Search

Background: Colorectal cancer (CRC) is one of the major causes of cancer-related mortality worldwide. The tumor microenvironment plays a significant role in CRC development, progression and metastasis. Oxidative stress in the colon is a major etiological factor impacting tumor progression. Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial member of the heat shock protein 90 (HSP90) family that is involved in modulating apoptosis in colon cancer cells under oxidative stress. We undertook this study to provide mechanistic insight into the role of TRAP1 under oxidative stress in colon cells., Methods: We first assessed the The Cancer Genome Atlas (TCGA) CRC gene expression dataset to evaluate the expression of TRAP1 and its association with oxidative stress and disease progression. We then treated colon HCT116 cells with hydrogen peroxide to induce oxidative stress and with the TRAP1 inhibitor gamitrinib-triphenylphosphonium (GTPP) to inhibit TRAP1. We examined the cellular proteomic landscape using liquid chromatography tandem mass spectrometry (LC-MS/MS) in this context compared to controls. We further examined the impact of treatment on DNA damage and cell survival., Results: TRAP1 expression under oxidative stress is associated with the disease outcomes of colorectal cancer. TRAP1 inhibition under oxidative stress induced metabolic reprogramming and heat shock factor 1 (HSF1)-dependent transactivation. In addition, we also observed enhanced induction of DNA damage and cell death in the cells under oxidative stress and TRAP1 inhibition in comparison to single treatments and the nontreatment control., Conclusions: These findings provide new insights into TRAP1-driven metabolic reprogramming in response to oxidative stress., Competing Interests: The authors declare no conflict of interest. Given her role as Guest Editor and Editorial Board Member, Ru Chen had no involvement in the peer-review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Alfonso Urbanucci., (© 2023 The Author(s). Published by IMR Press.)

The Hsp90 molecular chaperone is required for the function of hundreds of different cellular proteins. Hsp90 and a cohort of interacting proteins called cochaperones interact with clients in an ATP-dependent cycle. Cochaperone functions include targeting clients to Hsp90, regulating Hsp90 ATPase activity, and/or promoting Hsp90 conformational changes as it progresses through the cycle. Over the last 20 years, the list of cochaperones identified in human cells has grown from the initial six identified in complex with steroid hormone receptors and protein kinases to about fifty different cochaperones found in Hsp90-client complexes. These cochaperones may be placed into three groups based on shared Hsp90 interaction domains. Available evidence indicates that cochaperones vary in client specificity, abundance, and tissue distribution. Many of the cochaperones have critical roles in regulation of cancer and neurodegeneration. A more limited set of cochaperones have cellular functions that may be limited to tissues such as muscle and testis. It is likely that a small set of cochaperones are part of the core Hsp90 machinery required for the folding of a wide range of clients. The presence of more selective cochaperones may allow greater control of Hsp90 activities across different tissues or during development.

Fragment-Based Drug Discovery (FBDD) approaches have gained popularity not only in industry but also in academic research institutes. However, the computational prediction of the binding mode adopted by fragment-like molecules within a protein binding site is still a very challenging task. One of the most crucial aspects of fragment binding is related to the large amounts of bound waters in the targeted binding pocket. The binding affinity of fragments may not be sufficient to displace the bound water molecules. In the present work, we confirmed the importance of the bound water molecules in the correct prediction of the fragment binding mode. Moreover, we investigate whether the use of methods based on explicit solvent molecular dynamics simulations can improve the accuracy of fragment posing. The protein chosen for this study is HSP-90.

Unicellular organisms live under diverse stressful conditions and must respond and adapt quickly to these stresses. When these stresses persist, cells favor a transition to quiescence. There are changes to many processes when cells begin their entry into quiescence. It has been reported that Hsp82 plays an important role in several such processes, and its distribution and activity change according to nutrient conditions. In this study, we found that the subcellular distribution of Hsp82 is regulated by its co-chaperone Ppt1. Under starvation conditions, Ppt1 expression was significantly reduced by a TOR-independent pathway. Furthermore, we found that Ppt1 regulates Hsp82 distribution in the cytoplasm and nucleus by dephosphorylating the S485 residue on Hsp82. The Hsp82 S485A strain has impaired membrane-related protein transport, and its cell size did not become larger in quiescence compared to log phase, resulting in failure to survive during starvation., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Among various solid tumours, gastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide. Expansion into the peritoneal cavity, which results from dissemination of diffuse cancer cells, is the main cause of mortality in gastric adenocarcinoma patients. Therefore, investigation of putative biomarkers involved in metastasis is prerequisite for GC management. In an effort to discover potential tumour markers associated with peritoneal metastasis of GC, a semi-synthetic human scFv library (Tomlinson I) was used to isolate novel antibody fragments recognizing MKN-45, a poorly differentiated diffuse gastric adenocarcinoma cell line. Four rounds of subtractive selection each consisting of extensive pre-absorption of phage library with NIH-3T3 murine embryonic fibroblasts and AGS (a well-differentiated intestinal gastric adenocarcinoma) cell line were carried out prior to positive selection on MKN-45 target cells. ELISA-based screening of 192 phage-displayed scFv clones indicated 21 high-affinity binders with specific staining of MKN-45 compared with AGS cells. Diversity analysis of the selected phage-scFvs resulted in five distinct sequences with multiple frequency. Further analysis by ELISA and flow cytometry verified three clones that specifically recognized MKN-45 cells. Liquid chromatography-mass spectrometry analysis of the scFv-immunoprecipitated proteins has led to identification of c-Met, HSP90 α and HSP90 β as candidate biomarkers associated with diffuse GC. Immunohistochemistry revealed the capability of purified scFvs to differentiate diffuse and intestinal gastric adenocarcinoma. Taken together, the isolated MKN-45-specific scFv fragments and their cognate antigens would be beneficial in screening and management as well as targeting and therapy of the diffuse gastric adenocarcinoma., (© 2019 John Wiley & Sons Ltd.)

