Lecture 13
Introduction to Proteostasis and Autophagy
Proteostasis refers to the dynamic regulation of the protein complement within the cell. It involves a delicate balance between biosynthetic pathways and degradative pathways.
Biosynthetic pathways include protein expression, folding, and trafficking.
Autophagy acts as the counterpoint to biosynthesis, serving as a major degradative pathway alongside the proteasomal degradation system (often taught in earlier years).
While proteasomal degradation typically handles individual proteins, autophagy is often a bulk degradation process, though it can also be highly specific.
Triggers for autophagic pathways include:
ER stress (Endoplasmic Reticulum stress).
Starvation and nutrient availability within the cell.
Cell maintenance and organelle turnover.
Immune responses (clearing bacteria or viruses).
Cell differentiation and developmental remodeling (e.g., the digestion of mitochondria in developing erythrocytes to form mature red blood cells).
The word "autophagy" translates literally to "self-eating."
The process is highly conserved among eukaryotes.
The Central Role of the Lysosome
The lysosome is the central organelle for all types of autophagic digestion. It sits at the junction of the endocytic and secretory pathways.
Lysosomal enzymes (hydrolases) are produced in the ER and processed in the Golgi apparatus. They are tagged for delivery to the late endosome via clathrin-coated vesicles.
Enzyme Types: The lysosome contains various hydrolytic enzymes, including:
Nucleases.
Glycosidases.
Lipases and Phospholipases.
Phosphatases.
Proteases.
Acidification and Activation:
Enzymes are transported in an inactive state and only become active in the low pH environment of the lysosome.
The acidic environment is generated by a -type ATPase, which uses ATP to pump protons () into the lysosomal lumen.
To maintain the voltage gradient across the membrane during proton pumping, the cell utilizes the efflux of calcium () or the influx of chlorine () ions.
As the pH drops, enzymes undergo autocatalytic activation.
Chaperone-Mediated Autophagy (CMA)
CMA involves the direct targeting of soluble proteins to the lysosome for translocation across the membrane and subsequent digestion.
The KFERQ Motif:
Proteins targeted for CMA contain a specific pentapeptide motif known as the KFERQ motif.
This motif is broadly defined and consists of:
Two positively charged residues (Lysine/K, Arginine/R).
Two hydrophobic residues (Isoleucine/I, Phenylala nine/F, Valine/V, Leucine/L).
One negatively charged residue (Aspartic Acid/D, Glutamic Acid/E).
A single Glutamine (Q) residue at either the C- or N-terminus of the motif.
These residues can appear in any order.
Approximately of mammalian proteins contain this motif.
Post-translational modifications, such as the acetylation of Lysine to mimic Glutamine, can expand the range of proteins targeted by CMA.
The CMA Mechanism:
Targeting: The chaperone HSC70 identifies and binds the KFERQ motif on the substrate protein in the cytosol.
Binding: HSC70 delivers the protein to the C-terminus of LAMP2A (Lysosome-Associated Membrane Protein 2A) on the lysosomal surface.
Multimerization: With the assistance of lysosomal HSP90 (on the luminal side), LAMP2A forms a trimer, creating a translocation channel.
Translocation: Lysosomal HSC70 (within the lumen) acts as a molecular motor to pull the substrate protein through the LAMP2A channel.
Degradation: The protein is digested by lysosomal enzymes.
Recycling: The LAMP2A trimer dissociates for reuse. Eventually, LAMP2A is degraded by metalloproteinases in lipid-rich regions of the lysosomal membrane to regulate its levels.
Regulation of Chaperone-Mediated Autophagy
The CMA process is regulated by nutrient sensors, specifically mTORC2 (Mammalian Target of Rapamycin Complex 2).
High Nutrient Conditions:
mTORC2 is active and activates the kinase AKT1.
AKT1 phosphorylates GFAP (Glial Fibrillary Acidic Protein).
Phosphorylated GFAP blocks the activity of LAMP2A, inhibiting CMA.
Low Nutrient Conditions (Starvation):
mTORC2 is inactive; AKT1 is inactive.
GFAP remains unphosphorylated.
