Lysosomal Degradation: Autophagy

Question 1

Cellular and foreign cargo

reach the lysosome (for degradation) via:

Answer 1

• Endocytosis/phagocytosis (Heterophagy–foreign items)

* Autophagy (from within cell)

Question 2

Subtypes of Autophagy

Answer 2

• chaperone mediated autophagy (CMA)
• microautophagy
• macroautophagy

Question 3

______ are the major degradative

compartments of eukaryotic cells

Answer 3

Lysosomes

Question 4

Lysosomes are important as seen through

Answer 4

• The lysosomal pathway is evolutionarily conserved and strictly regulated
• The lysosomal pathway operates at low levels under normal conditions (always working at some level)

Question 5

The lysosomal pathway mediates the degradation of:

Answer 5

• cytoplasmic components /expired intracellular organelles (ER, mitochondria)
• protein aggregates
• intracellular pathogens

Question 6

Autophagy is Crucial for ______

Answer 6

• Quality Control of Proteins and Organelles

- Constitutive, basal autophagy maintains proteostasis together with UPS

Question 7

How proteins are damaged

Answer 7

Proteins altered by mutations (such as polyglutamine expansion tracts), PTMs, or
stress (oxidative stress, UV irradiation, toxins) undergo a conformational change

Question 8

What happens to damaged proteins

Answer 8

Molecular chaperones recognize damaged
proteins and:
• refold and repair them or
• deliver them to protein degradation systems (usually UPS or CMA)

IF degradation systems are impaired, autophagy becomes the removal route

Question 9

Impaired autophagy leads to

Answer 9

Impaired autophagy is associated with the

formation of protein aggregates and increased neurodegeneration

Question 10

Autophagy occurs when…

Answer 10

• Oligomeric complexes that cannot be recognized by the UPS or CMA are removed mainly by autophagy
• If degradation systems (UPS, CMA) are impaired, autophagy becomes the removal route

Question 11

Canonical Autophagy

Answer 11

• Involves formation of a phagophore formed at the PAS (phagophore assembly site)
• Requires 15 autophagy-related (ATG) proteins acting hierarchically to construct the autophagosome

Question 12

AUTOPHAGY INITIATION

Answer 12

1) ULK1 complex is recruited to phagophore
2) ULK1 complex is negatively and positively regulated by upstream kinases that sense cellular nutrient and energy status
3) ULK1 complex activates the Class III PI3K complex
4) Class III PI3K complex generates PI3P
at the phagophore
5) PI3P recruits PI3P-binding proteins including proteins involved in the ‘‘elongation reaction’’

Question 13

PHAGOPHORE ELONGATION

Answer 13

1) PI3P recruits two Ub-like conjugation systems responsible for phagophore elongation
2) light chain 3 (LC3), is conjugated to phosphatidylethanolamine (PE)
3) PE-conjugated LC3 (LC3II) is found on autophagosomal membranes

Question 14

AUTOPHAGOSOME-LYSOSOME FUSION

Answer 14

1) Autophagosomes move in direction of lysosomes and fuse with them
2) Contents sequestered inside the autolysosome are degraded and released into the cytoplasm for recycling
- -> releases aa’s that can be used for protein synth etc.

Question 15

Canonical autophagy process

Answer 15

1–AUTOPHAGY INITIATION
2–PHAGOPHORE ELONGATION
3–AUTOPHAGOSOME-LYSOSOME FUSION

Question 16

We can detect autophagy through

Answer 16

• western blot

- fluorescence

Question 17

Use of fluorescence to detect autophagy

Answer 17

Use 2 fluorophores one is red and the other is green

• red and green expressed in autophagy initiation = yellow colour
• only red expressed during fusion = red
• no fluorescence once autolysosome is formed

Question 18

Cargo Sequestration: Bulk Autophagy

Answer 18

• Catabolizes cytoplasmic components non-selectively
  • Occurs constitutively at low levels under nutrient-rich conditions –> Mediates global turnover of cytoplasmic materials
  • Activated by nutrient deprivation

Question 19

Defective bulk autophagy leads to:

Answer 19

– amino acid insufficiency (as autophagy releases AAs to build more proteins)
– LEADS to impaired protein synthesis for adaptation to starvation and energy production for cell survival

Question 20

Selective Autophagy

Answer 20

• Sequestration of autophagic cargo is selective
• Selectivity is provided by specialized factors: the autophagy receptors
• Autophagy receptors bind to the phagophore & cargo on either sides of the receptor

Question 21

Selective Autophagy can be ___ or ____

Answer 21

Ub-dependent or UB-independent

Question 22

Ub-dependent: autophagy receptors contain:

Answer 22

– a Ub-binding domain(UBD) that recognize
Ub in cargo
– a LC3 interaction region (LIR)–which brings cargo to phagophore

Question 23

How can we have selectivity without Ub (in UB-independent)

Answer 23

autophagy receptors recognize specific types of protein-, lipid-, or sugar-based signals

Question 24

Examples of Selective Autophagy

Answer 24

• Mitophagy
• Aggrephagy
• Pexophagy
• Xenophagy

Question 25

Mitophagy

Answer 25

type of selective autophagy | elimination of old or damaged mitochondria

Question 26

Aggrephagy

Answer 26

type of selective autophagy | elimination of certain protein aggregates

Question 27

Pexophagy

Answer 27

type of selective autophagy | removal of peroxisomes

Question 28

Xenophagy

Answer 28

type of selective autophagy | Removal of intracellular pathogens

Question 29

mTOR role in autophagy

Answer 29

- acts as the Gatekeeper of Autophagy Signaling - mTOR is a highly conserved serine/threonine protein kinase - mTOR serves as a master regulator of autophagy

Question 30

How does mTOR regulate autophagy

Answer 30

* It acts as a central sensor | * senses and integrates signals from numerous sources (growth factors, amino acids, hypoxia, and energy levels)

Question 31

mTOR is activated by

Answer 31

Amino acids growth factors glucose

Question 32

Amino acids activate mTOR through

Answer 32

lysosomal mechanisms

Question 33

Growth factors activate mTOR through

Answer 33

PI3K class I-Akt signaling

Question 34

Glucose activates mTOR by

Answer 34

inhibiting the kinase AMPK | - note: AMPK is activated by a decrease in ATP/AMP ratio

Question 35

AMPK and mTOR

Answer 35

AMPK represses mTOR by: • Direct phosphorylation of mTORC1 • Inhibition of PI3K/AKT pathway •AMPK activates ULK complex

Question 36

Active mTORC1 suppresses autophagy by:

Answer 36

• Phosphorylation and inhibition of the ULK1 and VPS34 complexes • Inhibition of TFEB nuclear translocation (by phosphorylation TFEB --> inhibits actions by preventing its entry into the nucleus)

Question 37

When nutrients are abundant mTORC1 is

Answer 37

ACTIVATED | autophagy inhibited b/c don't need to make nutrients

Question 38

mTOR is a ___ modulator of autophagy

Answer 38

NEGATIVE

Question 39

The inositol signaling pathway _______ regulates autophagy

Answer 39

negatively (i.e. inositol and IP3 inhibits autophagy)

Question 40

IP3 stimulation

Answer 40

1) Stimulation of G-protein-coupled receptor causes elevation of cAMP and activation of PLC 2) PLC hydrolyses PIP2 to form IP3 and DAG 3) IP3 inhibits autophagy (through intracellular calcium release)

Question 41

IP3 and calcium interaction

Answer 41

IP3 binds to IP3Rs on the ER to release stored Ca2+ – Ca2+ impairs autophagosome maturation by blocking autophagosome-lysosome fusion – Ca2+ activates calpains (enzymes that decrease autophagy)

Question 42

IP3 degradation

Answer 42

``` IP3 is degraded to IP2 and IP which is further hydrolyzed by IMPase into free inositol Inositol inhibits autophagosome assembly ```

Question 43

Calcium regulation of autophagy

Answer 43

High levels of cytosolic Ca2+ activate calpains which block autophagy • Calpain activates Gsa, increasing AC activity that generates cAMP to suppress autophagy • Increase of Ca2+ cytosolic levels by influx through L-type Ca2+ channels also inhibits autophagy

Question 44

Only ____ proteins can be degraded by UPS

Answer 44

Only soluble proteins can be degraded by UPS • Natively soluble proteins are usually tagged by Lys48 and Lys11 chains • Soluble misfolded proteins are tagged with mainly Lys48-linked polyub

Question 45

Aggresomes

Answer 45

Inclusion bodies of aggregated proteins | associated with HDAC6 and the MTOC located at the centriole

Question 46

Clearance of toxic proteins HOW

Answer 46

Clearance of toxic protein aggregates is achieved through aggresomes and selective autophagy

Question 47

K63 polyubiquitination

Answer 47

K63 polyubiquitination may promote binding to p62 and facilitate the recruitment of autophagic membrane to the aggresome for the formation of an autophagosome

Question 48

Crosstalk between UPS and | Autophagy occurs through

Answer 48

the aggresome Ubiquitination and sequestration of protein in aggresomes and cytoplasmic inclusions may represent a mechanism of cell protection