Abundant Chaperone Research Articles

Hold Me Tight

Hold Me Tight

Targeting secreted heat shock protein-90alpha (Hsp90α) to prevent breast cancer progression

Heat shock protein 90 (Hsp90) has been historically known as an abundant and intracellular ATP-driven chaperone with more than 100 target proteins that often play critical roles in various branches of the signaling networks for control of cell metabolism, survival, growth and differentiation. In many cancers, Hsp90 is either over-expressed or over-activated and it, therefore, becomes a target for anti-cancer drugs with a hope to simultaneously shut down multiple constitutively activated oncogene products in cancer cells. Despite of the high expectations, the toxicity of geldanamycin (GM)-derived inhibitors in humans has been a long-standing hurdle. However, studies of past few years have demonstrated a surprising need for cancer cells to secrete Hsp90α for tissue invasion and metastasis. I will present here new evidence that the metastatic breast cancer cells, MDA-MB-231, constitutively secrete Hsp90α due to their constitutive expression of the hypoxia-inducible factor-1alpha (HIF1α). Down-regulation of either HIF1α or HIF1β completely blocked Hsp90α secretion. The main function of the secreted Hsp90α is to promote breast cancer cell motility via binding to the cell surface lipoprotein receptor LRP1 (LDL Receptor-Related Protein-1). We have mapped this important extracellular Hsp90α function to a 27-amino acid peptide located at the boundary between the linker region (LR) and the middle domain (M) of Hsp90α. Selective inhibition of the extracellular action of Hsp90 or down-regulation of LRP-1 receptor blocked breast cancer invasion in an in vitro matrigel assay and tumor formation in nude mice. Most intriguingly, since secretion of Hsp90α was only found to occur under pathological conditions such as cancer and is not part of normal cells’ life, anti-cancer drugs targeting the 27-amino acid peptide of the extracellular Hsp90α should be more effective and less toxic in humans. We propose that the novel “secreted Hsp90-LRP1 Receptor” autocrine signaling plays a role in breast cancer progression and the extracellular, not intracellular, Hsp90α is an effective and safer target for anti-cancer therapies.

Neuroligin Trafficking Deficiencies Arising from Mutations in the α/β-Hydrolase Fold Protein Family

Despite great functional diversity, characterization of the alpha/beta-hydrolase fold proteins that encompass a superfamily of hydrolases, heterophilic adhesion proteins, and chaperone domains reveals a common structural motif. By incorporating the R451C mutation found in neuroligin (NLGN) and associated with autism and the thyroglobulin G2320R (G221R in NLGN) mutation responsible for congenital hypothyroidism into NLGN3, we show that mutations in the alpha/beta-hydrolase fold domain influence folding and biosynthetic processing of neuroligin3 as determined by in vitro susceptibility to proteases, glycosylation processing, turnover, and processing rates. We also show altered interactions of the mutant proteins with chaperones in the endoplasmic reticulum and arrest of transport along the secretory pathway with diversion to the proteasome. Time-controlled expression of a fluorescently tagged neuroligin in hippocampal neurons shows that these mutations compromise neuronal trafficking of the protein, with the R451C mutation reducing and the G221R mutation virtually abolishing the export of NLGN3 from the soma to the dendritic spines. Although the R451C mutation causes a local folding defect, the G221R mutation appears responsible for more global misfolding of the protein, reflecting their sequence positions in the structure of the protein. Our results suggest that disease-related mutations in the alpha/beta-hydrolase fold domain share common trafficking deficiencies yet lead to discrete congenital disorders of differing severity in the endocrine and nervous systems.

