Histone Deacetylase6Regulates Cellular Autophagy Via Modulating The Acetylation Of LC3

Aim:In eukaryotic cells, autophagy is the evolutionarily conserved pathway fordegradation of damaged organelles and misfolded proteins, and is induced by nutrientwithdrawal, including amino acid deficiency and serum deprivation. Althoughacetylated modification of many autophagy-related proteins has been determined toinfluence the autophagic flux, the regulation of acetylated microtubule-associatedprotein1light chain3(LC3) is not completely understood. LC3B is a key componentof the double-membrane autophagosome, and can mediate the autophagic degradation.In this study, we primarily detected the change of LC3B acetylation bypan-acetylation antibody after autophagy induction. Furthermore, we aimed to figureout the influence of LC3B acetylation on the autophagy process by analyzing theregulating mechanism of LC3B acetylation after autophagy induction.Method:1. HeLa cells (human cervical carcinoma cell line) maintained in DMEMsupplemented with10%FBS at37oC in a humidified incubator with5%CO2.2. Serum-free DMEM medium was used to induce autophagy in HeLa cells.3. Western blot analysis was used to detect the change of LC3B accumulation.Autophagosome formation was detected by immunofluorescence.4. The deacetylase activity of HDAC6was inhibited by Tubacin treatment inHeLa cells, and autophagy induction was assayed.5. LC3B in HeLa cells treated with different stimuli was purified byimmunoprecipitation, and used to detect the change of its acetylation.6. As a marker of autophagic degradation, p62/SQSTM1was used to reveal theautophagic completion in HeLa cells treated with different stimuli.7. After co-staining of LC3B/LAMP2antibodies in HeLa cells,autophagosome-lysosome fusion was detected by confocal microscopy. Result:1. Upon serum starvation, which induced autophagy in HeLa cells, theacetylation levels of phosphotidylethanolamine (PE)-conjugated LC3B (LC3B-II)were markedly decreased. Such deacetylation of LC3B-II during autophagy inductioncould not be attenuated by pretreatment with lysosomal inhibitor chloroquine.2. Under normal culture medium, we observed increased levels of acetylatedLC3B-II in cells treated with tubacin, a specific inhibitor of histone deacetylase6(HDAC6). Compared with tubacin treatment, co-treatment with tubacin andserum-free medium attenuated LC3B-II acetylation. The deacetylation of LC3B-IIwas also suppressed by HDAC6knockdown, suggesting that HDAC6is the regulatingprotein of LC3B-II deacetylation.3. In HeLa cells cultured in serum free medium, tubacin-induced increase inLC3B-II acetylation during autophagy induction was associated with p62/SQSTM1accumulation. HDAC6knockdown also restricted the autophagic degradation ofp62/SQSTM1.4. Tubacin treatment inhibited the fusion between autophagosome andlysosome.Conclusion:1. In HeLa cells maintained in normal medium, LC3B-II was acetylated. Afterautophagy induction by serum-free medium in HeLa cells, acetylation levels ofLC3B-II were decreased. Such decrease was not a result of LC3B-II degradation, andwas regulated by HDAC6.2. HDAC6is not the only deacetylase regulated the deacetylation of LC3B-IIduring serum-starvation induced autophagy.3. The inhibition of autophagosome-lysosome fusion as well as autophagicdegradation was correlated with the blockage of LC3B-II deacetylation. Acetylationgroup may function as an obstacle to the interaction between LC3B-II and otherproteins mediated the fusion between autophagosome and lysosome, finally resulting in the separation of autophagosome and lysosome.