Lysosomal Degradation: Autophagy Flashcards
Cellular and foreign cargo
reach the lysosome (for degradation) via:
- Endocytosis/phagocytosis (Heterophagy–foreign items)
* Autophagy (from within cell)
Subtypes of Autophagy
- chaperone mediated autophagy (CMA)
- microautophagy
- macroautophagy
______ are the major degradative
compartments of eukaryotic cells
Lysosomes
Lysosomes are important as seen through
- The lysosomal pathway is evolutionarily conserved and strictly regulated
- The lysosomal pathway operates at low levels under normal conditions (always working at some level)
The lysosomal pathway mediates the degradation of:
- cytoplasmic components /expired intracellular organelles (ER, mitochondria)
- protein aggregates
- intracellular pathogens
Autophagy is Crucial for ______
- Quality Control of Proteins and Organelles
- Constitutive, basal autophagy maintains proteostasis together with UPS
How proteins are damaged
Proteins altered by mutations (such as polyglutamine expansion tracts), PTMs, or
stress (oxidative stress, UV irradiation, toxins) undergo a conformational change
What happens to damaged proteins
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
Impaired autophagy leads to
Impaired autophagy is associated with the
formation of protein aggregates and increased neurodegeneration
Autophagy occurs when…
- 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
Canonical Autophagy
- Involves formation of a phagophore formed at the PAS (phagophore assembly site)
- Requires 15 autophagy-related (ATG) proteins acting hierarchically to construct the autophagosome
AUTOPHAGY INITIATION
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’’
PHAGOPHORE ELONGATION
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
AUTOPHAGOSOME-LYSOSOME FUSION
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.
Canonical autophagy process
1–AUTOPHAGY INITIATION
2–PHAGOPHORE ELONGATION
3–AUTOPHAGOSOME-LYSOSOME FUSION
We can detect autophagy through
- western blot
- fluorescence
Use of fluorescence to detect autophagy
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
Cargo Sequestration: Bulk Autophagy
- Catabolizes cytoplasmic components non-selectively
• Occurs constitutively at low levels under nutrient-rich conditions –> Mediates global turnover of cytoplasmic materials
• Activated by nutrient deprivation
Defective bulk autophagy leads to:
– 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
Selective Autophagy
- 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
Selective Autophagy can be ___ or ____
Ub-dependent or UB-independent
Ub-dependent: autophagy receptors contain:
– a Ub-binding domain(UBD) that recognize
Ub in cargo
– a LC3 interaction region (LIR)–which brings cargo to phagophore
How can we have selectivity without Ub (in UB-independent)
autophagy receptors recognize specific types of protein-, lipid-, or sugar-based signals
Examples of Selective Autophagy
- Mitophagy
- Aggrephagy
- Pexophagy
- Xenophagy
Mitophagy
type of selective autophagy
elimination of old or damaged mitochondria
Aggrephagy
type of selective autophagy
elimination of certain protein aggregates
Pexophagy
type of selective autophagy
removal of peroxisomes
Xenophagy
type of selective autophagy
Removal of intracellular pathogens
mTOR role in autophagy
- acts as the Gatekeeper of Autophagy Signaling
- mTOR is a highly conserved serine/threonine protein kinase
- mTOR serves as a master regulator of autophagy
How does mTOR regulate autophagy
- It acts as a central sensor
* senses and integrates signals from numerous sources (growth factors, amino acids, hypoxia, and energy levels)
mTOR is activated by
Amino acids
growth factors
glucose
Amino acids activate mTOR through
lysosomal mechanisms
Growth factors activate mTOR through
PI3K class I-Akt signaling
Glucose activates mTOR by
inhibiting the kinase AMPK
- note: AMPK is activated by a decrease in ATP/AMP ratio
AMPK and mTOR
AMPK represses mTOR by:
• Direct phosphorylation of mTORC1
• Inhibition of PI3K/AKT pathway
•AMPK activates ULK complex
Active mTORC1 suppresses autophagy by:
• 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)
When nutrients are abundant mTORC1 is
ACTIVATED
autophagy inhibited b/c don’t need to make nutrients
mTOR is a ___ modulator of autophagy
NEGATIVE
The inositol signaling pathway _______ regulates autophagy
negatively (i.e. inositol and IP3 inhibits autophagy)
IP3 stimulation
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)
IP3 and calcium interaction
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)
IP3 degradation
IP3 is degraded to IP2 and IP which is further hydrolyzed by IMPase into free inositol Inositol inhibits autophagosome assembly
Calcium regulation of autophagy
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
Only ____ proteins can be degraded by UPS
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
Aggresomes
Inclusion bodies of aggregated proteins
associated with HDAC6 and the MTOC located at the centriole
Clearance of toxic proteins HOW
Clearance of toxic protein aggregates is achieved through aggresomes and selective autophagy
K63 polyubiquitination
K63 polyubiquitination may promote binding to p62 and facilitate the recruitment of autophagic membrane to the aggresome for the formation of an autophagosome
Crosstalk between UPS and
Autophagy occurs through
the aggresome
Ubiquitination and sequestration of protein in aggresomes and cytoplasmic inclusions may represent a mechanism of cell protection