Arabidopsis Stromal-derived Factor2 (SDF2) Is a Crucial Target of the Unfolded Protein Response in the Endoplasmic Reticulum

Stresses increasing the load of unfolded proteins that enter the endoplasmic reticulum (ER) trigger a protective response termed the unfolded protein response (UPR). Stromal cell-derived factor2 (SDF2)-type proteins are highly conserved throughout the plant and animal kingdoms. In this study we have characterized AtSDF2 as crucial component of the UPR in Arabidopsis thaliana. Using a combination of biochemical and cell biological methods, we demonstrate that SDF2 is induced in response to ER stress conditions causing the accumulation of unfolded proteins. Transgenic reporter plants confirmed induction of SDF2 during ER stress. Under normal growth conditions SDF2 is highly expressed in fast growing, differentiating cells and meristematic tissues. The increased production of SDF2 due to ER stress and in tissues that require enhanced protein biosynthesis and secretion, and its association with the ER membrane qualifies SDF2 as a downstream target of the UPR. Determination of the SDF2 three-dimensional crystal structure at 1.95 A resolution revealed the typical beta-trefoil fold with potential carbohydrate binding sites. Hence, SDF2 might be involved in the quality control of glycoproteins. Arabidopsis sdf2 mutants display strong defects and morphological phenotypes during seedling development specifically under ER stress conditions, thus establishing that SDF2-type proteins play a key role in the UPR.

Secreted Heat Shock Protein-90α: A More Effective and Safer Target for Anti-Cancer Drugs?

Until recently, heat shock protein 90 (Hsp90) has been mostly known as an abundant intracellular chaperone with more than 100 target proteins often involved in control of cell metabolism, survival, growth and differentiation. Hsp90 has been a target for anti-cancer drugs, since it was found either overexpressed or overactive in cancer cells. For more than a decade, geldanamycin (GM)-derived inhibitors of Hsp90s ATPase have entered various clinical trials around the world. Despite of high expectations, the efficacy of these inhibitors in humans has been less than what was hoped for. While newer generations of GM inhibitors are being developed and tested in several ongoing clinical trials, recent studies have discovered a surprising need for cancer cells to constitutively secrete Hsp90α for invasion and metastasis. A main function for secreted Hsp90 is to promote cell motility via the cell surface receptor LRP1 and/or secreted MMP2. Distinct from its intracellular chaperone function that requires the N-terminal ATPase and the C-terminal dimerization, the pro-motility activity of extracellular Hsp90α resides in a highly charged peptide between the linker region and the middle domain in Hsp90α. Selective inhibition of secreted Hsp90α blocks tumor cell invasion in vitro and in vivo. More importantly, since Hsp90α secretion does not occur in normal cells under physiological conditions, drugs that selectively target the secreted Hsp90α at its pro-motility region may prove to be more effective and less toxic for treatment of cancer patients. Keywords: HSP90, secretion, cell migration, cancer progression, anti-cancer target

Abstract 428: Targeting secreted heat shock protein-90alpha (hsp90α) to prevent breast cancer progression

Abstract Targeting secreted heat shock protein-90alpha (Hsp90α) to prevent breast cancer progression Z. Zhao, C. Cheng, R. Kim, M. Chen, D. T. Woodley and W. Li* Department of Dermatology and the USC-Norris Comprehensive Cancer Center, the University of Southern California Keck School of Medicine, Los Angeles, California, 90033, USA Until recently, heat shock protein 90 (Hsp90) has been mostly known as an abundant, intracellular and ATP-driven chaperone with more than 100 target proteins that often play critical roles in various branches of the signaling networks for control of cell metabolism, survival, growth and differentiation. Since Hsp90 has been found either over-expressed or over-activated in cancer cells than in normal cells, this intracellular protein has become a target for anti-cancer drugs with a hope to simultaneously shut down multiple constitutively activated oncogene products in cancer cells. Despite of the high expectations, the toxicity of geldanamycin (GM)-derived inhibitors in humans has been a long-standing hurdle. Newer generations of GM inhibitors are still being tested currently in at least eight clinical trials. However, studies of past few years have demonstrated a surprising need for cancer cells to secrete Hsp90α for tissue invasion and metastasis. The mechanism of the extracellar Hsp90α action has since been actively investigated. We present here new evidence that the metastatic breast cancer cells, MDA-MB-231, constitutively secrete Hsp90α due to their constitutive expression of the hypoxia-inducible factor-1alpha (HIF1α). Down-regulation of either HIF1α or HIF1β or replacing them with their nuclear tanslocation-defective mutants completely blocked Hsp90α secretion. The main function of the secreted Hsp90α was to promote breast cancer cell motility via binding to the cell surface lipoprotein receptor LRP1 (LDL Receptor-Related Protein-1). Unlike its intracellular chaperone function that requires the N-terminal ATPase and the C-terminal dimerization domains, this extracellular Hsp90α promotes cell motility via a 27-amino acid peptide located at the boundary between the linker region (LR) and the middle domain (M) of Hsp90α. These studies led to the notion that a novel “secreted Hsp90α-LRP1 Receptor” autocrine signaling plays a role in breast cancer progression. Indeed, selective inhibition of the extracellular action of Hsp90 or down-regulation of LRP-1 receptor blocked breast cancer invasion in an in vitro matrigel assay and tumor formation in nude mice. Most intriguingly, since secretion of Hsp90α was only found to occur under pathological conditions such as cancer and is not part of normal cells’ life, anti-cancer drugs targeting the 27-amino acid peptide of the extracellular Hsp90α should be more effective and less toxic in humans. We propose here that the extracellular, not intracellular, Hsp90α is an effective and safer target for anti-cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 428.

