Hsp90 Complex Research Articles

Hsp90 Directly Modulates the Spatial Distribution of AF9/MLLT3 and Affects Target Gene Expression

AF9/MLLT3 contributes to the regulation of the gene encoding the epithelial sodium channel alpha, ENaCalpha, in renal tubular cells. Specifically, increases in AF9 protein lead to a reduction in ENaCalpha expression and changes in AF9 activity appear to be an important component of aldosterone signaling in the kidney. Whereas AF9 is found in the nucleus where it interacts with the histone H3 lysine 79 methyltransferase, Dot1, AF9 is also present in the cytoplasm. Data presented in this report indicate that the heat shock protein Hsp90 directly and specifically interacts with AF9 as part of an Hsp90-Hsp70-p60/Hop chaperone complex. Experimental manipulation of Hsp90 function by the inhibitor novobiocin, but not 17-AAG, results in redistribution of AF9 from a primarily nuclear to cytoplasmic location. Knockdown of Hsp90 with siRNA mimics the effect elicited by novobiocin. As expected, a shift in AF9 from the nucleus to the cytoplasm in response to Hsp90 interference leads to increased ENaCalpha expression. This is accompanied by a decrease in AF9 occupancy at the ENaCalpha promoter. Our data suggest that the interaction of Hsp90, Hsp70, and p60/Hop with AF9 is necessary for the proper subnuclear localization and activity of AF9. AF9 is among a growing number of nuclear proteins recognized to rely on the Hsp90 complex for nuclear targeting.

Hsp110 Chaperones Control Client Fate Determination in the Hsp70–Hsp90 Chaperone System

Heat shock protein 70 (Hsp70) plays a central role in protein homeostasis and quality control in conjunction with other chaperone machines, including Hsp90. The Hsp110 chaperone Sse1 promotes Hsp90 activity in yeast, and functions as a nucleotide exchange factor (NEF) for cytosolic Hsp70, but the precise roles Sse1 plays in client maturation through the Hsp70-Hsp90 chaperone system are not fully understood. We find that upon pharmacological inhibition of Hsp90, a model protein kinase, Ste11DeltaN, is rapidly degraded, whereas heterologously expressed glucocorticoid receptor (GR) remains stable. Hsp70 binding and nucleotide exchange by Sse1 was required for GR maturation and signaling through endogenous Ste11, as well as to promote Ste11DeltaN degradation. Overexpression of another functional NEF partially compensated for loss of Sse1, whereas the paralog Sse2 fully restored GR maturation and Ste11DeltaN degradation. Sse1 was required for ubiquitinylation of Ste11DeltaN upon Hsp90 inhibition, providing a mechanistic explanation for its role in substrate degradation. Sse1/2 copurified with Hsp70 and other proteins comprising the "early-stage" Hsp90 complex, and was absent from "late-stage" Hsp90 complexes characterized by the presence of Sba1/p23. These findings support a model in which Hsp110 chaperones contribute significantly to the decision made by Hsp70 to fold or degrade a client protein.

S100 proteins regulate the interaction of Hsp90 with Cyclophilin 40 and FKBP52 through their tetratricopeptide repeats

