The Biological Role Of Nuclear Beclin1

Objective:To explore new function of nuclear Beclinl.Methods:(1) The liver of C57BL/6J mice was surgically excised and cryostat section were cut to observe the subcellular localization of Beclin1in development by confocal microscopy;(2) Under basal and starvation induced autophagy level, we used immunoprecipitation to analyze the interaction of Beclin1with PI3KC3and Bcl-2in NIH3T3cell line;(3) Talen strategy was used to construct beclin1knockout cell line, and we used PI staining, cell counting, and Western blotting to analyze the consequences of Beclin1knockdown on cell cycle;(4) NIH3T3cells were irradiated with y ray, and the cytoplasmic and nucleic proteins from the cells were then separated to examine DNA damage response to radiation exposure;(5) To explore the non-classic nuclear localization sequence of Beclin1, we deleted each major domain of Beclin1by means of Overlap PCR, and then transfected the beclin1mutants into NIH3T3cells. After obtaining the stable cell lines with G418selection, we separated the cytoplasmic and nucleic proteins from cells to check which domain is responsible for Beclin1nuclear localization;(6) We use Pull Down assay and LC-MS/MS mass spectrum to explore the candidate proteins interact with Beclin1in nuclear.Results:(1) Beclin1is relocalized to the nucleus during mouse developmental progression;(2) Nuclear Beclin1may play a role different from its cytoplasmic counterpart in autophagy cascade;(3) Beclin1may play a role in cell cycle regulation, and knockdown Beclin1will induce cell cycle arrest, increase the percentage of cells in G2/M phase;(4) Beclin1may play a role in DNA damage repair, and y ray will upregulate Beclin1level in nuclear, but has no influence on cytoplasma and total Beclin1level;(5) Amino acid from188-241and267-325of Beclin1may be responsible for its nuclear localization; (6) Mass spectrum result shows that DNA topoisomerase Ⅱ-beta may interact with Beclin1in nuclear.Conclusion:The present study reveals that Beclin1contains a non-classic nuclear localization sequence, and its role in nuclear may correlate to cell cycle regulation, y ray induced DNA damage repair.