Extracellular Chaperones.

The maintenance of the levels and correct folding state of proteins (proteostasis) is a fundamental prerequisite for life. Life has evolved complex mechanisms to maintain proteostasis and many of these that operate inside cells are now well understood. The same cannot yet be said of corresponding processes in extracellular fluids of the human body, where inappropriate protein aggregation is known to underpin many serious diseases such as Alzheimer's disease, type II diabetes and prion diseases. Recent research has uncovered a growing family of abundant extracellular chaperones in body fluids which appear to selectively bind to exposed regions of hydrophobicity on misfolded proteins to inhibit their toxicity and prevent them from aggregating to form insoluble deposits. These extracellular chaperones are also implicated in clearing the soluble, stabilized misfolded proteins from body fluids via receptor-mediated endocytosis for subsequent lysosomal degradation. Recent work also raises the possibility that extracellular chaperones may play roles in modulating the immune response. Future work will better define the in vivo functions of extracellular chaperones in proteostasis and immunology and pave the way for the development of new treatments for serious diseases.

Abstract B81: AUY922, a heat shock protein 90 inhibitor, actively inhibits human gastric cancer cells via down regulation of growth factor receptors, and acts synergistically with relevant chemotherapeutic agents

Abstract Heat shock protein 90 (Hsp90) is the most abundant cellular chaperone and has nearly 100 pivotal client proteins, which are commonly dysregulated in cancers. The positive rate of Hsp90beta in gastric cancer tissue was approximately 30%, and was higher than non-cancerous gastric mucosa. Hsp90 inhibitors should have great potentials in the exploration for treatment strategy in gastric cancer (GC). AUY922, kindly provided by Novartis Pharma AG, is a novel resorcinylic isoxazole amide Hsp90 inhibitor. A panel of human GC cell lines (N87, AGS, SNU-1, SNU-16, KATO-III from ATCC) was used in this study. Using 72-hour MTT colorimetric assay, we demonstrated that AUY922 has highly active growth inhibitory effects as a single agent of IC50 in the ranges between 8.1 ± 0.2 and 14.7 ± 0.8 nM (except the c-met-overexpressing KATO-III cells with a IC50 of 158.1 ± 5.1 nM), which is more active than 17-AAG (17-allylamino-17-demethoxygeldanamycin) and 17-DMAG (17-dimethylaminoethyl-amino-17-demethoxygeldanamycin) of IC50 between 17.2 ± 0.6 and 61.3 ± 1.9 nM, and IC50 between 10.5 ± 0.5 and 24.3 ± 1.0 nM, respectively. By MTT colorimetric assay and median effect analysis, AUY922 has significant synergistic cytotoxicity with cisplatin, oxaliplatin, 5-fluorouracil (5-FU), and irinotecan, while acts antagonistically with paclitaxel and docetaxel in GC cells. Several important growth factor receptors are highly expressed in GC, namely 50-60% of EGFR (epidermal growth factor receptor, HER1), 15–22% of HER-2/neu, and 32–58% of c-met (hepatocyte growth factor receptor, HGF receptor), respectively. By immunoblotting analysis, AUY922 effectively down-regulates growth factor receptors (c-met, EGFR, and HER-2/neu) with a concentration of 25nM, 25nM, and 125nM in KATO-III GC cells, respectively. The c-met-overexpressing KATO-III cells are highly resistant to AUY922. In KATO-III cells, knocking-down of c-met by siRNA, then examining by 48-hour MTT colorimetric assay may partly abrogate the AUY922 resistance and increase sensitivity to AUY922 by decreasing IC50 from 790.0 ± 10.6 nM to 350.8 ± 5.4 nM. Our data indicate that AUY922 actively inhibits human GC cells via down regulation of growth factor receptors, and acts synergistically with various relevant chemotherapeutic agents. It is reasonable to use AUY922 alone, or to combine AUY922 with relevant chemotherapeutic agents (cisplatin, oxaliplatin, 5-FU, or irinotecan) in future development of clinical trials for gastric cancers. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B81.

