Autophagy Biochemistry Lecture Summary PDF

Summary of the Biochemistry lecture, „Autophagy” Dr. Endre Kristóf Intracellular protein breakdown occurs in several cell compartments, but the two most important ones are the lysosomes where individual proteases degrade proteins appearing in this acid...

Summary of the Biochemistry lecture, „Autophagy” Dr. Endre Kristóf Intracellular protein breakdown occurs in several cell compartments, but the two most important ones are the lysosomes where individual proteases degrade proteins appearing in this acidified compartment, and the cytosol (or the nucleus), where the ubiquitin-dependent proteasome system degrades various types of proteins. These results in the complete degradation of proteins into amino acids. Autophagy is cell biology process conserved during evolution. Constituents of the cytoplasm are surrounded by double membranes forming vesicles which then are delivered to the lysosomes where they are degraded. Not only not properly folded proteins or proteins at the end of their life-span but also excess or damaged organelles or even microorganisms reaching inside of the cells can be eliminated by this process. Three different types of autophagy have been described in mammalian cells: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). In macroautophagy intracellular components are sequestered by a limiting membrane to form an autophagic vacuole that then fuses with lysosomes. In microautophagy, substrates are directly internalized through invaginations of the lysosomal membrane. In contrast to this “in-bulk” degradation, in CMA, selective substrate proteins are translocated into the lysosomes one by one after binding to a lysosomal receptor (LAMP-2A). Upon nutrient deprivation, autophagy catabolizes cytoplasmic components non- selectively into autophagosomes and mediates recycling and global turnover of cytoplasmic materials. In this case, it can be considered as an adaptive response to generate energy source in starvation and under stress conditions. In selective autophagy particular substrates are targeted into the autophagosome by selective autophagy receptors. The targeted cargo includes protein aggregates, damaged mitochondria or pathogens, such as bacteria. The selection of the specific substrate is based on the ubiquitination of the target and binding of autophagy receptors (such as p62, NBR1, NDP52, Optineurin) containing both ubiquitin binding domain (UBD domain) and LC3 binding domain (LIR domain), bringing targets into the autophagosome. This contributes to the quality control of intracellular homeostasis. Multiple forms of stress activate autophagy. Degradation of proteins, lipids, carbohydrates, and nucleic acids liberates amino acids, fatty acids, sugars, and nucleosides that are released into the cytoplasm for reutilization. Sugars, including glucose released from glycogen granules by glycogenolysis or autophagy, are catabolized by glycolysis and the pentose phosphate pathway (PPP) to generate ATP, and pyruvate for subsequent citric acid cycle cycle metabolism. Nucleosides are used for new nucleic acid synthesis and catabolized by the combined action of the PPP and glycolysis. Amino acids are used as building blocks for new protein synthesis, for ATP production by central carbon metabolism, and in liver as substrates for gluconeogenesis. They also can be combined to yield citrate, which drives lipid synthesis and membrane biogenesis. Catabolism of amino acids yields ammonia, an activator of autophagy. Fatty acids released from lipolysis or from autophagy of membranes or lipid droplets yield acetyl-CoA, which feeds the citric acid cycle, supporting ATP production and citrate generation. Macroautophagy is the process of engulfment of cytoplasmic material, including organelles and protein aggregates, into a double membrane vesicle, the autophagsosome. Induction of macroautophagy is initiated by activation of the Atg1 complex (Atg1/Atg13/Atg17 and other components). Autophagosome nucleation requires class-III phosphatidylinositol-3- kinase (Vps34) and Beclin-1/Atg6 to recruit proteins and lipids required for autophagosome formation. Elongation and completion are mediated by two-ubiquitin-like systems, which result in lipidated LC3 binding to the autophagosome membrane. The completed autophagosome then fuses with the lysosome, where the autophagosome contents are degraded. Macroautophagy occurs at a basal level and can be induced in response to environmental signals including nutrients and hormones, and also microbial pathogens. The best-characterized regulatory pathway includes the TOR which act to inhibit autophagy. Autophagosome formation can be initiated via TOR inhibition or AMPK activation. This results in the phosphorylation of ULK1 at sites that activate it and catalyze phosphorylation of other components of the Atg1–ULK complex (composed of ULK1, ULK2, Atg13, FIP200 and Atg101). ULK1 also phosphorylates AMBRA, a component of the class-III phosphatidylinositol-3-kinase complex I (Vps34, Vps15, Atg14, and beclin-1), enabling it to relocate from the cytoskeleton to the isolation membrane. Phosphatidylinositol 3-phosphate (PI3P), generated by Vps34 activity, specifically binds the PI3P effectors WD repeat domain phosphoinositide-interacting (WIPI) 1 and WIPI2 and catalyzes the first of two types of ubiquitination-like reactions that regulate isolation membrane elongation. In this first reaction, Atg5 and Atg12 are conjugated to each other in the presence of Atg7 and Atg10. Attachment of the fully formed complex containing Atg5, Atg12 and Atg16L on the isolation membrane induces the second complex to covalently conjugate phosphatidylethanolamine to LC3, which facilitates closure of the isolation membrane. Atg9 (the Atg9–Atg2–Atg18 complex), another factor essential for this event, cycles between endosomes, the Golgi and the phagophore, possibly carrying lipid components for membrane expansion. Atg4 removes LC3- II from the outer surface of newly formed autophagosomes, and LC3 on the inner surface is eventually degraded when the autophagosome fuses with lysosomes. With regard to medical practice, autophagy has important roles in pathologic conditions (infections, cancer, neurodegeneration, ageing etc.). For example, proteins associated with Parkinson's disease pathology are the kinase PINK1 and the ubiquitin ligase Parkin. PINK1 activates Parkin, and Parkin subsequently ubiquitinates VDAC1 and other mitochondrial proteins, leading to the selective removal of mitochondria via autophagy (mitophagy) and preserving cells from the damage cause by reactive oxygen species generated by nonfunctional mitochondria. Because of the mitochondrial content is regulated by a balance between mitochondrial biogenesis and degradation, the absence of functional PINK1 or Parkin disrupts mitochondrial dynamics and contributes to the development of Parkinson's disease.