Mechanism Of Action Of VSV G Transmembrane Region During Fusion And Sodium Selenite Induced Up-regulation Of ROS By P70S6K/p53/ULK1 Pathway To Inhibit Protective Autophagy

Part oneMembrane fusion is an essential physiological process that involved in multiple cellular processes including cell fusion,vesicle trafficking,organelle dynamics and virus infection.Almost all fusion events mentioned above are driven by a special kind of membrane proteins,membrane fusion proteins,also known as fusogens.The well known fusion proteins,SNARE complex,which mediate vesicle fusion events,have been well elucidated in the past two decades.Another crutial fusion process is enveloped viral fusion which is mediated by different kinds of viral fusion proteins.Viral fusion proteins localize on viral envelope and dominate a crucial step for virus infection,the uncoating step,that is,the fusion of the envelope with host cellular membrane.Different kinds of fusion proteins induced similar membrane fusion process by distinct mechanisms.Unlike SNARE complex,viral fusion proteins have more complex conformations resulting in more complex fusion mechanisms.In this study,we focus on the role of transmembrane(TM)domain in VSV G(Vesicular Stomatitis Virus G protein)induced fusion process.We set up a novel cell-cell fusion assay to monitor VSV G induced fusion events in real time.This assay also facilitates the quantitative analysis of VSV G fusion activity.Based on this assay,we found that VSV G induced cell-cell fusion quickly,within 1-2min.To investigate the role of TM domain,we replaced TM of VSV G with those from other fusion proteins.Our results indicated that TM replacement significantly reduced fusion capacity of VSV G and VSV G induced lentivirus infection as well.Considering the intact extracellular domain of VSV G,we supposed that it could innitiated fusion process as normal but block the transition of hemifusion to full tusion.We set up another cell-cell fusion assay to test this hypothesis and found that TM replacement led to hemifusion blockage.Finally,we screened the key residues in TM domain of VSV G for fusion activity and found that the role of TM domain in VSV G infuced fusion event is sequence dependent.In this sutdy,we found the TM of VSV G protein is essential to its fusion capacity.Replacement of VSV G TM reduce fusion capacity by block fusion process at hemifusion stage.Our study provided some new clues for the mechanism of VSV G mediated membrane fusion and find a potential target for anti-viral prevention and treatment.Part twoSelenium,an essential trace element,is of fundamental importantce to human health and the prevention of tumors.Large amount of evidence indicates that supra-nutritional selenium is able to induce apoptosis in diverse tumor cell lines,such as prostate cancer,bladder cancer,thyroid cancer,esophageal cancer,liver cancer,colon cancer,lung cancer and leukemia?As a result,making a thorough inquiry into the molecular mechanisms of selenium induced leukemia cell apoptosis would be of great significance for the development of drugs containing selenium and the treatment of leukemia with new ideas and theoretical basis.In this study,we investigated the mechanism of selenite induced inhibition of protective autophagy and apoptosis in NB4 cells.At first,we confirmed that the decrease of autophagy and the induction of apoptosis after selenite treatment by immunofluorescence,electronic microscopy and flow cytometry.Furthermore,we found that reactive oxygen species(ROS)increased significantly after treated with selenite,and the inhibition of autophagy was reversed after treated with ROS scavenger.So we purposed that the inhibition of autophagy induced by selenite was mediated by ROS.Furthermore,we found that ROS induced inhibition of autophagy was related to ULK1,which was transcriptional regulated by p-p53(Ser392),confirmed by ChIP.Screening the kinases,we established that p70S6K was the kinase that was responsible for the phosphorylation of p53(Ser392).And the interaction of p70S6K and p53 was confirmed by immunofluorescence and IP assay.Therefore,we have concluded that selenite induced ROS can inhibit protective autophagy and induce apoptosis through p70S6K/p53/ULKl pathway.