S100 proteins are a subfamily of the EF-hand type calcium sensing proteins, the exact biological functions of which have not been clarified yet. In this work, we have identified Cyclophilin 40 (CyP40) and FKBP52 (called immunophilins) as novel targets of S100 proteins. These immunophilins contain a tetratricopeptide repeat (TPR) domain for Hsp90 binding. Using glutathione-S transferase pull-down assays and immunoprecipitation, we have demonstrated that S100A1 and S100A2 specifically interact with the TPR domains of FKBP52 and CyP40 in a Ca2+-dependent manner, and lead to inhibition of the CyP40–Hsp90 and FKBP52–Hsp90 interactions. These findings have suggested that the Ca2+/S100 proteins are TPR-targeting regulators of the immunophilins–Hsp90 complex formations. Structured summaryMINT-7710442: FKBP52 (uniprotkb:Q02790) physically interacts (MI:0915) with S100A6 (uniprotkb:P06703) by competition binding (MI:0405)MINT-7710192: Cyp40 (uniprotkb:P26882) binds (MI:0407) to S100A1 (uniprotkb:P35467) by pull down (MI:0096)MINT-7710412: Cyp40 (uniprotkb:P26882) physically interacts (MI:0915) with S100A2 (uniprotkb:P29034) by competition binding (MI:0405)MINT-7710374: FKBP52 (uniprotkb:Q02790) binds (MI:0407) to S100A2 (uniprotkb:P29034) by pull down (MI:0096)MINT-7710452: Cyp40 (uniprotkb:P26882) physically interacts (MI:0914) with S100A2 (uniprotkb:P29034) and Hsp90 (uniprotkb:P07900) by anti tag coimmunoprecipitation (MI:0007)MINT-7710387: FKBP52 (uniprotkb:Q02790) binds (MI:0407) to S100A6 (uniprotkb:P06703) by pull down (MI:0096)MINT-7710279: FKBP52 (uniprotkb:Q02790) physically interacts (MI:0915) with S100A1 (uniprotkb:P35467) by competition binding (MI:0405)MINT-7710224: FKBP52 (uniprotkb:Q02790) binds (MI:0407) to Hsp90 (uniprotkb:P07900) by pull down (MI:0096)MINT-7710464: Cyp40 (uniprotkb:P26882) physically interacts (MI:0914) with S100A6 (uniprotkb:P06703) and Hsp90 (uniprotkb:P07900) by anti tag coimmunoprecipitation (MI:0007)MINT-7710249: Cyp40 (uniprotkb:P26882) binds (MI:0407) to Hsp90 (uniprotkb:P07900) by pull down (MI:0096)MINT-7710422: Cyp40 (uniprotkb:P26882) physically interacts (MI:0915) with S100A6 (uniprotkb:P06703) by competition binding (MI:0405)MINT-7710348: Cyp40 (uniprotkb:P26882) binds (MI:0407) to S100A2 (uniprotkb:P29034) by pull down (MI:0096)MINT-7710208: FKBP52 (uniprotkb:Q02790) binds (MI:0407) to S100A1 (uniprotkb:P35467) by pull down (MI:0096)MINT-7710265: Cyp40 (uniprotkb:P26882) physically interacts (MI:0915) with S100A1 (uniprotkb:P35467) by competition binding (MI:0405)MINT-7710361: Cyp40 (uniprotkb:P26882) binds (MI:0407) to S100A6 (uniprotkb:P06703) by pull down (MI:0096)MINT-7710476: FKBP52 (uniprotkb:Q02790) physically interacts (MI:0914) with S100A2 (uniprotkb:P29034) and Hsp90 (uniprotkb:P07900) by anti tag coimmunoprecipitation (MI:0007)MINT-7710316: FKBP52 (uniprotkb:Q02790) physically interacts (MI:0914) with S100A1 (uniprotkb:P35467) and Hsp90 (uniprotkb:P07900) by anti tag coimmunoprecipitation (MI:0007)MINT-7710432: FKBP52 (uniprotkb:Q02790) physically interacts (MI:0915) with S100A2 (uniprotkb:P29034) by competition binding (MI:0405)MINT-7710488: FKBP52 (uniprotkb:Q02790) physically interacts (MI:0914) with S100A6 (uniprotkb:P06703) and Hsp90 (uniprotkb:P07900) by anti tag coimmunoprecipitation (MI:0007)MINT-7710329: S100A6 (uniprotkb:P14069) physically interacts (MI:0914) with FKBP52 (uniprotkb:P30416) and Cyp40 (uniprotkb:Q08752) by anti bait coimmunoprecipitation (MI:0006)MINT-7710295: Cyp40 (uniprotkb:P26882) physically interacts (MI:0914) with Hsp90 (uniprotkb:P07900) and S100A1 (uniprotkb:P35467) by anti tag coimmunoprecipitation (MI:0007)

A Proteomic Investigation of Ligand-dependent HSP90 Complexes Reveals CHORDC1 as a Novel ADP-dependent HSP90-interacting Protein

