagy regulation

AMPK promotes (+)

autophagy upon its activation under conditions of energy deficiency (elevated AMP/ATP ratio)

[AMP/ATP] = low ratio →

a lot of energy

[AMP/ATP] = high ratio →

low energy state

mTOR inhibits (-)

autophagy

mTOR integrates signals from

GFs, stress, energy status, oxygen, and AAs

mTOR

ser/thr and thy protein kinase that regulates cell growth, proliferation, movement, survival, protein synthesis, transcription, and autophagy

mTOR is the central regulator of

mammalian metabolism and physiology (e.g., diabetes, obesity, and cancers)

What are mTOR catalytic subunits?

• mTORC1

• mTORC2

mTORC1

regulate protein synthesis and cell growth

mTORC2

responsive to GFs, involved in metabolic regulation, associates with ribosomes

mTOR inhibits (-) autophagy via

the inhibition of ULK1 (Unc-51 like autophagy activating kinase)

Rapamycin

inhibits mTORC1 and has shown to extend lifespan in vertebrates

Calorie restriction has been shown to

reduce mTOR activity

Would rapamycin promote or inhibit autophagy?

promote

Would rapamycin extend or shorten lifespan?

extend

Example experiment for rapamycin:

flies → rapamycin → lysosome tracking via Lysotracker (proxy for autophagy)

• more rapamycin = more autophagy

Atg5 is involved in the early stages of

autophagosome formation

What do you think would happenn if ATG is downregulated and flies are treated with rapamycin?

there would be a minimal increase in lifespan

Conclusion of experiment for rapamycin

inhibition of mTOR signaling via rapamycin extends lifespan via an increase in autophagy in flies

AMPK

enzyme that plays a role in cellular energy homeostasis

Activation of AMPK signifies low energy within

the cell

When cellular energy levels are low

AMPK activated glucose and fatty acid uptake and oxidation

inhibits mTORC1 →

inhibition (-) of protein synthesis

AMPK activates

autophagy by activating ULK1

AMPK activity increases with

exercise

Selective Autophagy

removes and recycles harmful or unneeded materials from the cell

Examples of Autophagy

• protein aggregates

• damaged mitochondria

• unneeded peroxisomes

• excess ribosomes

• ER and endosomes

• lipid droplets

• intracellular pathogens

The general process of selective autophagy

• cargo recognition

• coupling of cargo to the phagosome

• degradation of cargo

Selective autophagy uses

selective autophagy receptors

Bulk Autophagy

critical for maintaining a cellular supply of lipids, amino acids, carbohydrates, and nucleotides

Bulk autophagy is largely triggered by

starvation

Mitophagy consists of the autophagic turnover of

old or dysfunctional mitochondria

Mitophagy can be induced as an

adaptive metabolic response to prevent the build-up of ROS under prolonged hypoxia

The proper balance between mitophagy and mitochondrial biogenesis is important for homeostasis. Why?

it keeps healthy mitochondria alive

Damaged mitochondria leads to a depletion of ATP and release of cytochrome c →

caspases and apoptosis

impaired and mitophagy is observed in

Parkinson’s Disease (PD) and Alzheimer’s Disease (AD)

How do cells know the difference between damaged and healthy mitochondria?

• PINK1

• PINK1 transverses from outer membrane of mitochondria to the inner membrane to monitor quality of organelle

• in damaged mitochondria, the inner membrane becomes depolarized → PINK1 cannot enter

• PINK1 recruits Parkin (an E3 ubiquitin ligase)

PINK1 (PTEN-induced kinase 1)

can detect mitochondrial quality

Loss of function in either PINK1 or Parkin in neurons can lead to

Parkinson’s Disease