Background: Calcific aortic valve disease (CAVD) is an atheroinflammatory process; finally it leads to progressive calcification of the valve. There is no effective pharmacological treatment for CAVD and many of the underlying molecular mechanisms remain unknown. We conducted a proteomic study to reveal novel factors associated with CAVD., Methods: We compared aortic valves from patients undergoing valvular replacement surgery due to non-calcified aortic insufficiency (control group, n = 5) to a stenotic group (n = 7) using two-dimensional difference gel electrophoresis (2D-DIGE). Protein spots were identified with mass spectrometry. Western blot and immunohistochemistry were used to validate the results in a separate patient cohort and Ingenuity Pathway Analysis (IPA) was exploited to predict the regulatory network of CAVD., Results: We detected an upregulation of complement 9 (C9), serum amyloid P-component (APCS) and transgelin as well as downregulation of heat shock protein (HSP90), protein disulfide isomerase A3 (PDIA3), annexin A2 (ANXA2) and galectin-1 in patients with aortic valve stenosis. The decreased protein expression of HSP90 was confirmed with Western blot., Conclusions: We describe here a novel data set of proteomic changes associated with CAVD, including downregulation of the pro-inflammatory cytosolic protein, HSP90.

Purpose: Myocardial dysfunction is common in sepsis but optimal treatment strategies are unclear. The inodilator, levosimendan was suggested as a possible therapy; however, the levosimendan to prevent acute organ dysfunction in Sepsis (LeoPARDS) trial found it to have no benefit in reducing organ dysfunction in septic shock. In this study we evaluated the effects of levosimendan in patients with and without biochemical cardiac dysfunction and examined its non-inotropic effects., Methods: Two cardiac biomarkers, troponin I (cTnI) and N-terminal prohormone of brain natriuretic peptide (NT-proBNP), and five inflammatory mediators were measured in plasma from patients recruited to the LeoPARDS trial at baseline and over the first 6 days. Mean total Sequential Organ Failure Assessment (SOFA) score and 28-day mortality were compared between patients with normal and raised cTnI and NT-proBNP values, and between patients above and below median values., Results: Levosimendan produced no benefit in SOFA score or 28-day mortality in patients with cardiac dysfunction. There was a statistically significant treatment by subgroup interaction (p = 0.04) in patients with NT-proBNP above or below the median value. Those with NT-proBNP values above the median receiving levosimendan had higher SOFA scores than those receiving placebo (mean daily total SOFA score 7.64 (4.41) vs 6.09 (3.88), mean difference 1.55, 95% CI 0.43-2.68). Levosimendan had no effect on the rate of decline of inflammatory biomarkers., Conclusion: Adding levosimendan to standard care in septic shock was not associated with less severe organ dysfunction nor lower mortality in patients with biochemical evidence of cardiac dysfunction.

Kinases, which function in numerous cell signaling processes, are among the best characterized groups of client proteins for the 90-kDa heat shock protein (HSP90), a molecular chaperone that suppresses the aggregation and maintains the proper folding of its substrate proteins (i.e., clients). No high-throughput proteomic method, however, has been developed for the characterizations of the interactions between HSP90 and the human kinome. Herein, by employing a parallel-reaction monitoring (PRM)-based targeted proteomic method, we found that 99 out of the 249 detected kinase proteins display diminished expression in cultured human cells upon treatment with ganetespib, a small-molecule HSP90 inhibitor. PRM analysis of kinase proteins in the affinity pull-down samples showed that 86 out of the 120 detected kinases are enriched from the CRISPR-engineered cells where a tandem affinity tag was conjugated with the C-terminus of endogenous HSP90β protein over the parental cells. Together, our results from the two complementary quantitative proteomic experiments offer systematic characterizations about the HSP90-kinase interactions at the entire proteome scale and reveal extensive interactions between HSP90 and kinase proteins in human cells.

The anti-malarial drug artemisinin (ART) possesses potent anti-inflammatory activity, yet its underlying mechanism of action has remained elusive. Here we employed quantitative chemical proteomics to in situ profile the cellular targets of ART and identified heat shock protein 90 (HSP90) as a direct target. Further study revealed that ART suppressed the production of nitric oxide (NO) in macrophages via inhibiting the interaction between HSP90 and inducible NO synthase (iNOS).

Background: Carcinogenesis occurs in elastin-rich tissues and leads to local inflammation and elastolytic proteinase release. This contributes to bioactive matrix fragment (Matrikine) accumulation like elastin degradation products (EDP) stimulating tumour cell invasive and metastatic properties. We previously demonstrate that EDPs exert protumoural activities through Hsp90 secretion to stabilised extracellular proteinases., Methods: EDP influence on cancer cell blebbing and extracellular vesicle shedding were examined with a videomicroscope coupled with confocal Yokogawa spinning disk, by transmission electron microscopy, scanning electron microscopy and confocal microscopy. The ribosomal protein SA (RPSA) elastin receptor was identified after affinity chromatography by western blotting and cell immunolocalisation. mRNA expression was studied using real-time PCR. SiRNA were used to confirm the essential role of RPSA., Results: We demonstrate that extracellular matrix degradation products like EDPs induce tumour amoeboid phenotype with cell membrane blebbing and shedding of extracellular vesicle containing Hsp90 and proteinases in the extracellular space. EDPs influence intracellular calcium influx and cytoskeleton reorganisation. Among matrikines, VGVAPG and AGVPGLGVG peptides reproduced EDP effects through RPSA binding., Conclusions: Our data suggests that matrikines induce cancer cell blebbing and extracellular vesicle release through RPSA binding, favouring dissemination, cell-to-cell communication and growth of cancer cells in metastatic sites.