Structural studies of the chaperone HSP90 have revealed that nucleotide and drug ligands induce several distinct conformational states; however, little is known how these conformations affect interactions with co-chaperones and client proteins. Here we use tandem affinity purification and LC-MS/MS to investigate the proteome-wide effects of ATP, ADP, and geldanamycin on the constituents of the human HSP90 interactome. We identified 52 known and novel components of HSP90 complexes that are regulated by these ligands, including several co-chaperones. Interestingly, our results also show that geldanamycin treatment causes HSP90 complexes to become significantly enriched for core transcription machinery, suggesting that HSP90 inhibition may have broad based effects on transcription and RNA processing. We further characterized a novel ADP-dependent HSP90 interaction with the cysteine- and histidine-rich domain (CHORD)-containing protein CHORDC1. We show that this interaction is stimulated by high ADP:ATP ratios in cell lysates and in vitro with purified recombinant proteins. Furthermore, we demonstrate that this interaction is dependent upon the ability of HSP90 to bind nucleotides and requires the presence of a linker region between the CHORD domains in CHORDC1. Together these findings suggest that the HSP90 interactome is dynamic with respect to nucleotide and drug ligands and that pharmacological inhibition of HSP90 may stimulate the formation of specific complexes.

The Hsp90 Cochaperone, FKBP51, Increases Tau Stability and Polymerizes Microtubules

Imbalanced protein load within cells is a critical aspect for most diseases of aging. In particular, the accumulation of proteins into neurotoxic aggregates is a common thread for a host of neurodegenerative diseases. Our previous work demonstrated that age-related changes to the cellular chaperone repertoire contributes to abnormal buildup of the microtubule-associated protein tau that accumulates in a group of diseases termed tauopathies, the most common being Alzheimer's disease. Here, we show that the Hsp90 cochaperone, FK506-binding protein 51 (FKBP51), which possesses both an Hsp90-interacting tetratricopeptide domain and a peptidyl-prolyl cis-trans isomerase (PPIase) domain, prevents tau clearance and regulates its phosphorylation status. Regulation of the latter is dependent on the PPIase activity of FKBP51. FKB51 enhances the association of tau with Hsp90, but the FKBP51/tau interaction is not dependent on Hsp90. In vitro FKBP51 stabilizes microtubules with tau in a reaction depending on the PPIase activity of FKBP51. Based on these new findings, we propose that FKBP51 can use the Hsp90 complex to isomerize tau, altering its phosphorylation pattern and stabilizing microtubules.

Phosphorylated hamartin–Hsp70 complex regulates apoptosis via mitochondrial localization

The products of the tuberous sclerosis complex (TSC) genes, hamartin and tuberin, form a heterodimer. Recently we reported that hamartin directly interacted with Hsp70. However, the physiological implications of this interaction have not yet been clearly defined. Here we show that hamartin localized to the outer membrane of the mitochondria in an Hsp70-dependent manner. Moreover, phosphorylation of the T417 residue of hamartin was required for its localization to the mitochondria as well as its interaction with Hsp70. A non-phosphorylatable hamartin mutant at residue T417 was unable to localize to the mitochondria and suppress apoptosis, whereas non-phosphorylatable hamartin mutants T357A and T390A localized to the mitochondria and suppressed apoptosis. Importantly, non-phosphorylatable mutants (T357A, T390A and T417A) promoted apoptosis after treatment with Hsp 70-inhibitor KNK437. We conclude that hamartin inhibited apoptosis by localizing to the mitochondria and that its phosphorylation and binding to Hsp70 was required for facilitation of this process.

FKBPL Regulates Estrogen Receptor Signalling and Determines Response to Endocrine Therapy.

