Transcriptional activation of endoplasmic reticulum chaperone GRP78 by HCMV IE1-72 protein

Glucose-regulated protein 78 (GRP78), a key regulator of endoplasmic reticulum (ER) stress, facilitates cancer cell growth and viral replication. The mechanism leading to grp78 gene activation during viral infection is largely unknown. In this study, we show that the immediate-early 1 (IE1-72) protein of the human cytomegalovirus (HCMV) is essential for HCMV-mediated GRP78 activation. IE1-72 upregulated grp78 gene expression depending on the ATP-binding site, the zinc-finger domain and the putative leucine-zipper motif of IE1-72, as well as the ER stress response elements (ERSEs) on the grp78 promoter. The purified IE1-72 protein bound to the CCAAT box within ERSE in vitro, whereas deletion mutants of IE1-72 deficient in grp78 promoter stimulation failed to do so. Moreover, IE1-72 binding to the grp78 promoter in infected cells accompanied the recruitment of TATA box-binding protein-associated factor 1 (TAF1), a histone acetyltransferase, and the increased level of acetylated histone H4, an indicator of active-state chromatin. These results provide evidence that HCMV IE1-72 activates grp78 gene expression through direct promoter binding and modulation of the local chromatin structure, indicating an active viral mechanism of cellular chaperone induction for viral growth.     

ATF6 as a transcription activator of the endoplasmic reticulum stress element: thapsigargin stress-induced changes and synergistic interactions with NF-Y and YY1

ATF6, a member of the leucine zipper protein family, can constitutively induce the promoter of glucose-regulated protein (grp) genes through activation of the endoplasmic reticulum (ER) stress element (ERSE). To understand the mechanism of grp78 induction by ATF6 in cells subjected to ER calcium depletion stress mediated by thapsigargin (Tg) treatment, we discovered that ATF6 itself undergoes Tg stress-induced changes. In nonstressed cells, ATF6, which contains a putative short transmembrane domain, is primarily associated with the perinuclear region. Upon Tg stress, the ATF6 protein level dropped initially but quickly recovered with the additional appearance of a faster-migrating form. This new form of ATF6 was recovered as soluble nuclear protein by biochemical fractionation, correlating with enhanced nuclear localization of ATF6 as revealed by immunofluorescence. Optimal ATF6 stimulation requires at least two copies of the ERSE and the integrity of the tripartite structure of the ERSE. Of primary importance is a functional NF-Y complex and a high-affinity NF-Y binding site that confers selectivity among different ERSEs for ATF6 inducibility. In addition, we showed that YY1 interacts with ATF6 and in Tg-treated cells can enhance ATF6 activity. The ERSE stimulatory activity of ATF6 exhibits properties distinct from those of human Ire1p, an upstream regulator of the mammalian unfolded protein response. The requirement for a high-affinity NF-Y site for ATF6 but not human Ire1p activity suggests that they stimulate the ERSE through diverse pathways.     

Lead-induced endoplasmic reticulum (ER) stress responses in the nervous system

Lead (Pb) poisoning continues to be a significant health risk because of its pervasiveness in the environment, its known neurotoxic effects in children, and potential endogenous exposure from Pb deposited in bone. New information about mechanisms by which Pb enters cells and its organelle targets within cells are briefly reviewed. Toxic effects of Pb on the endoplasmic reticulum (ER) are considered in detail, based on recent evidence that Pb induces the expression of the gene for 78-kD glucose-regulated protein (GRP78) and other ER stress genes. GRP78 is a molecular chaperone that binds transiently to proteins traversing through the ER and facilitates their folding, assembly, and transport. Models are presented for the induction of ER stress by Pb in astrocytes, the major cell type of the central nervous system, in which Pb accumulates. A key feature of the models is disruption of GRP78 function by direct Pb binding. Possible pathways by which Pb-bound GRP78 stimulates the unfolded protein response (UPR) in the ER are discussed, specifically transduction by IRE1/ATF6 and/or IRE1/JNK. The effect of Pb binding to GRP78 in the ER is expected to be a key component for understanding mechanisms of Pb-induced ER stress gene expression.