The early diagnosis of liver cancer by target biomarkers is of great significance for improving the survival rate of cancer patients. However, it is still a challenging task to sensitively detect circulating protein biomarkers due to decreased binding activity of antibodies originating from uncontrolled orientation of immobilization on the surface of a solid matrix. In this work, a novel immunoaffinity probe, Fe 3 O 4 @TpBD-DSS-Ab-MEG, based on magnetic COFs with ordered arrangement of anchored antibodies has been developed and applied for the first time to detection of a cancer biomarker, heat shock protein 90alpha (Hsp90α). The fabricated composites possess favorable features from magnetic cores and COF shells, including strong magnetic responses (7.96 emu g -1 ), ordered active groups, a large amount of immobilized antibodies (111.7 μg/mg), good solvent and thermal stability. Fe 3 O 4 @TpBD-DSS-Ab-MEG demonstrated low detection limit (50 pg/mL), high selectivity (Hsp90α:BSA = 1:1000), desirable repeatability and good stability for Hsp90α immunocapture. Compared with other immunoprobes, our materials showed higher selectivity and sensitivity, which were mainly attributed to regular arrays of surface antibodies. Furthermore, samples containing Hsp90α at the concentration of 1 µg/mL in human plasma were used to test our immunoprobe, and 2 peptides of Hsp90α were successfully observed. The proposed non-invasive immunoassay strategy offers enhanced ability to control the orientation of immobilized antibodies and great promise for accurate analysis of the liver cancer biomarker Hsp90α in a complicated biological matrix. In addition, the facile preparation of magnetic COFs support and the satisfactory analytical performance made the newly developed immunoprobe a potential tool for sensitive detection of other cancer biomarkers in clinical diagnosis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Heat shock protein 90 is an ATP-dependent molecular chaperone important for folding, maturation and clearance of aberrantly expressed proteins and is abundantly expressed (1-2% of all proteins) in the cytosol of all normal cells. In some tumour cells, however, strong expression of HSP90 is also observed on the cell membrane and in the extracellular matrix and the affinity of tumoural HSP90 for ATP domain inhibitors was reported to increase over 100-fold compared to that of HSP90 in normal cells. Here, we explore [ 11 C]NMS-E973 as a PET tracer for in vivo visualisation of HSP90 and as a potential tool for in vivo quantification of occupancy of HSP90 inhibitors. Methods: HSP90 expression was biochemically characterized in a panel of established cell lines including the melanoma line B16.F10. B16.F10 melanoma xenograft tumour tissue was compared to non-malignant mouse tissue. NMS-E973 was tested in vitro for HSP90 inhibitory activity in several tumour cell lines. HSP90-specific binding of [ 11 C]NMS-E973 was evaluated in B16.F10 melanoma cells and B16.F10 melanoma, prostate cancer LNCaP and PC3, SKOV-3 xenograft tumour slices and in vivo in a B16.F10 melanoma mouse model. Results: Strong intracellular upregulation and abundant membrane localisation of HSP90 was observed in the different tumour cell lines, in the B16.F10 tumour cell line and in B16.F10 xenograft tumours compared to non-malignant tissue. NMS-E973 showed HSP90-specific inhibition and reduced proliferation of cells. [ 11 C]NMS-E973 showed strong binding to B16.F10 melanoma cells, which was inhibited by 200 µM of PU-H71, a non-structurally related HSP90 inhibitor. HSP90-specific binding was observed by in vitro autoradiography of murine B16.F10 melanoma, LNCaP and PC3 prostate cancer and SKOV-3 ovary carcinoma tissue slices. Further, B16.F10 melanoma-inoculated mice were subjected to a µPET study, where the tracer showed fast and persistent tumour uptake. Pretreatment of B16.F10 melanoma mice with PU-H71 or Ganetespib (50 mg/kg) completely blocked tumour accumulation of [ 11 C]NMS-E973 and confirmed in vivo HSP90 binding specificity. HSP90-specific binding of [ 11 C]NMS-E973 was observed in blood, lungs and spleen of tumour-bearing animals but not in control animals. Conclusion: [ 11 C]NMS-E973 is a PET tracer for in vivo visualisation of tumour HSP90 expression and can potentially be used for quantification of HSP90 occupancy. Further translational evaluation of [ 11 C]NMS-E973 is warranted., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Background: Hsp90-beta has been investigated to be correlated with the occurrence and development of tumor. The intention of this research was to test the level of Hsp90-beta in malignant pleural effusion (MPE) of patients with lung cancer and disclose the clinical significance of Hsp90-beta as a potential tumor marker for differential diagnosis of pleural effusion caused by lung cancer., Methods: The level of Hsp90-beta was determined using enzyme-linked immunosorbent assay. Calculations of the Hsp90-beta threshold, the sensitivity and specificity for distinguishing MPE from benign pleural effusion were performed using receiver operator characteristic curve., Results: The level of Hsp90-beta in MPE of lung cancer patients was higher than that in control individuals (P < 0.05) and increased MPE Hsp90-beta was correlated with the pathological differentiation, tumor size and lymphatic metastasis (P < 0.05). The cutoff value of Hsp90-beta produced by receiver operator characteristic curve for distinguishing lung cancer from control individuals were 1.659 ng/mL and the sensitivity and specificity were 93.46 and 79%., Conclusions: Increased Hsp90-beta in MPE was correlated with malignant biological behavior of lung cancer patients, indicating that the level of Hsp90-beta could be a tool of referential value for differential diagnosis of pleural effusion caused by lung cancer.

The 90-kDa heat shock protein (HSP90) is a molecular chaperone that maintains the proper folding of its client proteins including protein kinases and steroid hormone receptors. Helicases are a group of nucleic acid-binding ATPases that can unwind DNA and/or RNA and function in almost every aspect of nucleic acid metabolism. Not much, however, is known about the interactions between HSP90 and helicase proteins. Herein, we developed a parallel-reaction monitoring (PRM)-based targeted proteomic method that allows for quantifying >80% of the human helicase proteome. By employing this method, we demonstrated that a large number of helicase proteins exhibited diminished expression in cultured human cells upon treatment with two small-molecule inhibitors of HSP90. We further introduced a tandem affinity tag to the C-terminus of endogenous HSP90β protein by using the CRISPR-Cas9 genome editing method. Affinity purification followed by LC-PRM analysis revealed an enrichment of 40 out of the 66 quantified helicases from the lysate of cells expressing tagged HSP90β. Together, we developed a high-throughput targeted proteomic method for assessing quantitatively the human helicase proteome, and our results support that helicases may constitute an important group of client proteins for HSP90.

Quantum dots (QDs) have significant potentials in biomedical applications of bioimaging and biosensing. Spontaneous adsorption of proteins on QDs surface is a common phenomenon, which occurred to serum proteins in biological samples, and has been observed to enhance QDs fluorescence. In this study, fluorescence alteration of 3-mercaptopropionic acid (MPA) capped CdSe quantum dots by four individual biomarker proteins was investigated. By monitoring the fluorescence emission of QDs, the biomarker protein adsorbed spontaneously on QDs surface was recognized and quantified. When alpha fetoprotein (AFP) or heat shock protein 90 alpha (HSP90α) were present, the QDs became brighter. The presence of cytochrome C (CytoC) or lysozyme (Lyz) made the QDs dimmer first, and then brighter. Within five minutes response time all four biomarker proteins were detected individually with the estimated detection limit in the range of 1-10 ng/mL and good linear dynamic ranges. The results suggested that the fluorescence of QDs was responsive to not only serum proteins but also biomarker proteins. The fluorescence response was able to correlate quantitatively with the amount of biomarker proteins in relatively low concentrations. These results provide more information to understand QDs and support their applications in biomedical fields., (Published by Elsevier Inc.)