Abstract Approximately 40% of patients with ER+ breast cancers do not respond to endocrine therapies; furthermore, most tumours eventually become resistant. We have identified a novel Hsp90 co-chaperone and immunophilin, FKBPL, that binds to Hsp90 and affects the stability and signalling of the estrogen receptor (ER) with implications for breast cancer growth and sensitivity to endocrine therapies.Using co-immunoprecipitations, we have demonstrated that FKBPL interacts within Hsp90 complexes associated with the estrogen receptor in MCF-7 cells. Cells stably overexpressing FKBPL become dependent on estrogen for their growth and even in its presence, FKBPL over-expression slows the rate of proliferation of these cells. More importantly, this dependence on estrogen, rendered FKBPL over-expressing cells dramatically more sensitive (up to 90%) to the anti-estrogens, tamoxifen and fulvestrant. Furthermore, knock-down of FKBPL using a targeted siRNA approach, dramatically increased the resistance of these cells to tamoxifen, supporting a role for FKBPL as a determinant of response to endocrine therapies.We have also identified putative estrogen responsive elements within FKBPL's promoter and show that FKBPL is upregulated in response to estrogen suggesting that FKBPL itself is an estrogen responsive gene. As FKBPL levels increased in response to estrogen, ER levels fell; implicating FKBPL in the stabilisation of ER. This is supported by data demonstrating that FKBPL over-expressing cells exhibit decreased levels of ER and cathepsin D, an estrogen responsive gene, critical to breast cancer growth, survival and invasion. Furthermore, knockdown of FKBPL using an siRNA approach increased ER and cathepsin D levels. The regulation of this ER-responsive gene supports a functional role for FKBPL in physiological ER-mediated signalling. FKBPL is also important for the stabilisation of the cyclin dependent kinase inhibitor, p21 (Jascur et al., 2005). We have seen a dramatic fall in p21 levels when FKBPL is knocked down with a targeted siRNA. Loss of p21 has been associated with a tamoxifen growth inducing phenotype and hyperphosphorylation of ER at S118, and with subsequent increased expression of ER-regulated genes. Here we show that ER phosphorylation is increased in FKBPL knockdown cells and decreased in FKBPL over-expressing cells. Together, these data support a model in which high levels of FKBPL would stabilise p21, reducing ER phosphorylation, abrogating tamoxifen-induced agonist potency and so increase sensitivity to the drug.Finally, having established that FKBPL expression may result in p21-mediated growth arrest and sensitisation to endocrine therapies, we hypothesised that FKBPL may have prognostic value that might impact upon tumour proliferative capacity and improve outcome independent of ER status. This was verified in two publically available breast cancer patient cohorts where we demonstrate that high FKBPL expression was correlated with increased overall survival and distant metastasis-free survival. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5126.

Role of oxidative stress in geldanamycin-induced cytotoxicity and disruption of Hsp90 signaling complex

Heat shock protein 90 (Hsp90) is a chaperone protein regulating PC-12 cell survival by binding and stabilizing Akt, Raf-1, and Cdc37. Hsp90 inhibitor geldanamycin (GA) cytotoxicity has been attributed to the disruption of Hsp90 binding, and the contribution of oxidative stress generated by its quinone group has not been studied in this context. Reactive oxygen species (ROS) and cell survival were assessed in PC-12 cells exposed to GA or menadione (MEN), and Akt, Raf-1, and Cdc37 expression and binding to Hsp90 were determined. GA disrupted Hsp90 binding and increased ROS production starting at 1 h, and cell death occurred at 6 h, inhibited by N-acetylcysteine (NAC) without preventing dissociation of proteins. At 24 h, NAC prevented cytotoxicity and Hsp90 complex disruption. However, MnTBAP antioxidant treatment failed to inhibit GA cytotoxicity, suggesting that NAC acts by restoring glutathione. In contrast, 24 h MEN treatment induced cytotoxicity without disrupting Hsp90 binding. GA and MEN decreased Hsp90-binding protein expression, and proteasomal inhibition prevented MEN-, but not GA-induced degradation. In conclusion, whereas MEN cytotoxicity is mediated by ROS and proteasomal degradation, GA-induced cytotoxicity requires ROS but induces Hsp90 complex dissociation and proteasome-independent protein degradation. These differences between MEN- and GA-induced cytotoxicity may allow more specific targeting of cancer cells.

