The The Man Lc3b-beta And Identification Of Negative Feedback Regulation Mechanism Of Lc3b-alpha

Rat microtubule-associate proteins 1 light chain 3(LC3) undergoes a series of post-translational modifications, and finally localizes to autophagic membranes. A lipidated form of LC3, LC3-II, has been shown to be an autophagosomal marker in mammals, and has been used to study autophagy in physiological and pathologic processes. We have reported three human members of LC3 family, named LC3A, LC3B, and LC3C. LC3A and LC3C undergo the same post-translational modification as yeast Atg8 and rat LC3, which entails the characteristic C-terminal cleavage after a conserved glycine residue, but this C-terminal cleavage was not observed in LC3B. We detected a novel type of modification for LC3B, and the site of post-translational modification for LC3B is Lysl22 rather than the conserved Glyl20 in the other two proteins. However, a recent study reported that the C-terminus of LC3B could be cleaved to expose the conserved Glyl20 for further ubiquitylation-like reactions.To clarify the two inconsistent observations, we proved that alternative splicing leads to two human LC3B isoforms with and without Arg70 (designated as LC3B-α and LC3B-β, respectively). It was further demonstrated that LC3B-α undergoes the characteristic modifications triggered by C-terminal cleavage after Glyl20 and localizes to autophagosomal membranes like rat LC3, whereas LC3B-β has not, suggesting that the Arg70 controls the C-terminal cleavage and cellular localization of human LC3. Through mass spectrum analysis, we found although the Lysl21 mutation of LC3B-p can affect the electrophoresis on SDS-PAGE, the molecular weights of LC3B-β and LC3B-β-K121A are almost identical, suggesting that there is no modification on LC3B-β. To gain the mechanistic insight into the distinct C-terminus cleavage of LC3B-α andLC3B-β, we performed molecular dynamics simulations to investigate the effects of Arg70 on the conformation of the C-termini. It was revealed that the C-terminus of LC3B-α is flexible and disordered, whereas that of LC3B-β is tightly bound to the main body of the protein, explaining the inability of LC3B-β to undergo C-terminal cleavage. Both LC3B-α and LC3B-β can interact with ATG4B, the cysteine proteases that cleave the C-terminal of LC3. Moreover, LC3B-β can inhibit the post-translational modification of LC3B-α, suggesting that LC3B-β can negative regulate autophagy.Additionally, we found rat LC3A and LC3B, two novel isoforms of rat LC3, undergo the same post-translational modifications and localize to autophagic membranes as rat LC3. Moreover the conserved Gly120 residues of rat LC3A and LC3B are also essential for their characteristic C-terminal cleavage and localization to autophagic membranes. Present data suggested that rat LC3A and LC3B could also be used as two novel autophagosomal markers.