![]() The autophagic system also harnesses proteins known as autophagy receptors, which increase the selectivity of the autophagic process by facilitating the engulfment of certain cargoes by the growing autophagosomes 4. The lipidated LC3, known as LC3-II, has a faster mobility than LC3-I on SDS PAGE and is relatively specifically associated with autophagosomes and autolysosomes (in the absence of conditions stimulating LC3-associated phagocytosis). ATG7, the E1-like enzyme, ATG3, an E2-like enzyme, and the ATG5-12-16L1 complex as the E3-like enzyme then conjugate LC3 family members to phosphatidylethanolamine (PE) on the surface of nascent autophagosomes 1. ![]() LC3-I is generated by proteolytic cleavage of pro-LC3 by ATG4, which exposes a C-terminal glycine that is amenable to conjugation. The second set of reactions involves the ubiquitin-like LC3 protein family. This conjugate then forms a complex with ATG16L1 3. The first reaction employs the E1-like ATG7 and the E2-like ATG10 enzymes, which conjugate the ubiquitin-like ATG12 to ATG5. The double-membraned autophagosomes form from cup-shaped structures known as phagophores and are responsible for the engulfment of cargoes that are subsequently degraded after fusion with lysosomes 1.Īutophagosome formation involves two successive ubiquitin-like reactions. This process is conserved across eukaryotes and involves the formation of double-membraned structures known as autophagosomes. It targets substrates like long-lived proteins, aggregate-prone proteins, and damaged organelles for lysosomal degradation to maintain cellular homeostasis 1, 2. Similar content being viewed by othersĪutophagy is a process that has been widely implicated in the pathogenesis of various conditions, such as neurodegenerative diseases, cancer, and inflammation. This observation questions the reliability of LC3-immunofluorescence assays in cells with compromised autophagy. This phenomenon is due to LC3-I sequestration to p62 aggregates, which accumulate when autophagy is impaired. This occurs even with transient and incomplete inhibition of autophagosome biogenesis. Here we find that the endogenous LC3-positive puncta become larger in cells where autophagosome formation is abrogated, and are prominent even when LC3-II is not formed. As LC3-II is relatively specifically associated with autophagosomes and autolysosomes (in the absence of conditions stimulating LC3-associated phagocytosis), quantification of LC3-positive puncta is considered as a gold-standard assay for assessing the numbers of autophagosomes in cells. LC3-II, a standard marker for autophagosomes, is generated by the conjugation of cytosolic LC3-I to phosphatidylethanolamine (PE) on the surface of nascent autophagosomes. ![]() The process involves the formation of double-membraned autophagosomes that engulf the cargoes destined for degradation, sometimes with the help of autophagy receptors like p62, which are themselves autophagy substrates. Autophagy is an evolutionarily conserved process across eukaryotes that degrades cargoes like aggregate-prone proteins, pathogens, damaged organelles and macromolecules via delivery to lysosomes. ![]()
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