Opposite roles of metastasin (S100A4) in two potentially tumoricidal mechanisms involving human lymphocyte protein Tag7 and Hsp70

We compare the physical and functional interactions between three widespread multifunctional proteins [metastasin (Mts1/S100A4), innate immunity-related Tag7/PGRP-S, and Hsp70] in two experimental models relevant to host-tumor relationships on humoral and cellular levels. (i) Tag7 and Hsp70 in solution or in a lymphocyte make a stable binary complex that is highly cytotoxic for some tumor cells. Here, we show that Mts1 prevents Tag7.Hsp70 assembly in solution, and an excess of Mts1 disrupts the existing Tag7.Hsp70 complex; accordingly, Tag7.Hsp70 cytotoxicity (exemplified with L929 cells) is diminished in the presence of excess Mts1. (ii) Tag7 exposed on a specialized subset of lymphokine-activated killer cells makes specific contact with Hsp70 exposed on some HLA-negative tumor cells, thus enabling FasL/Fas-mediated induction of apoptosis. Here, we show that some CD4(+)CD25(+) cells coexpose Mts1 with Tag7 and FasL, that Mts1 and Tag7 closely contact the same Hsp70 molecule on the target K562 cell (as evidenced by cross-linking), and that killing of such targets is abolished by Mts1-specific antibodies (or selective removal of Mts1-exposing lymphocytes). Thus, this phenotype active against immunoevasive cancerous cells is defined as CD4(+)CD25(+), FasL(+), Tag7(+)Mts1(+) (approximately 0.5% of total lymphocytes in culture). Remarkably, similar effectors with at least the same activity are often found in fresh donor blood samples (approximately 10(4) effectors/mL). Thus, our models suggest that interactions between the three proteins in different situations may have opposite functional outcomes as regards antitumor defense, immune escape, and metastasis.

Rescue of atypical protein kinase C in epithelia by the cytoskeleton and Hsp70 family chaperones

Atypical PKC (PKC iota) is a key organizer of cellular asymmetry. Sequential extractions of intestinal cells showed a pool of enzymatically active PKC iota and the chaperone Hsp70.1 attached to the apical cytoskeleton. Pull-down experiments using purified and recombinant proteins showed a complex of Hsp70 and atypical PKC on filamentous keratins. Transgenic animals overexpressing keratin 8 displayed delocalization of Hsp70 and atypical PKC. Two different keratin-null mouse models, as well as keratin-8 knockdown cells in tissue culture, also showed redistribution of Hsp70 and a sharp decrease in the active form of atypical PKC, which was also reduced by Hsp70 knockdown. An in-vitro turn motif rephosphorylation assay indicated that PKC iota is dephosphorylated by prolonged activity. The Triton-soluble fraction could rephosphorylate PKC iota only when supplemented with the cytoskeletal pellet or filamentous highly purified keratins, a function abolished by immunodepletion of Hsp70 but rescued by recombinant Hsp70. We conclude that both filamentous keratins and Hsp70 are required for the rescue rephosphorylation of mature atypical PKC, regulating the subcellular distribution and steady-state levels of active PKC iota.

CUDC-305, a Novel Synthetic HSP90 Inhibitor with Unique Pharmacologic Properties for Cancer Therapy

We designed and synthesized CUDC-305, an HSP90 inhibitor of the novel imidazopyridine class. Here, we report its unique pharmacologic properties and antitumor activities in a variety of tumor types. The potency of the compound was analyzed by fluorescence polarization competition binding assay. Its antiproliferative activities were assessed in 40 human cancer cell lines. Its pharmacologic properties and antitumor activities were evaluated in a variety of tumor xenograft models. CUDC-305 shows high affinity for HSP90alpha/beta (IC(50), approximately 100 nmol/L) and HSP90 complex derived from cancer cells (IC(50), 48.8 nmol/L). It displays potent antiproliferative activity against a broad range of cancer cell lines (mean IC(50), 220 nmol/L). CUDC-305 exhibits high oral bioavailability (96.0%) and selective retention in tumor (half-life, 20.4 hours) compared with normal tissues. Furthermore, CUDC-305 can cross blood-brain barrier and reach therapeutic levels in brain tissue. CUDC-305 exhibits dose-dependent antitumor activity in an s.c. xenograft model of U87MG glioblastoma and significantly prolongs animal survival in U87MG orthotopic model. CUDC-305 also displays potent antitumor activity in animal models of erlotinib-resistant non-small cell lung cancer and induces tumor regression in animal models of MDA-MB-468 breast cancer and MV4-11 acute myelogenous leukemia. Correlating with its efficacy in these various tumor models, CUDC-305 robustly inhibits multiple signaling pathways, including PI3K/AKT and RAF/MEK/ERK, and induces apoptosis. In combination studies, CUDC-305 enhances the antitumor activity of standard-of-care agents in breast and colorectal tumor models. CUDC-305 is a promising drug candidate for the treatment of a variety of cancers, including brain malignancies.

