The PTPIP51 TPR-Domain: a novel lipid transfer domain?

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Advances towards understanding the mechanism of action of the Hsp90 Complex

Hsp90 (Heat Shock Protein 90) is an ATP (Adenosine triphosphate) molecular chaperone responsible for the activation and maturation of client proteins. The mechanism by which Hsp90 achieves such activation, involving structurally diverse client proteins, has remained enigmatic. However, recent advances using structural techniques, together with advances in biochemical studies, have not only defined the chaperone cycle but have shed light on its mechanism of action. Hsp90 hydrolysis of ATP by each protomer may not be simultaneous and may be dependent on the specific client protein and co-chaperone complex involved. Surprisingly, Hsp90 appears to remodel client proteins, acting as a means by which the structure of the client protein is modified to allow its subsequent refolding to an active state, in the case of kinases, or by making the client protein competent for hormone binding, as in the case of the GR (glucocorticoid receptor). This review looks at selected examples of client proteins, such as CDK4 (cyclin-dependent kinase 4) and GR, which are activated according to the so-called ‘remodelling hypothesis’ for their activation. A detailed description of these activation mechanisms is paramount to understanding how Hsp90-associated diseases develop

Editorial: the role of heat shock proteins in neuroprotection

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Co-crystalization and in vitro biological characterization of 5-Aryl-4-(5-substituted-2-4-dihydroxyphenyl)-1,2,3-thiadiazole Hsp90 inhibitors

A potential therapeutic strategy for targeting cancer that has gained much interest is the inhibition of the ATP binding and ATPase activity of the molecular chaperone Hsp90. We have determined the structure of the human Hsp90α N-terminal domain in complex with a series of 5-aryl-4-(5-substituted-2-4-dihydroxyphenyl)-1,2,3-thiadiazoles. The structures provide the molecular details for the activity of these inhibitors. One of these inhibitors, ICPD 34, causes a structural change that affects a mobile loop, which adopts a conformation similar to that seen in complexes with ADP, rather than the conformation generally seen with the pyrazole/isoxazole-resorcinol class of inhibitors. Competitive binding to the Hsp90 N-terminal domain was observed in a biochemical assay, and these compounds showed antiproliferative activity and induced apoptosis in the HCT116 human colon cancer cell line. These inhibitors also caused induction of the heat shock response with the upregulation of Hsp72 and Hsp27 protein expression and the depletion of Hsp90 clients, CRAF, ERBB2 and CDK4, thus confirming that antiproliferative activity was through the inhibition of Hsp90. The presence of increased levels of the cleavage product of PARP indicated apoptosis in response to Hsp90 inhibitors. This work provides a framework for the further optimization of thiadiazole inhibitors of Hsp90. Importantly, we demonstrate that the thiadiazole inhibitors display a more limited core set of interactions relative to the clinical trial candidate NVP-AUY922, and consequently may be less susceptible to resistance derived through mutations in Hsp9

HECTD3 mediates an HSP90-dependent degradation pathway for protein kinase clients

Inhibition of the ATPase cycle of the HSP90 chaperone promotes ubiquitylation and proteasomal degradation of its client proteins, which include many oncogenic protein kinases. This provides the rationale for HSP90 inhibitors as cancer therapeutics. However, the mechanism by which HSP90 ATPase inhibition triggers ubiquitylation is not understood, and the E3 ubiquitin ligases involved are largely unknown. Using a siRNA screen, we have identified components of two independent degradation pathways for the HSP90 client kinase CRAF. The first requires CUL5, Elongin B, and Elongin C, while the second requires the E3 ligase HECTD3, which is also involved in the degradation of MASTL and LKB1. HECTD3 associates with HSP90 and CRAF in cells via its N-terminal DOC domain, which is mutationally disrupted in tumor cells with activated MAP kinase signaling. Our data implicate HECTD3 as a tumor suppressor modulating the activity of this important oncogenic signaling pathway

Dihydropyridines allosterically modulate Hsp90 providing a novel mechanism for heat shock protein co-induction and neuroprotection

Chaperones play a pivotal role in protein homeostasis, but with age their ability to clear aggregated and damaged protein from cells declines. Tau pathology is a driver of a variety of neurodegenerative disease and in Alzheimer's disease (AD) it appears to be precipitated by the formation of amyloid-β (Aβ) aggregates. Aβ-peptide appears to trigger Tau hyperphosphorylation, formation of neurofibrillary tangles and neurotoxicity. Recently, dihydropyridine derivatives were shown to upregulate the heat shock response (HSR) and provide a neuroprotective effect in an APPxPS1 AD mouse model. The HSR response was only seen in diseased cells and consequently these compounds were defined as co-inducers since they upregulate chaperones and co-chaperones only when a pathological state is present. We show for compounds tested herein, that they target predominantly the C-terminal domain of Hsp90, but show some requirement for its middle-domain, and that binding stimulates the chaperones ATPase activity. We identify the site for LA1011 binding and confirm its identification by mutagenesis. We conclude, that binding compromises Hsp90's ability to chaperone, by modulating its ATPase activity, which consequently induces the HSR in diseased cells. Collectively, this represents the mechanism by which the normalization of neurofibrillary tangles, preservation of neurons, reduced tau pathology, reduced amyloid plaque, and increased dendritic spine density in the APPxPS1 Alzheimer's mouse model is initiated. Such dihydropyridine derivatives therefore represent potential pharmaceutical candidates for the therapy of neurodegenerative disease, such as AD

