|
The immunoglobulin heavy chain binding protein (BiP) is a Hsp70 homologue inthe endoplasmic reticulum (ER)lumen and alsr srgers to the glucose regulated
protien GRP78. Under normmal gorwht conditions. BiP is synthesized constitutively. It's expression can be further induced, manily ocntrolled at transcriptonal level,by a number of ER stress conditions such as treatment of cells with calciumionophore A23187. In yeast cells BiP is the product of the KAR2 gene and has a rolein the folding and assembly of nascent polypeptide in the ER lumen. BiP may also involve in translocaion of precuresors across the ER membrane and prevent the aggregation of malfolded protein before their degration. Analyses of the human BiP promoter sequenc have revealed that two major stress response elements (SRE),BiP33 and BiP36, are within -107 and -39 region containing two direct repeated CCAAT boxes. To ideintify a factor(s) which binds to the stress response element, A A23187 treated HeLa cDNA expression library was screened by using BiP36 pentamer probe (BiP36x5). After screening 1,000,000 clones ,I obtained ten putative positive clones which were confirmed through three successive screenings. DNA sequence analyses of the clones showed virtual identity ot six genes for 23 kDa HBP, XPE, HSP86, TCB, IAP, and Myosin VIIa. In our previously studies, YB-1 and XPE were isolated as a stress response element binding protein and DNA lesion- recongnizing protein, respectively. The Southwestern blot analysis suggested that bacteriallyproduced YB-1 possessed binding affinity with single-stranded SRE and HBP was capable bound to double-stranded SRE. In cell lysates with different ER stress treatments, the various expression of protiens specific to anti-BiP, YB-1 and XPE.antisera could be observed. To evaluate further the binding activity and specificity, electrophoretic mobility shift assays (EMSA) were performmed for YB-1 and XPE. These results showed that in vitro translated YB-1 and XPE proteins using rabbit reticulocyte lysates bound preferentially to the anti-sense strand SRE oligonucleotide.In transient transfection experimetns, YB-1 repressed the transcriptional activationof the BiP promoter under A23187 stimulus. However, XPE has minimal effect on the ER stress inducion of the BiP promoter.
|