Rationale: Pulmonary arterial hypertension (PAH) is a vascular remodeling disease with a poor prognosis and limited therapeutic options. Although the mechanisms contributing to vascular remodeling in PAH are still unclear, several features, including hyperproliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs), have led to the emergence of the cancer-like concept. The molecular chaperone HSP90 (heat shock protein 90) is directly associated with malignant growth and proliferation under stress conditions. In addition to being highly expressed in the cytosol, HSP90 exists in a subcellular pool compartmentalized in the mitochondria (mtHSP90) of tumor cells, but not in normal cells, where it promotes cell survival., Objectives: We hypothesized that mtHSP90 in PAH-PASMCs represents a protective mechanism against stress, promoting their proliferation and resistance to apoptosis., Methods: Expression and localization of HSP90 were analyzed by Western blot, immunofluorescence, and immunogold electron microscopy. In vitro, effects of mtHSP90 inhibition on mitochondrial DNA integrity, bioenergetics, cell proliferation and resistance to apoptosis were assessed. In vivo, the therapeutic potential of Gamitrinib, a mitochondria-targeted HSP90 inhibitor, was tested in fawn-hooded and monocrotaline rats., Measurements and Main Results: We demonstrated that, in response to stress, HSP90 preferentially accumulates in PAH-PASMC mitochondria (dual immunostaining, immunoblot, and immunogold electron microscopy) to ensure cell survival by preserving mitochondrial DNA integrity and bioenergetic functions. Whereas cytosolic HSP90 inhibition displays a lack of absolute specificity for PAH-PASMCs, Gamitrinib decreased mitochondrial DNA content and repair capacity and bioenergetic functions, thus repressing PAH-PASMC proliferation (Ki67 labeling) and resistance to apoptosis (Annexin V assay) without affecting control cells. In vivo, Gamitrinib improves PAH in two experimental rat models (monocrotaline and fawn-hooded rat)., Conclusions: Our data show for the first time that accumulation of mtHSP90 is a feature of PAH-PASMCs and a key regulator of mitochondrial homeostasis contributing to vascular remodeling in PAH.

Ras signaling in response to environmental cues is critical for cellular morphogenesis in eukaryotes. This signaling is tightly regulated and its activation involves multiple players. Sometimes Ras signaling may be hyperactivated. In C. albicans, a human pathogenic fungus, we demonstrate that dynamics of hyperactivated Ras1 (Ras1G13V or Ras1 in Hsp90 deficient strains) can be reliably differentiated from that of normal Ras1 at (near) single molecule level using fluorescence correlation spectroscopy (FCS). Ras1 hyperactivation results in significantly slower dynamics due to actin polymerization. Activating actin polymerization by jasplakinolide can produce hyperactivated Ras1 dynamics. In a sterol-deficient hyperfilamentous GPI mutant of C. albicans too, Ras1 hyperactivation results from Hsp90 downregulation and causes actin polymerization. Hyperactivated Ras1 co-localizes with G-actin at the plasma membrane rather than with F-actin. Depolymerizing actin with cytochalasin D results in faster Ras1 dynamics in these and other strains that show Ras1 hyperactivation. Further, ergosterol does not influence Ras1 dynamics.

Biological functions in organisms are usually controlled by a set of interacting proteins, and identifying the proteins that interact is useful for understanding the mechanism of the functions. Immunoprecipitation is a method that utilizes the affinity of an antibody to isolate and identify the proteins that have interacted in a biological sample. In this study, the FD-LC-MS/MS method, which involves fluorogenic derivatization followed by separation and quantification by HPLC and finally identification of proteins by HPLC-tandem mass spectrometry, was used to identify proteins in immunoprecipitated samples, using heat shock protein 90 (HSP90) as a model of an interacting protein in HepaRG cells. As a result, HSC70 protein, which was known to form a complex with HSP90, was isolated, together with three different types of HSP90-beta. The results demonstrated that the proposed immunoaffinity-FD-LC-MS/MS method could be useful for simultaneously detecting and identifying the proteins that interact with a certain protein., (Copyright © 2017 John Wiley & Sons, Ltd.)

The molecular chaperone Heat Shock Protein 90 (Hsp90) is essential in eukaryotes. Hsp90 chaperones proteins that are important determinants of multistep carcinogenesis. The chaperone function of Hsp90 is linked to its ability to bind and hydrolyze ATP. Co-chaperones as well as posttranslational modifications (phosphorylation, SUMOylation, and ubiquitination) are important for its stability and regulation of the ATPase activity. Both mammalian and yeast cells can be used to express and purify Hsp90 and also detect its posttranslational modifications by immunoblotting.

Heat Shock Protein 90 (Hsp90) is a ubiquitous molecular chaperone that comprises about 1-3% of the total cellular protein. Over the last decade, Hsp90 has been detected and studied in the extracellular space (extracellular or eHsp90) of normal and neoplastic cells. Once outside the cell, eHsp90 has been shown to interact with extracellular client proteins and promote their stabilization and function. Cell conditioned media are routinely collected to detect and quantify eHsp90, and determine its interactions with extracellular clients. Finally, targeting specifically the eHsp90 with pharmacologic inhibitors or antibodies that are unable to cross the plasma membrane has been beneficial in inhibiting tumor cell motility and invasion.

Bacterial Hsp90 is an ATP-dependent molecular chaperone involved in protein remodeling and activation. The E. coli Hsp90, Hsp90 Ec , collaborates in protein remodeling with another ATP-dependent chaperone, DnaK, the E. coli Hsp70. Both Hsp90 Ec and DnaK hydrolyze ATP and client (substrate) proteins stimulate the hydrolysis. Additionally, ATP hydrolysis by the combination of Hsp90 Ec and DnaK is synergistically stimulated in the presence of client (substrate). Here, we describe two steady-state ATPase assays used to monitor ATP hydrolysis by Hsp90 Ec and DnaK as well as the synergistic stimulation of ATP hydrolysis by the combination of Hsp90 Ec and DnaK in the presence of a client (substrate). The first assay is a spectrophotometric assay based on enzyme-coupled reactions that utilize the ADP formed during ATP hydrolysis to oxidize NADH. The second assay is a more sensitive method that directly quantifies the radioactive inorganic phosphate released following the hydrolysis of [γ- 33 P] ATP or [γ- 32 P] ATP.

