C-terminal modulators of heat shock protein of 90 kDa (HSP90): State of development and modes of action

Cells constantly need to adopt to changing environmental conditions, maintaining homeostasis and proteostasis. Heat shock proteins are a diverse class of molecular chaperones that assist proteins in folding to prevent stress-induced misfolding and aggregation. The heat shock protein of 90 kDa (HSP90) is the most abundant heat shock protein. While basal expression of HSP90 is essential for cell survival, in many tumors elevated HSP90 levels are found, which is often associated with bad prognosis. Therefore, HSP90 has emerged as a major target in tumor therapy. The HSP90 machinery is very complex in that it involves large conformational changes during the chaperoning cycle and a variety of co-chaperones. At the same time, this complexity offers a plethora of possibilities to interfere with HSP90 function. The best characterized class of HSP90 modulators are competitive inhibitors targeting the N-terminal ATP-binding pocket. Nineteen compounds of this class entered clinical trials. However, due to severe adverse effects, including induction of the heat shock response, no N-terminal inhibitor has been approved by the FDA so far. As alternatives, compounds commonly referred to as “C-terminal inhibitors” have been developed, either as natural product-based analogues or by rational design, which employ multiple mechanisms to modulate HSP90 function, including modulation of the interaction with co-chaperones, induction of conformational changes that influence the chaperoning cycle, or inhibition of C-terminal dimerization. In this review, we summarize the current development state of characteristic C-terminal inhibitors, with an emphasis on their (proposed) molecular modes of action and binding sites.     

Molecular-level interplay between intrinsically disordered clients and Hsp90

Proteostasis is maintained by a network of molecular chaperones, a prominent member of which is the 90-kilodalton heat shock protein Hsp90. The chaperone function of Hsp90 has been extensively reviewed previously, emphasizing its ATPase activity and remodeling of folded client proteins. Experimental evidence implicating Hsp90 in neurodegenerative diseases has bolstered interest in the noncanonical chaperoning of intrinsically disordered protein (IDPs), however the interplay between Hsp90 and its disordered clients remains poorly understood. In this review we describe recent advances that have contributed to our understanding of the intricate mechanisms characterizing Hsp90-mediated chaperoning of the IDPs tau and α-synuclein and survey emerging insights into the modulation of the chaperone-client interplay in the context of neurodegeneration.     

Chaperoning checkpoint kinase 1 (Chk1), an Hsp90 client, with purified chaperones

Checkpoint kinase 1 (Chk1), a serine/threonine kinase that regulates DNA damage checkpoints, is destabilized when heat shock protein 90 (Hsp90) is inhibited, suggesting that Chk1 is an Hsp90 client. In the present work we examined the interplay between Chk1 and Hsp90 in intact cells, identified a source of unchaperoned Chk1, and report the in vitro chaperoning of Chk1 in reticulocyte lysates and with purified chaperones and co-chaperones. We find that bacterially expressed Chk1 is post-translationally chaperoned to an active kinase. This reaction minimally requires Hsp90, Hsp70, Hsp40, Cdc37, and the protein kinase CK2. The co-chaperone Hop, although not essential for the activation of Chk1 in vitro, enhanced the chaperoning process, whereas the co-chaperone p23 did not stimulate the chaperoning reaction. Additionally, we found that the C-terminal regulatory domain of Chk1 affects the association of Chk1 with Hsp90. Collectively these results provide new insights into Hsp90-dependent chaperoning of a client kinase and identify a novel, biochemically tractable model system that will be useful to further dissect the Hsp90-dependent chaperoning of this important and ubiquitous class of Hsp90 clients.     

Control of steroid receptor function and cytoplasmic-nuclear transport by heat shock proteins.

