Yair Argon

GRP94: An HSP90-like protein specialized for protein folding and quality control in the endoplasmic reticulum.

Glucose-regulated protein 94 is the HSP90-like protein in the lumen of the endoplasmic reticulum and therefore it chaperones secreted and membrane proteins. It has essential functions in development and physiology of multicellular organisms, at least in part because of this unique clientele. GRP94 shares many biochemical features with other HSP90 proteins, in particular its domain structure and ATPase activity, but also displays distinct activities, such as calcium binding, necessitated by the conditions in the endoplasmic reticulum. GRP94s mode of action varies from the general HSP90 theme in the conformational changes induced by nucleotide binding, and in its interactions with co-chaperones, which are very different from known cytosolic co-chaperones. GRP94 is more selective than many of the ER chaperones and the basis for this selectivity remains obscure. Recent development of molecular tools and functional assays has expanded the spectrum of clients that rely on GRP94 activity, but it is still not clear how the chaperone binds them, or what aspect of folding it impacts. These mechanistic questions and the regulation of GRP94 activity by other proteins and by post-translational modification differences pose new questions and present future research avenues. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).

GRP94 in ER quality control and stress responses

A system of endoplasmic reticulum (ER) chaperones has evolved to optimize the output of properly folded secretory and membrane proteins. An important player in this network is Glucose Regulated Protein 94 (GRP94). Over the last decade, new structural and functional data have begun to delineate the unique characteristics of GRP94 and have solidified its importance in ER quality control pathways. This review describes our current understanding of GRP94 and the four ways in which it contributes to the ER quality control: (1) chaperoning the folding of proteins; (2) interacting with other components of the ER protein folding machinery; (3) storing calcium; and (4) assisting in the targeting of malfolded proteins to ER-associated degradation (ERAD).

Molecular chaperones and the biosynthesis of antigen receptors

Oligomeric antigen receptors must fold and assemble in the endoplasmic reticulum (ER) before they can be expressed on the surface of lymphocytes. It is increasingly evident that these processes are facilitated by molecular chaperones. Here, Jeffrey Melnick and Yair Argon review the known ER chaperones, summarize their roles in the maturation of antigen receptors, and discuss how they may affect lymphocyte differentiation and function.

Radicicol-sensitive Peptide Binding to the N-terminal Portion of GRP94

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.

Development of a Grp94 inhibitor

Heat shock protein 90 (Hsp90) represents a promising therapeutic target for the treatment of cancer and other diseases. Unfortunately, results from clinical trials have been disappointing as off-target effects and toxicities have been observed. These detriments may be a consequence of pan-Hsp90 inhibition, as all clinically evaluated Hsp90 inhibitors simultaneously disrupt all four human Hsp90 isoforms. Using a structure-based approach, we designed an inhibitor of Grp94, the ER-resident Hsp90. The effect manifested by compound 2 on several Grp94 and Hsp90α/β (cytosolic isoforms) clients were investigated. Compound 2 prevented intracellular trafficking of the Toll receptor, inhibited the secretion of IGF-II, affected the conformation of Grp94, and suppressed Drosophila larval growth, all Grp94-dependent processes. In contrast, compound 2 had no effect on cell viability or cytosolic Hsp90α/β client proteins at similar concentrations. The design, synthesis, and evaluation of 2 are described herein.

Orchestration of secretory protein folding by ER chaperones.

The endoplasmic reticulum is a major compartment of protein biogenesis in the cell, dedicated to production of secretory, membrane and organelle proteins. The secretome has distinct structural and post-translational characteristics, since folding in the ER occurs in an environment that is distinct in terms of its ionic composition, dynamics and requirements for quality control. The folding machinery in the ER therefore includes chaperones and folding enzymes that introduce, monitor and react to disulfide bonds, glycans, and fluctuations of luminal calcium. We describe the major chaperone networks in the lumen and discuss how they have distinct modes of operation that enable cells to accomplish highly efficient production of the secretome. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum.