Context: Angelica sinensis (Oliv.) Diels (Umbelliferae) (AS) is a well-known Traditional Chinese Medicine (TCM) that enriches and regulates the blood., Objective: An integrated metabonomic and proteomic method was developed and applied to study the blood enrichment effects and mechanisms of AS on blood deficiency (BD) mouse model., Materials and Methods: Forty mice were randomly divided into the control, BD, High-dose of AS (ASH), Middle-dose of AS (ASM), and Low-dose of AS (ASL) groups. BD model mice were established by injecting N-acetylphenylhydrazine (APH) and cyclophosphamide (CTX) (ip). The aqueous extract of AS was administered at three dose of 20, 10, or 5 g/kg b. wt. orally for 7 consecutive days before/after APH and CTX administration. Gas chromatography-mass spectrometry (GC-MS) combined with pattern recognition method and 2D gel electrophoresis (2-DE) proteomics were performed in this study to discover the underlying hematopoietic regulation mechanisms of AS on BD mouse model., Results: Unlike in the control group, the HSP90 and arginase levels increased significantly (p < 0.05) in the BD group, but the levels of carbonic anhydrase, GAPDH, catalase, fibrinogen, GSTP, carboxylesterase and hem binding protein in the BD group decreased significantly (p < 0.05). Unlike the levels in the BD group, the levels of these biomarkers were regulated to a normal state near the control group in the ASM group. Unlike in the control group, l-alanine, arachidonic acid, l-valine, octadecanoic acid, glycine, hexadecanoic acid, l-threonine, butanoic acid, malic acid, l-proline and propanoic acid levels increased significantly (p < 0.05) in the BD group, the levels of d-fructose in the BD group decreased significantly (p < 0.05). The relative concentrations of 12 endogenous metabolites were also significantly affected by the ASL, ASM, and ASH treatments. Notably, most of the altered BD-related metabolites were restored to normal state after ASM administration., Conclusion: AS can promote hematopoietic activities, inhibit production of reactive oxygen species, regulate energy metabolism, increase antiapoptosis, and potentially contribute to the blood enrichment effects of AS against APH- and CTX-induced BD mice.

Boran (n=15) and Nguni (n=15) cows were used in a study to determine the effect of breed, age and coat colour on the concentration of heat shock protein 90 (HSP90AB1), physiological rectal and skin temperature, and markers of health. The cows were exposed to summer heat stress and Boran cows had higher significant (P<0.05) skin temperature (35.1±0.42°C) as compared to the Nguni cows (36.0±0.38°C). Nguni cows had higher body thermal gradients than the Boran cows. Boran cows had thicker skin (P<0.05) and longer hairs (24.3±2.26mm) than their Nguni counterparts (20.2±2.00mm). The HSP90AB1 concentration was increased in Boran cows, although breed had no significant (P>0.05) influence. Significantly (P<0.05) high urea and total cholesterol was recorded in Boran cows. Coat colour had a significant (P<0.05) effect on the weight and rectal temperature of the study animals. Coat colour and age had no significant effect (P>0.05) on the concentration of HSP90AB1, although older cows (≥9 years) had higher concentrations (5.4±1.29ng/ml). Age had a significant (P<0.05) effect on packed cell volume, neutrophil/lymphocyte, urea, total protein and gamma-glutamyl transferase whereas cows with ≥9 years had more concentrations than young ones. Age significantly (P<0.05) influenced hair length, skin temperature and the thermal gradients. Breed was positively correlated (P<0.001) to coat colour, age, body condition score, weight and temperature humidity index while negatively correlated to urea and total cholesterol. It was concluded that Nguni cows were more adaptable to hot environments than the Boran cows as the latter were unable to balance thermal load between their bodies and the environment., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Liver cancer is the fifth most common cancer with extremely low five year survival rate. Early diagnosis is of great importance for cancer therapy. In this work, stable isotope labeling-based relative quantitative proteomics and parallel reaction monitoring-based target proteomics were combined for cancer biomarker screening and validation. By using this strategy, 70 significantly changed proteins in hepatocellular carcinoma tissues were obtained, among which seven proteins were further validated. The validated proteins contain the clinically used hepatocellular carcinoma (HCC) biomarker alpha-fetoprotein (AFP) and the reported biomarker candidates Heat shock protein HSP 90-beta (HSP90), fatty acid-binding protein, epidermal (FABP5) and alcohol dehydrogenase 4 (ADH4), which demonstrated the robustness of the strategy. The proteins identified in this work could be benefit for further HCC biomarker screening and clinical validation. Moreover, this strategy could be further applied to other cancer types.

It is widely accepted that mature sperm contains RNA. The first hypothesis was that sperm RNAs have no functions of their own but are simply residues of spermatogenesis reflecting the events that occurred during their formation in the testes. More recently new discoveries have essentially expanded these views, showing that sperm mRNAs constitute a population of stable full-length transcripts, many of which are selectively retained during spermatogenesis and delivered to oocytes contributing to early embryo development. It is well known that semen quality can be influenced by occasional physical stress, infection, and variation in temperature and the definition of new markers for evaluation of semen could offer knowledge about the fertility potential of a semen sample. The aim of the present study was to evaluate the presence and the relative quantity of transcripts and protein of heat shock protein 70 (HSP70), 90 (HSP90) and clusterin (CLU) in Percoll-selected spermatozoa collected from seven adult boars of proven fertility routinely employed for artificial insemination. Our results showed the presence of HSP70, HSP90 and CLU transcripts with different level of expression: high for HSPs and low for CLU transcripts. The transcript level of both HSPs are similar among selected spermatozoa derived from high quality sperm with the exception of one boar that showed a reduced content of HSP70 and HSP90 mRNA together with a lower semen quality. At protein level, both HSPs were detected with similar amount among all seven boars whilst no band was evidenced for CLU protein.

Analysis of interactions between molecules is of fundamental importance in life science research. In this study, we applied weak affinity chromatography, based on high-performance liquid chromatography and mass spectrometry, as a powerful tool for direct analysis of the components of a chemical reaction mixture for their binding to a target protein. As a demonstration of the potential of this method, we analyzed the binding of the compounds of the reaction mixture to the chaperone heat shock protein 90 (Hsp90). It was possible to analyze quantitatively the binding of the components of the mixture to the target independently from each other without any preceding process such as purification. This feature has wide implications in biological sciences as crude mixtures, either natural or synthetic, can be analyzed directly for their possible binding to a target. This method could lead to savings in costs and labor through shortening chemical research project development time.