Non-phosphorylated GFAP binds to and stabilizes the LAMP2A trimer, promoting CMA.
Clinical Relevance:
Alzheimer’s Disease: Failure in CMA is linked to the accumulation of Tau protein tangles.
Parkinson’s Disease: Dysfunctional degradation of Alpha-synuclein is associated with CMA failure.
Cancer: Some cancers upregulate CMA to degrade pro-apoptotic signals, allowing tumor cells to persist.
Microautophagy
Microautophagy is less understood than other forms of autophagy.
It involves the direct invagination or protrusion of the lysosomal membrane to capture cargoes.
There is overlap with CMA: many microautophagy substrates also contain KFERQ motifs and rely on HSC70 for targeting to the lysosomal surface.
Instead of forming a channel (like LAMP2A in CMA), the binding triggers membrane remodeling to engulf the cargo.
Macroautophagy: The Canonical Pathway
Macroautophagy involves the formation of a unique double-membrane structure called the autophagosome.
Key Vocabulary:
Isolation Membrane / Phagophore: The initial small, double-membrane structure that grows to encapsulate cargo.
Autophagosome: The completed, closed double-membrane vesicle containing the cargo.
Amphisome: The structure formed when an autophagosome fuses with a late endosome.
Autolysosome: The final structure formed by the fusion of the autophagosome (or amphisome) with a lysosome.
Non-specific vs. Selective Macroautophagy:
Canonical/Bulk Autophagy: Non-specific capture of cytoplasmic contents during starvation.
Selective Autophagy: Specific targeting of substrates, often named based on the substrate:
Xenophagy: Digestion of bacteria/pathogens.
Mitophagy: Digestion of mitochondria.
Reticulophagy: Digestion of the Endoplasmic Reticulum.
Molecular Mechanism of Macroautophagy
1. Initiation:
Controlled by mTORC1 and the ULK1 complex (containing ULK1 and ATG13).
In high nutrient conditions, mTORC1 phosphorylates the ULK1 complex to inhibit it.
During starvation, the inhibition is lifted. The ULK1 complex becomes active and recruits ATG9 vesicles.
ATG9 vesicles are sourced primarily from the endosomal system (cycling between the plasma membrane and the Trans-Golgi Network).
2. Nucleation:
The ULK1 complex associates with the ER at a site called the Omegasome (so named because it looks like the Greek letter in 2D cross-sections).
ULK1 activates Beclin-1, which in turn activates Phosphatidylinositol 3-kinase (PI3K).
PI3K produces Phosphatidylinositol-3-phosphate (PI3P) on the membrane of the forming phagophore.
3. Elongation:
WIPI proteins bind to PI3P.
WIPI recruits the ATG16 complex, which facilitates the recruitment of LC3.
LC3 (Light Chain 3): Exists as LC3-I in the cytosol. When recruited to the membrane, it becomes LC3-II. The ratio of LC3-I to LC3-II is a standard laboratory measure of autophagic flux.
ATG2 forms a physical bridge/channel between the ER (Omegasome) and the phagophore. It acts as an escalator to shuttle lipids from the ER to the phagophore for rapid membrane expansion.
ATG9 acts as a flippase, redistributing lipids across the double membrane to allow even growth.
4. Closure:
Once the target is encapsulated, the membrane ends are joined.
This is facilitated by ESCRT proteins (Endosomal Sorting Complexes Required for Transport):
ESCRT-I acts as a bridge between the two membrane edges.
ESCRT-II acts as a "plug" to seal the membrane.
The recruitment of ESCRT proteins is facilitated by the ULK1 complex.
Cargo Targeting in Selective Autophagy
Specificity is achieved via receptor proteins that bridge the cargo and the LC3 proteins bound to the inner membrane of the phagophore.
P62: Contains a ubiquitin-binding domain and an LIR (LC3-Interacting Region). It targets ubiquitinated protein aggregates for degradation.
Optineurin: Recognizes bacterial pathogens on one side and binds LC3 on the other to promote xenophagy.
Failure of these selective processes results in chronic infection, accumulation of damaged organelles, and can ultimately trigger apoptosis (programmed cell death).