As targeted proteins that move within the cell, the steroid receptors have become very useful probes for understanding the linked phenomena of protein folding and transport. From the study of steroid receptor-associated proteins it has become clear over the past two years that these receptors are bound to a multiprotein complex containing at least two heat shock proteins, hsp90 and hsp56. Attachment of receptors to this complex in a cell-free system appears to require the protein unfolding/folding activity of a third heat shock protein, hsp70. Like the oncogenic tyrosine kinase pp60src, steroid receptors bind to this complex of chaperone proteins at the time of their translation. Binding of the receptor to the hsp90 component of the system occurs through the hormone binding domain and is under strict hormonal control. The hormone binding domain of the receptor acts as a transferable regulatory unit that confers both tight hormonal control and hsp90 binding onto chimaeric proteins. The model of folding and transport being developed for steroid receptors leads to some general suggestions regarding the folding and transport of targeted proteins in the cell.     

A highly charged sequence of chick hsp90: a good candidate for interaction with steroid receptors

The sequence of the entire chick 90 kDa heat shock protein (hsp90), the non hormone binding component of the heterooligomeric form of steroid receptors, is reported. A comparison of the amino acid sequence of the chick hsp90 to that of the homologous hsp90 from yeast to man, reveals 64-96% identity respectively, and even with E. coli hsp90 an identity of 44% is observed. Analysis of the sequence and a secondary structure prediction of chick hsp90 suggest that two hydrophilic regions A and B, predicted in alpha-helix may play a role in the interaction of hsp90 with other proteins such as steroid hormone receptors. While there are regions of the sequences completely conserved in all hsps90, the most negatively charged hydrophilic region (A) is absent in the E. coli protein.     

On the brotherhood of the mitochondrial chaperones mortalin and heat shock protein 60

The heat shock chaperones mortalin/mitochondrial heat shock protein 70 (mtHsp70) and Hsp60 are found in multiple subcellular sites and function in the folding and intracellular trafficking of many proteins. The chaperoning activity of these 2 proteins involves different structural and functional mechanisms. In spite of providing an excellent model for an evolutionarily conserved molecular "brotherhood", their individual functions, although overlapping, are nonredundant. As they travel to various locations, both chaperones acquire different binding partners and exert a more divergent involvement in tumorigenesis, cellular senescence, and immunology. An understanding of their functional biology may lead to novel designing and development of therapeutic strategies for cancer and aging.     

The role of stress proteins in prostate cancer

The development of therapeutic resistance, after hormone or chemotherapy for example, is the underlying basis for most cancer deaths. Exposure to anticancer therapies induces expression of many stress related proteins, including small heat shock proteins (HSPs). HSPs interact with various client proteins to assist in their folding and enhance the cellular recovery from stress, thus restoring protein homeostasis and promoting cell survival. The vents of cell stress and cell death are linked, as the induction of molecular chaperones appears to function at key regulatory points in the control of apoptosis. On the basis of these observations and on the role of molecular chaperones in the regulation of steroid receptors, kinases, caspases, and other protein remodelling events involved in chromosome replication and changes in cell structure, it is not surprising that molecular chaperones have been implicated in the control of cell growth and in resistance to various anticancer treatments that induce apoptosis. Recently, several molecular chaperones such as Clusterin and HSP27 have been reported to be involved in development and progression of hormone-refractory prostate cancer. In this review, we address some of the molecular and cellular events initiated by treatment induced stress, and discuss the potential role of chaperone proteins as targets for prostate cancer treatment.     

Molecular chaperones and subcellular trafficking of steroid receptors

Unliganded steroid receptors exist as heteromeric complexes comprised of heat shock and immunophilin proteins that associate either directly or indirectly with receptor carboxyl–terminal ligand-binding domains. Molecular chaperons, and other proteins associated with steroid receptors, play an important role in the maturation of receptors to a hormone-binding competent state. Steroid receptor-associated 90 and 70 kDa heat shock proteins, hsp90 and hsp70, respectively, have well established roles in protein folding in addition to participating in numerous subcellular trafficking pathways. In this review, we discuss the possible roles that molecular chaperons, such as hsp90, hsp70 and DnaJ proteins, have in steroid receptor trafficking within two distinct subcellular compartments, i.e. the cytoplasm and nucleus.     