A temperature sensitive mutant of heat shock protein 70 reveals an essential role during the early steps of tombusvirus replication

By co-opting host proteins for their replication, plus-stranded RNA viruses can support robust replication and suppress host anti-viral responses. Tomato bushy stunt virus (TBSV) recruit the cellular heat shock protein 70 (Hsp70), an abundant cytosolic chaperone, into the replicase complex. By taking advantage of yeast model host, we demonstrate that the four-member SSA subfamily of HSP70 genes is essential for TBSV replication. The constitutively expressed SSA1 and SSA2, which are resident proteins in the viral replicase, can be complemented by the heat-inducible SSA3 and/or SSA4 for TBSV replication. Using a yeast strain carrying a temperature sensitive ssa1(ts), but lacking functional SSA2/3/4, we show that inactivation of Ssa1p(ts) led to a defect in membrane localization of the viral replication proteins, resulting in cytosolic distribution of the viral proteins and lack of replicase activity. An in vitro replicase assembly assay with Ssa1p(ts) revealed that functional Ssa1p is required during the replicase assembly process, but not during minus- or plus-strand synthesis. Temperature shift experiments from nonpermissive to permissive in ssa1(ts) yeast revealed that the re-activated Ssa1p(ts) could promote efficient TBSV replication in the absence of other SSA genes. We also demonstrate that the purified recombinant Ssa3p can facilitate the in vitro assembly of the TBSV replicase on yeast membranes, demonstrating that Ssa3p can fully complement the function of Ssa1p. Taken together, the cytosolic SSA subfamily of Hsp70 proteins play essential and multiple roles in TBSV replication.

A Key Role for Heat Shock Protein 70 in the Localization and Insertion of Tombusvirus Replication Proteins to Intracellular Membranes

Plus-stranded RNA viruses coopt host proteins to promote their robust replication in infected hosts. Tomato bushy stunt tombusvirus (TBSV) is a model virus that can replicate a small replicon RNA in Saccharomyces cerevisiae and in plants. The tombusvirus replicase complex contains heat shock protein 70 (Hsp70), an abundant cytosolic chaperone, which is required for TBSV replication. To dissect the function of Hsp70 in TBSV replication, in this paper we use an Hsp70 mutant (ssa1 ssa2) yeast strain that supports a low level of TBSV replication. Using confocal laser microscopy and cellular fractionation experiments, we find that the localization of the viral replication proteins changes to the cytosol in the mutant cells from the peroxisomal membranes in wild-type cells. An in vitro membrane insertion assay shows that Hsp70 promotes the integration of the viral replication proteins into subcellular membranes. This step seems to be critical for the assembly of the viral replicase complex. Using a gene-silencing approach and quercetin as a chemical inhibitor to downregulate Hsp70 levels, we also confirm the significance of cytosolic Hsp70 in the replication of TBSV and other plant viruses in a plant host. Taken together, our results suggest that cytosolic Hsp70 plays multiple roles in TBSV replication, such as affecting the subcellular localization and membrane insertion of the viral replication proteins as well as the assembly of the viral replicase.