W1606 Eupatilin (5,7-Dihydroxy-3,4,6-Trimethoxyflavone) Inhibits the Inflammatory Effects Induced By B. Fragilis Enterotoxin via Dissociating the Complex of Hsp90 and Ikk-γ in Intestinal Epithelial Cells

W1606 Eupatilin (5,7-Dihydroxy-3,4,6-Trimethoxyflavone) Inhibits the Inflammatory Effects Induced By B. Fragilis Enterotoxin via Dissociating the Complex of Hsp90 and Ikk-γ in Intestinal Epithelial Cells

Cdc37 Regulates Ryk Signaling by Stabilizing the Cleaved Ryk Intracellular Domain

Ryk is a Wnt receptor that plays an important role in neurogenesis, neurite outgrowth, and axon guidance. We have reported that the Ryk receptor is cleaved by gamma-secretase and that its intracellular domain (ICD) translocates to the nucleus upon Wnt stimulation. Cleavage of Ryk and its ICD is important for the function of Ryk in neurogenesis. However, the question of how the Ryk ICD is stabilized and translocated into the nucleus remains unanswered. Here, we show that the Ryk ICD undergoes ubiquitination and proteasomal degradation. We have identified Cdc37, a subunit of the molecular chaperone Hsp90 complex, as a Ryk ICD-interacting protein that inhibits proteasomal degradation of the Ryk ICD. Overexpression of Cdc37 increases Ryk ICD levels and promotes its nuclear localization, whereas Cdc37 knockdown reduces Ryk ICD stability. Furthermore, we have discovered that the Cdc37-Ryk ICD complex is disrupted during neural differentiation of embryonic stem cells, resulting in Ryk ICD degradation. These results suggest that Cdc37 plays an essential role in regulating Ryk ICD stability and therefore in Ryk-mediated signal transduction.

The role of molecular chaperones in human misfolding diseases

Human misfolding diseases arise when proteins adopt non-native conformations that endow them with a tendency to aggregate and form intra- and/or extra-cellular deposits. Molecular chaperones, such as Hsp70 and TCP-1 Ring Complex (TRiC)/chaperonin containing TCP-1 (CCT), have been implicated as potent modulators of misfolding disease. These chaperones suppress toxicity of disease proteins and modify early events in the aggregation process in a cooperative and sequential manner reminiscent of their functions in de novo protein folding. Further understanding of the role of Hsp70, TRiC, and other chaperones in misfolding disease is likely to provide important insight into basic pathomechanistic principles that could potentially be exploited for therapeutic purposes.

The HSP90 binding mode of a radicicol-like E-oxime determined by docking, binding free energy estimations, and NMR 15N chemical shifts

We determine the binding mode of a macrocyclic radicicol-like oxime to yeast HSP90 by combining computer simulations and experimental measurements. We sample the macrocyclic scaffold of the unbound ligand by parallel tempering simulations and dock the most populated conformations to yeast HSP90. Docking poses are then evaluated by the use of binding free energy estimations with the linear interaction energy method. Comparison of QM/MM-calculated NMR chemical shifts with experimental shift data for a selective subset of backbone 15N provides an additional evaluation criteria. As a final test we check the binding modes against available structure–activity-relationships. We find that the most likely binding mode of the oxime to yeast HSP90 is very similar to the known structure of the radicicol–HSP90 complex.

