Study On The Nuclear Translocation Of NSREBP2

Sterol regulatory element binding proteins(SREBPs)are transcriptional factorsthat regulate intracellular cholesterol synthesis inthe endoplasmic reticulum.It plays a very important role inthe synthesis of cholesterol andthe waythat absorbs low density lipoprotein(LDL)to obtain cholesterol.SREBP2 is one member of SREBPs,and it is also responsible for regulatingthe transcription of cholesterol synthesis genes.Therefore,we studiedthe regulation of SREBPs onthe synthesis of cholesterol in humans mainly based onSREBP2.SREBPs require two cleavage steps to releasethe nuclear forms of SREBPs(nSREBPs)to enterthe nucleus and combine with sterol regulatory element(SRE)to regulatethe expression of cholesterol-related genes,thereby upregulating cholesterol levels.Studies have shownthat nSREBP2(nuclear forms of SREBP2,nSREBP2)binds tothe nuclear transporter receptor importin? in a dimeric manner andthen translocated intothe nucleus.However,the specific mechanism by which importin? directly recognizes nSREBPs is still unclear.Whether nSREBP2 can enterthe nucleus as an independent nuclear signal withoutthe help of importin? is also a possibility.The study ofthe SREBP2 nuclear import mechanism is of great significance for us to fully understandthe regulation of cholesterol metabolism inSREBPs.We usedthe method of lentivirus infection to overexpress nSREBP2 in He La cell.We foundthat most of cells were died.Andthen we finally isolated a monoclonal cell and named it nSREBP2-3.Further studies foundthat nSREBP2 did not enterthe nucleus in nSREBP2-3 cels,and its protein level did not increase,the expression level ofthe nSREBP2 target gene was also not up-regulated.We clonedthe exogenously expressed nSREBP2 fromthis cell and sequenced it.It was foundthat many ofthe adenines inthe posterior segment ofthe sequence were mutated into guanine.The reason forthe survival ofthis cell should be related tothe inability of nSREBP2 to completethe nuclear transport caused bythese mutations,and we will continue to explorethe specific causes ofthese mutations.Atthe same time,we usedthe mutated nSREBP2,which is fromthe nSREBP2-3 cells,asthe target gene to construct plasmid with overlapping PCR,andthen sequentially transfectedthe cels to observe whether nSREBP2 enters into nucleus,narrowingthe sequence rangethat affectsthe ability of entry nucleus.Finally,the amino acids inthis sequence were mutated by quickchange PCR.It was foundthat whenthe 371th arginine andthe 372th lysine were respectively mutated into acidic amino acids,the ability of nSREBP2 to entrythe nucleus was affected.Our results are different fromthe previously reportedthree leucine mutations inthe leucine zipper region of nSREBP2,which make it impossible to enterthe nucleus.And studies have shownthat dimerized nSREBP2 is embedded inthe nuclear transport protein importin?,only its 363th,365th,366th,and 371th amino acids interacted with importin?.However,it didn't affectthe ability of entrythe nucleus when we changedthe 363th and 365th amino acids,andthe 366th,371th amino acids did not have mutated in nSREBP2-3 cells,so we believethatthe mutation of 366th and 371th amino acids does not affect nSREBP2-3 enters into nucleus.Our work discoveredthe sequence of nSREBP2 transported intothe nucleus,revealing a new mechanism forthe transcription factor nSREBP2 to function.