Genetic approaches towards overcoming water deficit in plants - special emphasis on LEAs.

Water deficit arises as a result of low temperature, salinity and dehydration, thereby affecting plant growth adversely and making it imperative for plants to surmount such situations by acclimatizing/adapting at various levels. Water deficit stress results in significant changes in gene expression, mediated by interconnected signal transduction pathways that may be triggered by calcium, and regulated via ABA dependent and/or independent pathways. Hence, adaptation of plants to such stresses involves maintaining cellular homeostasis, detoxification of harmful elements and also growth alterations. Stress in general cause excess production of reactive oxygen species (ROS) and the plants overcome the same by either preventing the accumulation of ROS or by eliminating the ROS formed. Ion homeostasis includes processes such as cellular uptake, sequestration and export in conjunction with long distance transport. Requisite amounts of osmolytes are hence synthesized under stress to maintain turgor along with maintaining the macromolecular structures and also for scavenging ROS. Another noteworthy response is the accumulation of novel proteins, including enzymes involved in the biosynthesis of osmoprotectants, heat-shock proteins (HSPs), late embryogenesis abundant (LEA) proteins, antifreeze proteins, chaperones, detoxification enzymes, transcription factors, kinases and phosphatases. The LEAs belong to a redundant protein family and are highly hydrophilic, boiling-soluble, non-globular and therefore have been defined and classified accordingly. The precise function of LEAs is still unknown, but substantial evidence indicates their involvement in dessication tolerance as the expression of LEAs confers increased resistance to stress in heterologous yeast system and also significantly improves water deficit tolerance in transgenic plants. Genetic manipulation of plants towards conferring abiotic stress tolerance is a daunting task, as the abiotic stress tolerance mechanism is highly complex and various strategies have been exploited to address and evaluate the stress tolerance mechanism, and the molecular responses to water deficit via complex signaling networks. Genomic technologies have recently been useful in integrating the multigenicity of the plant stress responses through, transcriptomics, proteomics and metabolite profilling and their interactions. This review deals with the recent developments on genetic approaches for water stress tolerance in plants, with special emphasis on LEAs.

Immediate Protein Targets of Photodynamic Treatment in Carcinoma Cells

Oxidative stress induced in tumor cells undergoing photodynamic treatment (PDT) leads to extensive modification of many proteins in these cells. Protein oxidation mainly gives rise to formation of carbonyls and oxidized thiols. The immediate targets of PDT-induced protein oxidation in A431 tumor cells have been identified using a proteomic approach involving selective biotinylation, affinity purification and mass spectrometric identification of modified proteins. In all, 314 proteins were shown to undergo PDT-mediated oxidative modifications. While abundant structural proteins and chaperones represented a significant fraction of the carbonylated proteins, labeling of proteins containing oxidized thiols allowed identification of many proteins at low abundance and those involved in signaling and redox homeostasis. On the basis of the identification of these proteins, several likely mechanisms of PDT-induced triggering of apoptosis were put forward. This may not only lead to a further understanding of the complex network of cellular responses to oxidative stress, but it may also help in detailed targeting of photodynamic treatment applied to cancer.

Partners in surveillance and quality control

Partners in surveillance and quality control

Potential roles of abundant extracellular chaperones in the control of amyloid formation and toxicity

The in vivo formation of fibrillar proteinaceous deposits called amyloid is associated with more than 40 serious human diseases, collectively referred to as protein deposition diseases. In many cases the amyloid deposits are extracellular and are found associated with newly identified abundant extracellular chaperones (ECs). Evidence is presented suggesting an important regulatory role for ECs in amyloid formation and disposal in the body. A model is presented which proposes that, under normal conditions, ECs stabilize extracellular misfolded proteins by binding to them, and then guide them to specific cell receptors for uptake and subsequent degradation. Thus ECs and their receptors may be critical parts of a quality control system to protect the body against dangerously hydrophobic proteins/peptides. However, it also appears possible that in the presence of a high molar excess of misfolded protein, such as might occur during disease, the limited amounts of ECs available may actually exacerbate pathology. Further advances in understanding of the mechanisms that control extracellular protein folding are likely to identify new strategies for effective disease therapies.