Hsp90 and p23 facilitate steroid hormone receptor signaling in yeast

Steroid hormone receptors (SHRs) are among the best‐characterized "client" proteins of Hsp90 complexes. However, effects of Hsp90 and its co‐chaperone on SHR signal transduction have not been examined in a common system. We used a yeast system engineered to express human androgen, estrogen αand ?, glucocorticoid, mineralocorticoid, or progesterone receptors along with relevant reporter genes and tested whether Hsp90 isoproteins and p23 affected SHR signaling. Deletion of the endogenous HSC82 (homolog of human Hsp90 ?) or SBA1 (homolog of human p23) genes in this system strongly inhibited ligand‐mediated signaling of all SHRs in reporter gene assays. HSC82 gene deletion had greater impact on SHR signaling than did SBA1 gene deletions. Deletion of the HSP82 gene (homolog of human Hsp90α) did not affect SHR signaling, suggesting that the Hsc82p‐Sba1p complex specifically supports SHR signaling in this system. Effects of a lack of Hsc82p or Sba1p were restricted to SHR signaling because both cell growth and signaling by an unrelated reporter construct were unaffected by chaperone status. Geldanamycin and radicicol (Hsp90 inhibitors) strongly reduced SHR signaling at doses that did not affect cell growth. Collectively these results indicate that all SHRs, including estrogen receptorβ, are dependent on the Hsp90 chaperone system for proper function. Funded by NCI 1R33CA101622‐01A2.

Mitochondrial carrier protein biogenesis: role of the chaperones Hsc70 and Hsp90.

Metabolite carrier proteins of the mitochondrial inner membrane share homology in their transmembrane domains, which also carries their targeting information. In addition, some carriers have cleavable presequences which are not essential for targeting, but have some other function before import. The cytosolic chaperones Hsc70 (heat-shock cognate 70) and Hsp90 (heat-shock protein 90) complex with carrier precursors and interact specifically with the Tom (translocase of the mitochondrial outer membrane) 70 import receptor to promote import. We analysed how the presequences of the PiC (phosphate carrier) and CIC (citrate carrier) relate to the mechanisms of chaperone-mediated import. Deletion of the PiC presequence reduced the efficiency of import but, notably, not by causing aggregation. Instead, binding of the protein to Hsc70 was reduced, as well as the dependence on Hsc70 for import. Hsp90 binding and function in import was not greatly affected, but it could not entirely compensate for the lack of Hsc70 interaction. Deletion of the presequence from CIC was shown to cause its aggregation, but had little effect on the contribution to import of either Hsc70 or Hsp90. The presequence of PiC, but not that of CIC, conferred Hsc70 binding to dihydrofolate reductase fusion proteins. In comparison, OGC (oxoglutarate carrier) lacks a presequence and was more soluble, though it is still dependent on both Hsc70 and Hsp90. We propose that carrier presequences evolved to improve targeting competence by different mechanisms, depending on physical properties of the precursors in the cytosolic targeting environment.

Evaluation of the Hsp90 inhibitor NVP-AUY922 in multicellular tumour spheroids with respect to effects on growth and PET tracer uptake

