Lecture 13

• Autophagy


  Cellular process where cells degrade and recycle their own components
• Importance of autophagy

  • Removes protein aggregates, old and damaged organelles, and invading microbes
  • Involved in developmental remodeling and providing nutrients under low resource conditions
• Yoshinori Ohsumi awarded Nobel Prize for discoveries in mechanisms underlying autophagy in yeast


  2016
• Forms of autophagy

  • Macroautophagy
  • Microautophagy
  • Chaperone-Mediated Autophagy (CMA)
• Macroautophagy

  1. Formation of phagophore that engulfs cargo

  2. Phagophore forms double-membraned autophagosome

  3. Autophagosome fuses with lysosome to form autolysosome where contents are degraded and recycled
• Regulation of macroautophagy

  Involves complex regulatory mechanisms to ensure cells maintain homeostasis
• Microautophagy

  Direct engulfment of cargo by the lysosome itself, without the formation of an autophagosome
• Chaperone-Mediated Autophagy (CMA)

  1. Targets specific proteins for lysosomal degradation through a receptor-mediated mechanism

  2. Proteins with a recognized pentapeptide motif are translocated directly across the lysosomal membrane in a complex with chaperone proteins like Hsc70
• Lysosome

  • Acidic environment maintained at pH 4.5-5.0
  • Enzymes like cathepsins are activated in the acidic environment
• Lysosome formation and trafficking

  Lysosomal enzymes synthesized in ER as preproenzymes, processed in Golgi, and then directed to lysosomes
• Lysosomal pH maintenance

  • Acidic pH maintained by V-type ATPases that pump protons into the lumen
  • Transmembrane voltage generated is balanced by movement of other ions like Cl- and Ca2+
• Physiological roles of autophagy

  • Response to starvation
  • Quality control by removing old and malfunctioning organelles
• Autophagy plays a role in development by remodeling cellular structures and has implications in various diseases, particularly those related to protein aggregation like neurodegenerative disorders
• Regulation of autophagy

  Tightly controlled by signaling pathways sensitive to cellular energy levels, nutritional status, and stress signals
• KFERQ motif


  Motif recognized by chaperone protein Hsc70 in Chaperone-Mediated Autophagy (CMA)
• LAMP-2A


  Forms a channel through which proteins with the KFERQ motif are translocated into the lysosome
• Chaperone-Mediated Autophagy (CMA)

  1. Recognition of KFERQ motif by Hsc70 triggers delivery of substrate protein to LAMP-2A

  2. LAMP-2A facilitates translocation of substrate into lysosome where it is degraded
• Regulation of CMA

  • Activity and levels of LAMP-2A regulated by proteolysis
  • Nutrient-rich/high-fat diets and aging can inhibit CMA through stimulation of mTORC2 and Akt1
• Microautophagy

  Direct uptake of autophagic cargoes by late endosomes and lysosomes, sometimes involving substrates with KFERQ motifs
• Role of Hsc70 in microautophagy

  Binds directly to phosphatidylserine to deliver substrates to late endosome membrane for degradation
• Dysfunctional CMA


  Can lead to accumulation of neurodegenerative disease-related proteins
• CMA in cancer cells

  Can contribute to tumor growth and metastasis by degrading tumor suppressors and pro-apoptotic proteins
• Macroautophagy

  1. Initiation: Inhibition of mTORC activates ULK1 complexes

  2. Nucleation: PI3K complex activity leads to formation of omegasome

  3. Expansion: Phagophore expands through addition of membrane lipids

  4. Closure: Phagophore closes to form complete autophagosome

  5. Fusion with lysosome: Autophagosome fuses with lysosome to form autolysosome where contents are degraded
• Regulation of macroautophagy

  • mTORC inhibition triggers autophagy
  • ULK1 complex integrates signals from mTORC and AMPK pathways
• Biological significance of macroautophagy

  • Response to starvation
  • Quality control by removing damaged organelles and protein aggregates
  • Regulation of cellular processes including development, differentiation, and disease pathology
• LC3 proteins

  LC3A, LC3B, and LC3C isoforms play distinct roles in autophagy, associating with different cellular membranes
• LC3 processing

  1. LC3-I produced by proteolytic cleavage

  2. LC3-I conjugated to phosphatidylethanolamine to form LC3-II which associates with autophagosomal membranes
• Role of LC3-II in selective autophagy

  • Facilitates enclosure of autophagic cargo
  • Interacts with cargo receptors like p62/SQSTM1 to select cargo for degradation
• LC3-interacting region (LIR)

  Critical for interaction between cargo receptors and LC3-II, ensuring cargo is incorporated into autophagosomes
• Dysregulation of LC3 processing or cargo receptor function is linked to various diseases including neurodegenerative disorders and cancer