Toothpicks, Serendipity and the Emergence of the Escherichia coli DnaK (Hsp70) and GroEL (Hsp60) Chaperone Machines

THE purpose of this essay is to retrace some of the early steps that I and a few (then) young geneticists took in the late 1960s and early 1970s to define the Escherichia coli functions used by phage to properly execute their developmental cycle. Eventually, this led to the discovery and functional understanding of the so-called DnaK (Hsp70) and GroEL (Hsp60) molecular chaperone machines, universally conserved among the biological kingdoms (Lindquist and Craig 1988; Georgopoulos and Welch 1993). Now we know that these and other molecular chaperone machines are involved in a multitude of biological processes, including protection of nascent polypeptide chains from premature aggregation, disaggregation of protein aggregates, polypeptide transport across biological membranes, and proteolysis (Bukau and Horwich 1998; Hartl and Hayer-Hartl 2002; Craig et al. 2006). Because protein denaturation and aggregation are enhanced by various environmental stresses, e.g., an increase in temperature, it is not surprising that the DnaK (Hsp70) and GroEL (Hsp60) molecular chaperone machines were also discovered independently as “heat-shock” or “stress” proteins. Because of their very short growth cycles, phage have evolved a variety of strategies to subvert and customize host functions for their own use. Phage likely have a greater need than their hosts for quick and abundant chaperone power to carry out their developmental cycle in a timely fashion. If they fail to complete their cycle before the host lyses, infectious phage progeny will not be released into the medium, risking their extinction. This differential need for chaperone power likely explains why many of the bacterial mutations found to block phage development were eventually shown to be in genes encoding the GroEL and DnaK chaperone machines.

Monitoring chaperone engagement of substrates in the endoplasmic reticulum of live cells

The folding environment in the endoplasmic reticulum (ER) depends on multiple abundant chaperones that function together to accommodate a range of substrates. The ways in which substrate engagement shapes either specific chaperone dynamics or general ER attributes in vivo remain unknown. In this study, we have evaluated how changes in substrate flux through the ER influence the diffusion of both the lectin chaperone calreticulin and an inert reporter of ER crowdedness. During acute changes in substrate load, the inert probe revealed no changes in ER organization, despite significant changes in calreticulin dynamics. By contrast, inhibition of the lectin chaperone system caused rapid changes in the ER environment that could be reversed over time by easing new substrate burden. Our findings provide insight into the normal organization and dynamics of an ER chaperone and characterize the capacity of the ER to maintain homeostasis during acute changes in chaperone activity and availability.

Hsp90 Recognizes a Common Surface on Client Kinases

Hsp90 is a highly abundant chaperone whose clientele includes hundreds of cellular proteins, many of which are central players in key signal transduction pathways and the majority of which are protein kinases. In light of the variety of Hsp90 clientele, the mechanism of selectivity of the chaperone toward its client proteins is a major open question. Focusing on human kinases, we have demonstrated that the chaperone recognizes a common surface in the amino-terminal lobe of kinases from diverse families, including two newly identified clients, NFkappaB-inducing kinase and death-associated protein kinase, and the oncoprotein HER2/ErbB-2. Surface electrostatics determine the interaction with the Hsp90 chaperone complex such that introduction of a negative charge within this region disrupts recognition. Compiling information on the Hsp90 dependence of 105 protein kinases, including 16 kinases whose relationship to Hsp90 is first examined in this study, reveals that surface features, rather than a contiguous amino acid sequence, define the capacity of the Hsp90 chaperone machine to recognize client kinases. Analyzing Hsp90 regulation of two major signaling cascades, the mitogen-activated protein kinase and phosphatidylinositol 3-kinase, leads us to propose that the selectivity of the chaperone to specific kinases is functional, namely that Hsp90 controls kinases that function as hubs integrating multiple inputs. These lessons bear significance to pharmacological attempts to target the chaperone in human pathologies, such as cancer.