Chaperoning steroidal physiology: lessons from mouse genetic models of Hsp90 and its cochaperones

The molecular chaperone Hsp90 is abundant, ubiquitous, and catholic to biological processes in eukaryotes, controlling phosphorylation cascades, protein stability and turnover, client localization and trafficking, and ligand-receptor interactions. Not surprisingly, Hsp90 does not accomplish these activities alone. Instead, an ever-growing number of cochaperones have been identified, leading to an explosion of reports on their molecular and cellular effects on Hsp90 chaperoning of client substrates. Notable among these clients are many members of the steroid receptor family, such as glucocorticoid, androgen, estrogen and progesterone receptors. Cochaperones typically associated with the mature, hormone-competent states of these receptors include p23, the FK506-binding protein 52 (FKBP52), FKBP51, protein phosphatase 5 (PP5) and cyclophilin 40 (Cyp40). The ultimate relevance of these cochaperones to steroid receptor action depends on their physiological effects. In recent years, the first mouse genetic models of these cochaperones have been developed. This work will review the complex and intriguing phenotypes so far obtained in genetically-altered mice and compare them to the known molecular and cellular impacts of cochaperones on steroid receptors. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

Nuclear localization of two steroid receptor-associated proteins, hsp90 and p59

It has been proposed that the unliganded nontransformed form of steroid hormone receptor is a heterooligomer comprising, in addition to the hormone-binding subunit, two associated proteins: a heat shock protein of MW 90,000 (hsp90) and another protein of MW 59,000 (p59). Using monoclonal antibodies, we demonstrate immunocytochemically the presence of both hsp90 and p59 in cell nuclei of progesterone target cells of the rabbit uterus. While steroid receptors (e.g., progesterone receptors) appear to be exclusively nuclear, we find p59 predominantly in the cell nuclei and hsp90 in both the nucleus and the cytoplasm. In addition, Western blotting of high-salt extracts of nuclear proteins detects the presence of hsp90 and p59 in the nuclei of rabbit uterus. These observations are consistent with the presence of the untransformed heterooligomeric form of steroid hormone receptors in the nuclei of target cells.     

Transformation of glucocorticoid and progesterone receptors to the DNA-binding state

This brief review explores some recent observations relating to the structure of untransformed glucocorticoid and progesterone receptors and the mechanism by which the receptors are transformed to the DNA-binding state. In their molybdatestabilized, untransformed state, progesterone and glucocorticoid receptors exist as a heteromeric 8-9S complex containing one unit of steroid binding phosphoprotein and one or two units of the 90 kD heat shock protein hsp90. When the receptors are transformed, the steroid-binding protein dissociates from hsp90. In cytosol preparations, temperature-mediated dissociation proceeds much more rapidly in the presence of hormone. The dissociated receptor binds to DNA with high affinity, regardless of whether it is in the hormone-bound or the hormone-free state. These observations raise the possibility that the primary, and perhaps the only, role for the hormone is to promote dissociation of the receptor-hsp90 complex. Molybdate, vanadate, and tungstate inhibit receptor transformation to the DNA-binding form, an effect that appears to reflect the ability of these transition metal oxyanions to stabilize the complex between the steroid receptor and hsp90. By promoting the formation of disulfide bonds, hydrogen peroxide also stabilizes the glucocorticoid receptor-hsp90 complex and prevents receptor transformation. A small, heat-stable factor present in all cytosol preparations inhibits receptor transformation, and, when the factor is removed, glucocorticoid receptors are rapidly transformed. This ubiquitous factor has the physical properties of a metal anion, and it is proposed that molybdate and vanadate affect steroid receptor complexes by interacting with a metal anion-binding site that is normally occupied by this endogenous receptor-stabilizing factor.     

A subpopulation of the v-erb A oncogene protein, a derivative of a thyroid hormone receptor, associates with heat shock protein 90.

