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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.
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