Background: A 28-kDa HO-1 isoform is induced by oxidative stress and cancer and accumulates in the nucleus. Results: Nuclear HO-1 interacts with Nrf2 and alters expression of its target genes. Conclusion: HO-1 modulates Nrf2 function. Significance: Exploiting the synergistic benefits of the HO-1⅐Nrf2 protein complex is important for developing therapeutic strategies against oxidative stress or cancer.

Our findings suggest that increased activity of the ubiquitin-proteasome pathway, marked decreases in MyHCs, and atrophic AKT-FOXO signaling play important roles in eliciting the myofiber atrophy and decreases in diaphragm force generation associated with prolonged human diaphragm disuse.

GRP94 is a molecular chaperone that carries immunologically relevant peptides from cell to cell, transferring them to major histocompatibility proteins for presentation to T cells. Here we examine the binding of several peptides to recombinant GRP94 and study the regulation and site of peptide binding. We show that GRP94 contains a peptide-binding site in its N-terminal 355 amino acids. A number of peptides bind to this site with low on-and off-rates and with specificity that is distinct from that of another endoplasmic reticulum chaperone, BiP/GRP78. Binding to the N-terminal fragment is sufficient to account for the peptide binding activity of the entire molecule. Peptide binding is inhibited by radicicol, a known inhibitor of the chaperone activities of HSP90-family proteins. However, the peptide-binding site is distinct from the radicicol-binding pocket, because both can bind to the N-terminal fragment simultaneously. Furthermore, peptide binding does not cause the same conformational change as does binding of radicicol. When the latter binds to the N-terminal domain, it induces a conformational change in the downstream, acidic domain of GRP94, as measured by altered gel mobility and loss of an antibody epitope. These results relate the peptide-binding activity of GRP94 to its other function as a chaperone.

GRP94 (glucose-regulated protein of 94 kDa) is a major luminal constituent of the endoplasmic reticulum with known high capacity for calcium in vivo and a peptide-binding activity in vitro. In the present study, we show that Ca2+ regulates the ability of GRP94 to bind peptides. This effect is due to a Ca2+-binding site located in the charged linker domain of GRP94, which, when occupied, enhances the association of peptides with the peptide-binding site in the N-terminal domain of the protein. We further show that grp94-/- cells are hypersensitive to perturbation of intracellular calcium and thus GRP94 is important for cellular Ca2+ storage.

Aims: The response to oxidative stress and inflammation varies with diurnal rhythms. Nevertheless, it is not known whether circadian genes are regulated by these stimuli. We evaluated whether Rev-erba, a key circadian gene, was regulated by oxidative stress and/or inflammation in vitro and in a mouse model. Results: A unique sequence consisting of overlapping AP-1 and nuclear factor kappa B (NFjB) consensus sequences was identified on the mouse Rev-erba promoter. This sequence mediates Rev-erba promoter activity and transcription in response to oxidative stress and inflammation. This region serves as an NrF2 platform both to receive oxidative stress signals and to activate Rev-erba, as well as an NFjB-binding site to repress Rev-erba with inflammatory stimuli. The amplitude of the rhythmicity of Rev-erba was altered by pre-exposure to hyperoxia or disruption of NFjB in a cell culture model of circadian simulation. Oxidative stress overcame the inhibitory effect of NFjB binding on Rev-erba transcription. This was confirmed in neonatal mice exposed to hyperoxia, where hyperoxiainduced lung Rev-erba transcription was further increased with NFjB disruption. Interestingly, this effect was not observed in similarly exposed adult mice. Innovation: These data provide novel mechanistic insights into how key circadian genes are regulated by oxidative stress and inflammation in the neonatal lung. Conclusion: Rev-erba transcription and circadian oscillation are susceptible to oxidative stress and inflammation in the neonate. Due to Rev-erba's role in cellular metabolism, this could contribute to lung cellular function and injury from inflammation and oxidative stress. Antioxid. Redox Signal. 21, 17-32.