The v-erb A oncogene of avian erythroblastosis virus is derived from a host gene for a thyroid hormone receptor and is able to block differentiation of erythroid cells and modify the growth properties of fibroblasts. Unlike its host cell progenitor, the v-erb A protein is found in both cytoplasmic and nuclear fractions of the cell. I report here that the cytoplasmic form of the v-erb A protein is associated in a higher molecular weight complex with heat shock protein 90, the same polypeptide found in association with the unliganded steroid receptors and with the soluble forms of the src oncogene.     

Intracellular protozoan parasites of humans: the role of molecular chaperones in development and pathogenesis

Certain kinetoplastid (Leishmania spp. and Tryapnosoma cruzi) and apicomplexan parasites (Plasmodium falciparum and Toxoplasma gondii) are capable of invading human cells as part of their pathology. These parasites appear to have evolved a relatively expanded or diverse complement of genes encoding molecular chaperones. The gene families encoding heat shock protein 90 (Hsp90) and heat shock protein 70 (Hsp70) chaperones show significant expansion and diversity (especially for Leishmania spp. and T. cruzi), and in particular the Hsp40 family appears to be an extreme example of phylogenetic radiation. In general, Hsp40 proteins act as co-chaperones of Hsp70 chaperones, forming protein folding pathways that integrate with Hsp90 to ensure proteostasis in the cell. It is tempting to speculate that the diverse environmental insults that these parasites endure have resulted in the evolutionary selection of a diverse and expanded chaperone network. Hsp90 is involved in development and growth of all of these intracellular parasites, and so far represents the strongest candidate as a target for chemotherapeutic interventions. While there have been some excellent studies on the molecular and cell biology of Hsp70 proteins, relatively little is known about the biological function of Hsp70-Hsp40 interactions in these intracellular parasites. This review focuses on intracellular protozoan parasites of humans, and provides a critique of the role of heat shock proteins in development and pathogenesis, especially the molecular chaperones Hsp90, Hsp70 and Hsp40.     

Association of the heat shock protein hsp90 with steroid hormone receptors and tyrosine kinase oncogene products

Monospecific, polyclonal rabbit antibody raised against the 90-kd non-hormone binding component of molybdate-stabilized steroid hormone receptor specifically recognises the 90-kd molecular weight heat shock protein (hsp 90) in mink cell extracts. Partial proteolytic digestion experiments indicate that this protein is identical to the 90-kd phosphoprotein found in a highly stable complex with the protein products of at least three members of the tyrosine kinase family of oncogenes (src, fes, fgr).     

BAG-1 modulates the chaperone activity of Hsp70/Hsc70.

The 70 kDa heat shock family of molecular chaperones is essential to a variety of cellular processes, yet it is unclear how these proteins are regulated in vivo. We present evidence that the protein BAG-1 is a potential modulator of the molecular chaperones, Hsp70 and Hsc70. BAG-1 binds to the ATPase domain of Hsp70 and Hsc70, without requirement for their carboxy-terminal peptide-binding domain, and can be co-immunoprecipitated with Hsp/Hsc70 from cell lysates. Purified BAG-1 and Hsp/Hsc70 efficiently form heteromeric complexes in vitro. BAG-1 inhibits Hsp/Hsc70-mediated in vitro refolding of an unfolded protein substrate, whereas BAG-1 mutants that fail to bind Hsp/Hsc70 do not affect chaperone activity. The binding of BAG-1 to one of its known cellular targets, Bcl-2, in cell lysates was found to be dependent on ATP, consistent with the possible involvement of Hsp/Hsc70 in complex formation. Overexpression of BAG-1 also protected certain cell lines from heat shock-induced cell death. The identification of Hsp/Hsc70 as a partner protein for BAG-1 may explain the diverse interactions observed between BAG-1 and several other proteins, including Raf-1, steroid hormone receptors and certain tyrosine kinase growth factor receptors. The inhibitory effects of BAG-1 on Hsp/Hsc70 chaperone activity suggest that BAG-1 represents a novel type of chaperone regulatory proteins and thus suggest a link between cell signaling, cell death and the stress response.