HSP90 chaperones are essential regulators of cellular function, as they ensure the appropriate conformation of multiple key client proteins. Four HSP90 isoforms were identified in the protozoan parasite Theileria annulata. Partial characterization was undertaken for three and localization confirmed for cytoplasmic (TA12105), endoplasmic reticulum (TA06470), and apicoplast (TA10720) forms. ATPase activity and binding to the HSP90 inhibitor geldanamycin were demonstrated for recombinant TA12105, and all three native forms could be isolated to varying extents by binding to geldanamycin beads. Because it is essential, HSP90 is considered a potential therapeutic drug target. Resistance to the only specific Theileriacidal drug is increasing, and one challenge for design of drugs that target the parasite is to limit the effect on the host. An in vitro cell culture system that allows comparison between uninfected bovine cells and the T. annulata-infected counterpart was utilized to test the effects of geldanamycin and the derivative 17-AAG. T. annulata-infected cells had greater tolerance to geldanamycin than uninfected cells yet exhibited significantly more sensitivity to 17-AAG. These findings suggest that parasite HSP90 isoform(s) can alter the drug sensitivity of infected host cells and that members of the Theileria HSP90 family are potential targets worthy of further investigation., (©2016 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)

Background: Heat shock protein (HSP90) is a molecular chaperone involved in maintaining protein homeostasis by modulating stability of de novo synthesized proteins. Neoplastic cells with high metabolic rate have higher expression of HSP90 and develop so called "chaperone addiction". Specific inhibition of HSP90 has been therefore discussed as a viable therapeutic strategy and several inhibitors of HSP90 have already entered clinical trials. Recently, a novel role for HSP90 was found on plasma membrane of cancer cells. Since then, extracellular HSP90 has been implicated in increased tumor invasiveness and metastasis, but better understanding of its regulation is needed to fully explore its potential in early detection of malignity and import of specific HSP90 inhibitors. We have therefore analyzed correlation of extracellular HSP90 level with import of fluorescently-labeled inhibitor of HSP90 and total expression of HSP90., Methods: Flow cytometry was used to analyze cell uptake of FITC-Geldanamycin as well as level of extracellular HSP90, while total expression of HSP90 was analyzed by SDS-PAGE and subsequently Western blotting. Data was then subjected to statistical analysis to analyze possible correlation., Results: We have analyzed import of fluorescently labeled HSP90 inhibitor together with total and membrane level of HSP90 on a panel of selected breast carcinoma cell lines (BT-474, BT-549, BT-20, MCF-7, MDA-MB-468, SK-BR-3 a T-47D). Acquired data were subjected to statistical analysis that revealed a correlation between total and membrane level of HSP90 as well as correlation of ectopic HSP90 with uptake of HSP90 inhibitor., Conclusions: Our analysis has shown that import of HSP90 inhibitors is likely dependent on membrane level of HSP90 as well as its total expression, and therefore can potentially reflect HSP90 addiction of cancer cells.Key words: breast neoplasms - HSP90 - heat shock proteins - geldanamycin This work was supported by MEYS - NPS I - LO1413. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 13. 3. 2017Accepted: 26. 3. 2017.

Background: Colorectal cancer is the major cause of cancer mortality, despite development of therapeutic strategies. The novel marker tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial heat shock protein that has been related to drug resistance and protection from apoptosis in colorectal cancer. This study aims to delineate the clinicopathologic significance of TRAP1 expression in colorectal cancer., Methods: Seven-hundred and fourteen FFPE tissues were collected from colorectal cancer patients who underwent surgery from February 2002 to July 2011 at Dong-A University Medical Center, Busan, South Korea. We performed TRAP1 immunohistochemistry using tissue microarray, and divided into two groups, TRAP1 high expression group and low expression group. Statistical analysis was utilized to evaluate the association of TRAP1 with clinicopathologic characteristics and disease-specific survival of patients., Results: High TRAP1 expression was observed in 564 cases (79%) and low expression was 150 cases (21%). TRAP1 expression was significantly increased in colorectal cancer with advanced pathologic T-stage compared with that in early T-stage (p = 0.008). By univariate survival analysis, high TRAP1 expression was significantly associated with worse disease-specific survival (p = 0.01). But, TRAP1 expression was marginally associated with lymph node involvement and tumor differentiation (p = 0.085, p = 0.082, respectively). Multivariate analysis indicated that TRAP1 expression (hazard ratio, 1.947; 95% CI, 1.270 to 2.984; p = 0.002), and pathologic T stage (hazard ratio, 3.190; 95% CI, 1.275 to 7.983; p = 0.013) were independent prognostic factors for colorectal adenocarcinomas., Conclusions: Here, we found that overexpression of TRAP1 might contribute to tumor cell local invasion of colorectal cancer. The association between TRAP1 overexpression and worse disease-specific survival also suggested that TRAP1 protein expression might have oncogenic role. Consequently, our data demonstrated that TRAP1 expression was a good prognostic biomarker for depth of invasion and disease-specific survival in colorectal cancer.

Metabolic reprogramming is one of the most frequent stress-adaptive response of cancer cells to survive environmental changes and meet increasing nutrient requirements during their growth. These modifications involve cellular bioenergetics and cross talk with surrounding microenvironment, in a dynamic network that connect different molecular processes, such as energy production, inflammatory response, and drug resistance. Even though the Warburg effect has long been considered the main metabolic feature of cancer cells, recent reports identify mitochondrial oxidative metabolism as a driving force for tumor growth in an increasing number of cellular contexts. In recent years, oxidative phosphorylation has been linked to a remodeling of inflammatory response due to autocrine or paracrine secretion of interleukines that, in turn, induces a regulation of gene expression involving, among others, molecules responsible for the onset of drug resistance. This process is especially relevant in ovarian cancer, characterized by low survival, high frequency of disease relapse and chemoresistance. Recently, the molecular chaperone TRAP1 (tumor necrosis factor-associated protein 1) has been identified as a key junction molecule in these processes in ovarian cancer: in fact, TRAP1 mediates a metabolic switch toward oxidative phosphorylation that, in turn, triggers cytokines secretion, with consequent gene expression remodeling, finally leading to cisplatin resistance and epithelial-to-mesenchymal transition in ovarian cancer models. This review summarizes how metabolism, chemoresistance, inflammation, and epithelial-to-mesenchymal transition are strictly interconnected, and how TRAP1 stays at the crossroads of these processes, thus shedding new lights on molecular networks at the basis of ovarian cancer., (© 2017 Elsevier Inc. All rights reserved.)