Molecular targeting has become a prominent concept in cancer treatment and heat shock protein 90 (Hsp90) inhibitors are suggested as promising anticancer drugs. The Hsp90 complex is one of the chaperones that facilitate the refolding of unfolded or misfolded proteins and plays a role for key oncogenic proteins such as Her2, Raf-1, Akt/PKB, and mutant p53. NVP-AUY922 is a novel low-molecular Hsp90 inhibitor, currently under clinical development as an anticancer drug. Disruption of the Hsp90-client protein complexes leads to proteasome-mediated degradation of client proteins and cell death. The aim of the current study was to use a combination of the multicellular tumour spheroid (MTS) model and positron emission tomography (PET) to investigate the effects of NVP-AUY922 on tumour growth and its relation to PET tracer uptake for the selection of appropriate PET tracer. A further aim was to evaluate the concentration and time dependence in the relation between growth inhibition and PET tracer uptake as part of translational imaging activities. MTS of two breast cancer cell lines (MCF-7 and BT474), one glioblastoma cell line (U87MG) and one colon carcinoma cell line (HCT116) were prepared. Initially, we investigated MTS growth pattern and (3)H-thymidine incorporation in MTS after continuous exposure to NVP-AUY922 in order to determine dose response. Then the short-term effect of the drug on the four PET tracers 2-[(18)F] fluoro-2-deoxyglucose (FDG), 3'-deoxy-3'-fluorothymidine (FLT), methionine and choline was correlated to the long-term effect (changes in growth pattern) to determine the adequate PET tracer with high predictability. Next, the growth inhibitory effect of different dose schedules was evaluated to determine the optimal dose and time. Finally, the effect of a 2-h exposure to the drug on growth pattern and FDG/FLT uptake was evaluated. A dose-dependent inhibition of growth and decrease of (3)H-thymidine uptake was observed with 100% growth cessation in the dose range 7-52 nM and 50% (3)H-thymidine reduction in the range of 10-23 nM, with the most pronounced effect on BT474 cells. The effect of the drug was best detected by FLT. The results suggested that a complete cessation of growth of the viable cell volume was achieved with about 50% inhibition of FLT uptake 3 days after continuous treatment. Significant growth inhibition was observed at all doses and all exposure time spans. Two-hour exposure to NVP-AUY922 generated a growth inhibition which persisted dose dependently up to 10 days. The uptake of FDG per viable tumour volume was reduced by just 25% with 300 nM treatment of the drug, whereas the FLT uptake decreased up to 75% in correlation with the growth inhibition and recovery. Our results indicate a prolonged action of NVP-AUY922 in this cell culture, FLT is a suitable tracer for the monitoring of the effect and a FLT PET study within 3 days after treatment can predict the treatment outcome in this model. If relevant in vivo, this information can be used for efficient planning of animal PET studies and later human PET trial.

Cyclophilin 40: An Hsp90-cochaperone associated with apo-steroid receptors

Cyclophilin 40, a divergent loop cyclophilin first identified in association with the estrogen receptor α, contains a C-terminal tetratricopeptide repeat domain through which it shares structural identity with FK506-binding protein 52 (FKBP52) and other partner cochaperones in steroid receptor-heat shock protein 90 (Hsp90) complexes. By dynamically competing for Hsp90 interaction, the cochaperones allow the receptors to establish distinct Hsp90-chaperone complexes, with the potential to exert tissue-specific control over receptor activity. Cyclophilin 40 regulates Hsp90 ATPase activity during receptor-Hsp90 assembly. Functional deletion of the cyclophilin 40 yeast homologue, Cpr7, adversely affected estrogen receptor α and glucocorticoid receptor activity that could be fully restored, either with wild type Cpr7 or Cpr7 with a cyclophilin domain lacking isomerase activity. We draw parallels with the mechanism already established for FKBP52 and propose that the cyclophilin 40 divergent loop interfaces with a contact surface on the steroid receptor ligand-binding domain to achieve an optimal orientation for receptor activity.

From protein–protein interaction to therapy response: Molecular imaging of heat shock proteins

HSP70 promoter-driven gene therapy and inhibition of HSP90 activity with small molecule inhibitors are two shining points in a newly developed cohort of cancer treatment. For HSP70 promoters, high efficiency and heat inducibility within a localized region make it very attractive to clinical translation. The HSP90 inhibitors exhibit a broad spectrum of anticancer activities due to the downstream effects of HSP90 inhibition, which interfere with a wide range of signaling processes that are crucial for the malignant properties of cancer cells. In this review article, we summarize exciting applications of newly emerged molecular imaging techniques as they relate to HSP, including protein–protein interactions of HSP90 complexes, therapeutic response of tumors to HSP90 inhibitors, and HSP70 promoters-controlled gene therapy. In the HSPs context, molecular imaging is expected to play a vital role in promoting drug development and advancing individualized medicine.