Because the stress protein GRP94 can augment presentation of peptides to T cells, it is important to define how it, as well as all other HSP90 family members, binds peptides. Having previously shown that the N-terminal half of GRP94 can account for the peptide binding activity of the full-length protein, we now locate this binding site by testing predictions of a molecular docking model. The best predicted site was on the opposite face of the ␤ sheet from the pan-HSP90 radicicol-binding pocket, in close proximity to a deep hydrophobic pocket. The peptide and radicicol-binding sites are distinct, as shown by the ability of a radicicol-refractive mutant to bind peptide. When the fluorophore acrylodan is attached to Cys 117 within the hydrophobic pocket, its fluorescence is reduced upon peptide binding, consistent with proximity of the two ligands. Substitution of His 125 , which contacts the bound peptide, compromises peptide-binding activity. We conclude that peptide binds to the concave face of the ␤ sheet of the N-terminal domain, where binding is regulated during the action cycle of the chaperone.Glucose-regulated protein 94 (GRP94), 1 also known as gp96, is a member of the HSP90 family of molecular chaperones and can dramatically stimulate T cell responses by two mechanisms: enhancement of peptide presentation to the adaptive arm of the immune system (1) and stimulation of innate immunity (2). Because of these activities, tumor-derived GRP94 can be used to elicit immune response against the tumor and is potentially a powerful immunotherapeutic tool (3). The antigen-presentation activity was shown not to be due to any mutation in GRP94 that would enhance its immunogenicity, but rather due to its ability to bind peptides (1, 4). The GRP94-peptide complexes are known to be taken up by a subset of antigen-presenting cells via receptor-mediated endocytosis (5), and the chaperoned peptides are then re-presented on endogenous antigen-presenting cells MHC class I molecules on the cell surface. Although peptide binding is a general activity of many molecular chaperones, it has been argued that GRP94 is among the most effective of such chaperones in enhancing antigen presentation.Despite its importance, the GRP94-peptide interaction and the identity of the peptide-binding site have not been characterized in detail. They are crucial issues toward understanding the immunostimulatory action of GRP94. The mode of peptide binding by GRP94 may also inform about its activity as a chaperone of selected membrane-bound and secreted proteins (6), although the connection between the two activities is yet to be elucidated. The same questions are also unanswered for all other HSP90 chaperones, despite the central role of these cytosolic chaperones in organizing signaling complexes and regulating transcription factor (6). Several peptides derived from vesicular stomatitis virus (VSV) have been shown to bind GRP94. VSV8 is an octamer (RGYVYQGL) from the N protein of VSV and is the dominant T cell epitope of the virus, presented via MH...

SummaryER stress leads to upregulation of multiple folding and quality control components, known as the unfolded protein response (UPR). Glucose Regulated Protein 78 (GRP78) (also known as binding immunoglobulin protein, BiP, and HSPA5) and GRP94 are often upregulated coordinately as part of this homeostatic response. Given that endoplasmic reticulum (ER) chaperones have distinct sets of clients, we asked how cells respond to ablation of individual chaperones. The cellular responses to silencing BiP, GRP94, HSP47, PDIA6 and OS-9, were distinct. When BiP was silenced, a widespread UPR was observed, but when GRP94 was either inhibited or depleted by RNA interference (RNAi), the expression of only some genes was induced, notably those encoding BiP and protein disulfide isomerase A6 (PDIA6). Silencing of HSP47 or OS-9 did not lead to any compensatory induction of other genes. The selective response to GRP94 depletion was distinct from a typical ER stress response, both because other UPR target genes were not affected and because the canonical UPR signaling branches were not activated. The response to silencing of GRP94 did not preclude further UPR induction when chemical stress was imposed. Importantly, re-expression of wild-type GRP94 in the silenced cells prevented the upregulation of BiP and PDIA6, whereas re-expression of an ATPase-deficient GRP94 mutant did not, indicating that cells monitor the activity state of GRP94. These findings suggest that cells are able to distinguish among folding resources and generate distinct responses.

Cytokines play an important role in modulating the development and function of dendritic cells (DCs). Type I IFNs activate DCs and drive anti-viral responses, whereas IL-4 is the prototype of a Th2 cytokine. Evidence suggests that type I IFNs and IL-4 influence each other to modulate DC functions. We found that two type I IFNs, IFN-α and IFN-β, stimulated a similar costimulatory profile in myeloid resting DCs. IL-4 suppressed the response of myeloid DCs to both type I IFNs in vitro and in vivo by impairing the up-regulation of MHC and costimulatory molecules and the production of cytokines, such as IL-6 and IL-15, and anti-viral genes, such as Mx-1, upon type I IFN stimulation. In dissecting the mechanism underlying this inhibition, we characterized the positive feedback loop that is triggered by IFN-α in primary DCs and found that IL-4 inhibited the initial phosphorylation of STAT1 and STAT2 (the transducers of signaling downstream of IFN-α and -β receptors (IFNARs)) and reduced the up-regulation of genes involved in the amplification of the IFN response such as IRF-7, STAT1, STAT2, IFN-β, and the IFNARs in vitro and in vivo. Therefore, IL-4 renders myeloid DCs less responsive to paracrine type I IFNs and less potent in sustaining the autocrine positive loop that normally amplifies the effects of type I IFNs. This inhibition could explain the increased susceptibility to viral infections observed during Th2-inducing parasitoses.