Background/aims: Acetaminophen (APAP) refers to a medication used to manage pain and fever symptoms. Heat shock protein 90 (Hsp90) is to be expressed during various stresses, such as wound healing and tissue remodeling. Recently, it is discovered that Hsp90 is a potential modifier of cytogenesis. In comparison to clinical references of liver damage, this study was designed to assess the potential bioeffect of Hsp90 in APAP-treated livers without conspicuous drug induced liver injury (DILI)., Methods: In our current study, human plasma samples of APAP-used patients were collected for biochemical assays in clinical parameters. Adult male mice were used to investigate the biocharacterization of Hsp90 in APAP-treated livers through serological tests and immunoassays. Further, a mouse liver cell strain was employed in assessment of bioeffect of APAP on hepatocellular Hsp90 expression., Results: Correspondingly, the clinical data showed APAP-administered patients resulted in increased Hsp90 levels in serum when compared to other clinical parameters of liver injury. In adult mice study, APAP-treated livers showed unchanged hepatocellular and metabolic functions, as highlighted in biochemical analysis and immunoassay. Notably, Hsp90 expression in APAP-treated mice were elevated in the serum and liver samples. In quantitative western blot assay, the present data suggested that hepatocellular Hsp90 level was up-regulated followed by APAP treatment. In mouse cell strain study, APAP-treated liver cells had increased trend of aminotransferase contents and apoptotic counts. Further, endogenous Hsp90 expression in APAP-exposed cells was increased dose- and time-dependently., Conclusions: In conclusion, our current findings disclose that Hsp90 biomolecule may be a potential indicator for APAP-induced inconspicuous DILI, in which it seems to be characterized with more sensitive than other diagnosis criteria., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

Background: While there is compelling rationale to use heat shock protein 90 (Hsp90) inhibitors for treatment of advanced prostate cancer, agents that target the N-terminal ATP-binding site of Hsp90 have shown little clinical benefit. These N-terminal binding agents induce a heat shock response that activates compensatory heat shock proteins, which is believed to contribute in part to the agents' lack of efficacy. Here, we describe the functional characterization of two novel agents, SM253 and SM258, that bind the N-middle linker region of Hsp90, resulting in reduced client protein activation and preventing C-terminal co-chaperones and client proteins from binding to Hsp90., Methods: Inhibition of Hsp90 activity in prostate cancer cells by SM253 and SM 258 was assessed by pull-down assays. Cell viability, proliferation and apoptosis were assayed in prostate cancer cell lines (LNCaP, 22Rv1, PC-3) cultured with N-terminal Hsp90 inhibitors (AUY922, 17-AAG), SM253 or SM258. Expression of HSR heat shock proteins, Hsp90 client proteins and co-chaperones was assessed by immunoblotting. Efficacy of the SM compounds was evaluated in human primary prostate tumors cultured ex vivo by immunohistochemical detection of Hsp70 and Ki67., Results: SM253 and SM258 exhibit antiproliferative and pro-apoptotic activity in multiple prostate cancer cell lines (LNCaP, 22Rv1, and PC-3) at low micromolar concentrations. Unlike the N-terminal inhibitors AUY922 and 17-AAG, these SM agents do not induce expression of Hsp27, Hsp40, or Hsp70, proteins that are characteristic of the heat shock response, in any of the prostate cell lines analyzed. Notably, SM258 significantly reduced proliferation within 2 days in human primary prostate tumors cultured ex vivo, without the significant induction of Hsp70 that was caused by AUY922 in the tissues., Conclusions: Our findings provide the first evidence of efficacy of this class of C-terminal modulators of Hsp90 in human prostate tumors, and indicate that further evaluation of these promising new agents is warranted. Prostate 76:1546-1559, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

While the molecular chaperone heat shock protein 90 (HSP90) is involved in a multitude of physiological and pathological processes, its role relating to pulmonary arterial hypertension (PAH) remains unclear. In the present study, we investigated the effect in which HSP90 improves pulmonary arteriole remodeling, and explored the therapeutic utility of targeting HSP90 as therapeutic drug for PAH. By Elisa and immunohistochemistry, HSP90 was found to be increased in both plasma and membrane walls of pulmonary arterioles from PAH patients. Moreover, plasma HSP90 levels positively correlated with mean pulmonary arterial pressure and C-reactive protein. In a monocrotaline-induced rat model of PH, we found that 17-AAG, a HSP90-inhibitor, alleviated the progress of PH, demonstrated by lower pulmonary arterial pressure and absence of right ventricular hypertrophy. Immunohistochemical staining demonstrated that 17-AAG improved pulmonary arteriole remodeling on the basis of reduced wall thickness and wall area. The inflammatory response attributed to PH could be attenuated by 17-AAG through reduction of NF-κB signaling. Moreover, 17-AAG was found to suppress PDGF-stimulated proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) through induction of cell cycle arrest in the G1 phase. In conclusion, HSP90 inhibitor 17-AAG could improve pulmonary arteriole remodeling via inhibiting the excessive proliferation of PASMCs, and inhibition of HSP90 may represent a therapeutic avenue for the treatment of PAH.

The objective of this study was to analyze the expression and clinical role of 13 signaling molecules in a large cohort of breast carcinoma patients with long follow-up period. Breast carcinomas (n=410) were analyzed for protein expression of phosphorylated mitogen-activated protein kinases (p-ERK, p-JNK, p-p38) and phosphoinositide 3-kinase signaling pathway proteins (p-AKT, p-mTOR, p-p70S6K); the BAG family proteins BAG-1 and BAG-4/SODD; the antiapoptotic protein Bcl-2; the inhibitor of apoptosis family member Survivin; and the heat shock protein family members HSP27, HSP70, and HSP90. Protein expression was studied for association with clinicopathological parameters and survival. Significantly higher expression of p-AKT (P<.001), p-mTOR (P<.001), p-p70S6K (P<.001), Bcl-2 (P<.001), BAG-4/SODD (P<.001), HSP27 (P<.001), HSP70 (P=.012), HSP90 (P<.001), and Survivin (P=.004) was found in infiltrating ductal and lobular carcinomas compared to mucinous carcinomas. Bcl-2 expression was significantly higher in grades 1 and 2 compared to grade 3 carcinomas (P<.001). p-AKT expression was higher in tumors more than 2cm (P=.027), whereas p-mTOR expression was lowest in tumors more than 5cm (P=.019). Higher BAG-4/SODD, HSP70, and HSP90 expression was associated with poor overall survival (P=.016, P=.039, and P=.023, respectively) in univariate analysis, whereas the only independent prognosticator in Cox multivariate survival analysis was tumor diameter (P=.003). In conclusion, BAG-4/SODD, HSP70, and HSP90 are potential prognostic markers in node-negative breast carcinoma that merit further research., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The pathogenesis of many tumors, including brain tumors, has been associated with hypoxia, which induces the transcriptional activity of hypoxia-inducible Factor-1α (HIF-1α). HIF-1α is normally degradated by the von Hippel-Lindau protein (pVHL) but, in hypoxia, pVHL/HIF-1α interaction is inhibited resulting in the nuclear accumulation of HIF-1α. Hsp90 (Heat shock protein 90), as a chaperone protein, plays a critical role for both stabilization of HIF-1α and degradation of pVHL. The aim of this study was to estimate immunohistochemically the expression levels of HIF-1α and pVHL, in relation to Hsp90, in different types of human brain tumors (42 gliomas, 9 medulloblastomas, and 38 meningiomas) using specific antibodies. The tumors were further divided into two groups according to the age of patients (≥19 years old or ⟨19 years old). Nuclear, for HIF-1α, and cytoplasmic, for pVHL and Hsp90, localization was detected in a high percentage of tumor cells in the majority of tumors. In astrocytomas, a significant, grade-dependent relationship for HIF-1α immunoexpression was observed (p⟨0.05). Furthermore, there was a significant correlation between pVHL and Hsp90 immunoexpression (p⟨0.01). The group of ≥19 years old patients with glioblastomas (WHO grade IV) demonstrated significantly increased immunoexpression for HIF-1α compared to pVHL (p⟨0.0001) and Hsp90 expression (p⟨0.01). In medulloblastomas, a significant correlation of HIF-1α with Hsp90 immunoexpression (p⟨0.05) was found. In meningiomas, no significant correlation for the expression of the three proteins was detected (p≥0.05). These results indicate that HIF-1α/pVHL/Hsp90 interactions may be implicated in biology of different types of brain tumors through different signaling mechanisms.