Two-colour single-molecule photoinduced electron transfer fluorescence imaging microscopy of chaperone dynamics

Many proteins are molecular machines, whose function is dependent on multiple conformational changes that are initiated and tightly controlled through biochemical stimuli. Their mechanistic understanding calls for spectroscopy that can probe simultaneously such structural coordinates. Here we present two-colour fluorescence microscopy in combination with photoinduced electron transfer (PET) probes as a method that simultaneously detects two structural coordinates in single protein molecules, one colour per coordinate. This contrasts with the commonly applied resonance energy transfer (FRET) technique that requires two colours per coordinate. We demonstrate the technique by directly and simultaneously observing three critical structural changes within the Hsp90 molecular chaperone machinery. Our results reveal synchronicity of conformational motions at remote sites during ATPase-driven closure of the Hsp90 molecular clamp, providing evidence for a cooperativity mechanism in the chaperone’s catalytic cycle. Single-molecule PET fluorescence microscopy opens up avenues in the multi-dimensional exploration of protein dynamics and allosteric mechanisms

Expressed in the yeast Saccharomyces cerevisiae, human ERK5 is a client of the Hsp90 chaperone that complements loss of the Slt2p (Mpk1p) cell integrity stress-activated protein kinase

ERK5 is a mitogen-activated protein (MAP) kinase regulated in human cells by diverse mitogens and stresses but also suspected of mediating the effects of a number of oncogenes. Its expression in the slt2Delta Saccharomyces cerevisiae mutant rescued several of the phenotypes caused by the lack of Slt2p (Mpk1p) cell integrity MAP kinase. ERK5 is able to provide this cell integrity MAP kinase function in yeast, as it is activated by the cell integrity signaling cascade that normally activates Slt2p and, in its active form, able to stimulate at least one key Slt2p target (Rlm1p, the major transcriptional regulator of cell wall genes). In vitro ERK5 kinase activity was abolished by Hsp90 inhibition. ERK5 activity in vivo was also lost in a strain that expresses a mutant Hsp90 chaperone. Therefore, human ERK5 expressed in yeast is an Hsp90 client, despite the widely held belief that the protein kinases of the MAP kinase class are non-Hsp90-dependent activities. Two-hybrid and protein binding studies revealed that strong association of Hsp90 with ERK5 requires the dual phosphorylation of the TEY motif in the MAP kinase activation loop. These phosphorylations, at positions adjacent to the Hsp90-binding surface recently identified for a number of protein kinases, may cause a localized rearrangement of this MAP kinase region that leads to creation of the Hsp90-binding surface. Complementation of the slt2Delta yeast defect by ERK5 expression establishes a new tool with which to screen for novel agonists and antagonists of ERK5 signaling as well as for isolating mutant forms of ERK5

Multi-chaperone function modulation and association with cytoskeletal proteins are key features of the function of AIP in the pituitary gland

Despite the well-recognized role of loss-of-function mutations of the aryl hydrocarbon receptor interacting protein gene (AIP) predisposing to pituitary adenomas, the pituitary-speci c function of this tumor suppressor remains an enigma. To determine the repertoire of interacting partners for the AIP protein in somatotroph cells, wild-type and variant AIP proteins were used for pull-down/quantitative mass spectrometry experiments against lysates of rat somatotropinoma-derived cells; relevant ndings were validated by co-immunoprecipitation and co-localization. Global gene expression was studied in AIP mutation positive and negative pituitary adenomas via RNA microarrays. Direct interaction with AIP was con rmed for three known and six novel partner proteins. Novel interactions with HSPA5 and HSPA9, together with known interactions with HSP90AA1, HSP90AB1 and HSPA8, indicate that the function/ stability of multiple chaperone client proteins could be perturbed by a de cient AIP co-chaperone function. Interactions with TUBB, TUBB2A, NME1 and SOD1 were also identi ed. The AIP variants p.R304* and p.R304Q showed impaired interactions with HSPA8, HSP90AB1, NME1 and SOD1; p.R304* also displayed reduced binding to TUBB and TUBB2A, and AIP-mutated tumors showed reduced TUBB2A expression. Our ndings suggest that cytoskeletal organization, cell motility/adhesion, as well as oxidative stress responses, are functions that are likely to be involved in the tumor suppressor activity of AIP

Structural mechanism for regulation of the AAA-ATPases RUVBL1-RUVBL2 in the R2TP co-chaperone revealed by cryo-EM

The human R2TP complex (RUVBL1-RUVBL2-RPAP3-PIH1D1) is an HSP90 co-chaperone required for the maturation of several essential multiprotein complexes, including RNA polymerase II, small nucleolar ribonucleoproteins, and PIKK complexes such as mTORC1 and ATR-ATRIP. RUVBL1-RUVBL2 AAA-ATPases are also primary components of other essential complexes such as INO80 and Tip60 remodelers. Despite recent efforts, the molecular mechanisms regulating RUVBL1-RUVBL2 in these complexes remain elusive. Here, we report cryo-EM structures of R2TP and show how access to the nucleotide-binding site of RUVBL2 is coupled to binding of the client recruitment component of R2TP (PIH1D1) to its DII domain. This interaction induces conformational rearrangements that lead to the destabilization of an N-terminal segment of RUVBL2 that acts as a gatekeeper to nucleotide exchange. This mechanism couples protein-induced motions of the DII domains with accessibility of the nucleotide-binding site in RUVBL1-RUVBL2, and it is likely a general mechanism shared with other RUVBL1-RUVBL2-containing complexes