Consequences of ERp57 Deletion on Oxidative Folding of Obligate and Facultative Clients of the Calnexin Cycle

Members of the protein-disulfide isomerase superfamily catalyze the formation of intra- and intermolecular disulfide bonds, a rate-limiting step of protein folding in the endoplasmic reticulum (ER). Here we compared maturation of one obligate and two facultative calnexin substrates in cells with and without ERp57, the calnexin-associated, glycoprotein-specific oxidoreductase. ERp57 deletion did not prevent the formation of disulfide bonds during co-translational translocation of nascent glycopolypeptides in the ER. It affected, however, the post-translational phases of oxidative influenza virus hemagglutinin (HA) folding, resulting in significant loss of folding efficiency for this obligate calnexin substrate. Without ERp57, HA also showed reduced capacity to recover from an artificially induced aberrant conformation, thus revealing a crucial role of ERp57 during post-translational reshuffling to the native set of HA disulfides. ERp57 deletion did not affect maturation of the model facultative calnexin substrates E1 and p62 (and of most cellular proteins, as shown by lack of induction of ER stress). ERp72 was identified as one of the ER-resident oxidoreductases associating with the orphan ERp57 substrates to maintain their folding competence.

Regulation of the Dynamics of hsp90 Action on the Glucocorticoid Receptor by Acetylation/Deacetylation of the Chaperone

It is known that inhibition of histone deacetylases (HDACs) leads to acetylation of the abundant protein chaperone hsp90. In a recent study, we have shown that knockdown of HDAC6 by a specific small interfering RNA leads to hyperacetylation of hsp90 and that the glucocorticoid receptor (GR), an established hsp90 "client" protein, is defective in ligand binding, nuclear translocation, and gene activation in HDAC6-deficient cells (Kovacs, J. J., Murphy, P. J. M., Gaillard, S., Zhao, X., Wu, J-T., Nicchitta, C. V., Yoshida, M., Toft, D. O., Pratt, W. B., and Yao, T-P. (2005) Mol. Cell 18, 601-607). Using human embryonic kidney wild-type and HDAC6 (small interfering RNA) knockdown cells transiently expressing the mouse GR, we show here that the intrinsic properties of the receptor protein itself are not affected by HDAC6 knockdown, but the knockdown cytosol has a markedly decreased ability to assemble stable GR.hsp90 heterocomplexes and generate stable steroid binding activity under cell-free conditions. HDAC6 knockdown cytosol has the same ability to carry out dynamic GR.hsp90 heterocomplex assembly as wild-type cytosol. Addition of purified hsp90 to HDAC6 knockdown cytosol restores stable GR.hsp90 heterocomplex assembly to the level of wild-type cytosol. hsp90 from HDAC6 knockdown cytosol has decreased ATP-binding affinity, and it does not assemble stable GR.hsp90 heterocomplexes when it is a component of a purified five-protein assembly system. Incubation of knockdown cell hsp90 with purified HDAC6 converts the hsp90 to wild-type behavior. Thus, acetylation of hsp90 results in dynamic GR.hsp90 heterocomplex assembly/disassembly, and this is manifest in the cell as a approximately 100-fold shift to the right in the steroid dose response for gene activation.

The Structure and Function of Xenopus NO38-Core, a Histone Chaperone in the Nucleolus

Xenopus NO38 is an abundant nucleolar chaperone and a member of the nucleoplasmin (Np) family. Here, we report high-resolution crystal structures of the N-terminal domain of NO38, as a pentamer and a decamer. As expected, NO38 shares the Np family fold. In addition, NO38- and Np-core pentamers each use highly conserved residues and numerous waters to form their respective decamers. Further studies show that NO38 and Np each bind equal amounts of the four core histones. However, NO38 prefers the (H3-H4)(2) tetramer, while Np probably prefers H2A-H2B dimers. We also show that NO38 and Np will each bind noncognate histones when the preferred partner is absent. We suggest that these chaperones must form decamers in order to bind histones and differentiate between histone tetramers and dimers. When taken together, these data imply that NO38 may function as a histone chaperone in the nucleolus.