Protein Disulfide Isomerase A6 Controls the Decay of IRE1α Signaling via Disulfide-Dependent Association

The response to endoplasmic reticulum (ER) stress relies on activation of unfolded protein response (UPR) sensors, and the outcome of the UPR depends on the duration and strength of signal. Here, we demonstrate a mechanism that attenuates the activity of the UPR sensor inositol-requiring enzyme 1α (IRE1α). A resident ER protein disulfide isomerase, PDIA6, limits the duration of IRE1α activity by direct binding to cysteine 148 in the lumenal domain of the sensor, which is oxidized when IRE1 is activated. PDIA6-deficient cells hyperrespond to ER stress with sustained autophosphorylation of IRE1α and splicing of XBP1 mRNA, resulting in exaggerated upregulation of UPR target genes and increased apoptosis. In vivo, PDIA6-deficient C. elegans exhibits constitutive UPR and fails to complete larval development, a program that normally requires the UPR. Thus, PDIA6 activity provides a mechanism that limits UPR signaling and maintains it within a physiologically appropriate range.

The Chaperone Activity of GRP94 Toward Insulin-like Growth Factor II Is Necessary for the Stress Response to Serum Deprivation

Insulin-like growth factor (IGF)-II is a hormone with mitogenic activity for many cell types and tissues. We demonstrate that its intracellular processing and secretion strictly depend on the endoplasmic reticulum chaperone glucose-regulated protein (GRP) 94. GRP94 interacts physically and transiently with pro-IGF-II intermediates, and its activity is essential for secretion of active IGF-II, thus establishing IGF-II as a client of GRP94. Embryonic stem (ES) cells that lack GRP94 are hypersensitive to stress conditions such as serum deprivation and die by apoptosis because they cannot respond to the stress by producing active IGF-II. This chaperone-client interaction may explain the previously documented antiapoptotic activity of GRP94 in a number of stress responses.

Redox controls UPR to control redox

ABSTRACT In many physiological contexts, intracellular reduction–oxidation (redox) conditions and the unfolded protein response (UPR) are important for the control of cell life and death decisions. UPR is triggered by the disruption of endoplasmic reticulum (ER) homeostasis, also known as ER stress. Depending on the duration and severity of the disruption, this leads to cell adaptation or demise. In this Commentary, we review reductive and oxidative activation mechanisms of the UPR, which include direct interactions of dedicated protein disulfide isomerases with ER stress sensors, protein S-nitrosylation and ER Ca2+ efflux that is promoted by reactive oxygen species. Furthermore, we discuss how cellular oxidant and antioxidant capacities are extensively remodeled downstream of UPR signals. Aside from activation of NADPH oxidases, mitogen-activated protein kinases and transcriptional antioxidant responses, such remodeling prominently relies on ER–mitochondrial crosstalk. Specific redox cues therefore operate both as triggers and effectors of ER stress, thus enabling amplification loops. We propose that redox-based amplification loops critically contribute to the switch from adaptive to fatal UPR.

An essential role for ATP binding and hydrolysis in the chaperone activity of GRP94 in cells

Glucose-regulated protein 94 (GRP94) is an endoplasmic reticulum (ER) chaperone for which only few client proteins and no cofactors are known and whose mode of action is unclear. To decipher the mode of GRP94 action in vivo, we exploited our finding that GRP94 is necessary for the production of insulin-like growth factor (IGF)-II and developed a cell-based functional assay. Grp94−/− cells are hypersensitive to serum withdrawal and die. This phenotype can be complemented either with exogenous IGF-II or by expression of functional GRP94. Fusion proteins of GRP94 with monomeric GFP (mGFP) or mCherry also rescue the viability of transiently transfected, GRP94-deficient cells, demonstrating that the fusion proteins are functional. Because these constructs enable direct visualization of chaperone-expressing cells, we used this survival assay to assess the activities of GRP94 mutants that are defective in specific biochemical functions in vitro. Mutations that abolish binding of adenosine nucleotides cannot support growth in serum-free medium. Similarly, mutations of residues needed for ATP hydrolysis also render GRP94 partially or completely nonfunctional. In contrast, an N-terminal domain mutant that cannot bind peptides still supports cell survival. Thus the peptide binding activity in vitro can be uncoupled from the chaperone activity toward IGF in vivo. This mutational analysis suggests that the ATPase activity of GRP94 is essential for chaperone activity in vivo and that the essential protein-binding domain of GRP94 is distinct from the N-terminal domain.