Introduction and Objectives: HSP-90 is an intracellular protein that protects the cell from environmental stress situations. The overexpression of HSP-90 isoforms could serve as a mechanism of resistance to radiotherapy for tumour cells. We studied this effect in a sample of head and neck tumours., Methods: We included 87 patients diagnosed with oral cavity, oropharynx, larynx and hypopharynx tumours. We studied the expression of the HSP-90 isoforms by real-time PCR on pre-treatment biopsy samples. We analysed the relationship between HSP-90 expression levels and local relapse of the tumour with CRT decision trees., Results: The expression levels of the inducible citosolic isoform (HSP90AA) allowed the definition of 2 groups of patients with different rates of local relapse. The group with a low expression level showed a 2.9% local relapse rate, while the group with a high expression level showed a 38.2% rate. Survival curves showed differences in time to local relapse for both groups of patients. These differences did not reach statistical significance., Conclusions: Radiotherapy response was related to expression levels of HSP-90 in a sample of head and neck cancer patients. This result could prove useful in the selection of treatments for this group of patients., (Copyright © 2015 Elsevier España, S.L.U. y Sociedad Española de Otorrinolaringología y Cirugía de Cabeza y Cuello. All rights reserved.)

The genes encoding HSP70 and HSP90 proteins were isolated from kaluga by homologous cloning and rapid amplification of complementary DNA (cDNA) ends (RACE). HSP70 (GenBank accession no. KP050541) and HSP90 (GenBank accession no. KP050542) cDNAs were composed of 2275 and 2718 bp and encoded polypeptides of 650 and 725 amino acids, respectively. Basic Local Alignment Search Tool (BLAST) analysis showed that HSP70 and HSP90 of kaluga shared high identities with those of Acipenser ruthenus, Acipenser schrenckii, and Acipenser baerii (98-99 %). Fluorescent real-time RT-PCR under unstressed conditions revealed that HSP70 and HSP90 were expressed in 11 different tissues of kaluga. Messenger RNA (mRNA) expressions of both HSP70 and HSP90 were highest in the intestine and lowest in the muscle. In addition, the patterns of mRNA expression of HSP70 and HSP90 were similar, although the level of expression was more in HSP90 than in HSP70 (P < 0.05).We also analyzed patterns of HSP70 and HSP90 expression in the muscle, gill, and liver of kaluga under different combinations of temperature and salinity stress, including temperatures of 4,10, 25, and 28 °C at 0 ppt salinity, and salinities of 10, 20, 30, and 40 ppt at 16 °C, where 16 °C at 0 ppt (parts per thousand) served as the control. We found that levels of mRNA expression of both HSP70 and HSP90 were highest at 4 °C in the muscle, gill, and liver and changed little with salinity stress. These results increase understanding of the mechanisms of stress response of cold freshwater fish.

Land snails are exposed to conditions of high ambient temperature and low humidity, and their survival depends on a suite of morphological, behavioral, physiological, and molecular adaptations to the specific microhabitat. We tested in six populations of the land snail Theba pisana whether adaptations to different habitats affect their ability to cope with thermal stress and their strategies of heat shock protein (HSP) expression. Levels of Hsp70 and Hsp90 in the foot tissue were measured in field-collected snails and after acclimation to laboratory conditions. Snails were also exposed to various temperatures (32 up to 54 °C) for 2 h and HSP messenger RNA (mRNA) levels were measured in the foot tissue and survival was determined. To test whether the physiological and molecular data are related to genetic parameters, we analyzed T. pisana populations using partial sequences of nuclear and mitochondrial DNA ribosomal RNA genes. We show that populations collected from warmer habitats were more thermotolerant and had higher constitutive levels of Hsp70 isoforms in the foot tissue. Quantitative real-time polymerase chain reaction (PCR) analysis indicated that hsp70 and hsp90 mRNA levels increased significantly in response to thermal stress, although the increase in hsp70 mRNA was larger compared to hsp90 and its induction continued up to higher temperatures. Generally, warm-adapted populations had higher temperatures of maximal induction of hsp70 mRNA synthesis and higher upper thermal limits to HSP mRNA synthesis. Our study suggests that Hsp70 in the foot tissue of T. pisana snails may have important roles in determining stress resistance, while Hsp90 is more likely implicated in signal transduction processes that are activated by stress. In the phylogenetic analysis, T. pisana haplotypes were principally divided into two major clades largely corresponding to the physiological ability to withstand stress, thus pointing to genetically fixed tolerance.

The chaperome is a large and diverse protein machinery composed of chaperone proteins and a variety of helpers, such as the co-chaperones, folding enzymes, and scaffolding and adapter proteins. Heat shock protein 90s and 70s (HSP90s and HSP70s), the most abundant chaperome members in human cells, are also the most complex. As we have learned to appreciate, their functions are context dependent and manifested through a variety of conformations that each recruit a subset of co-chaperone, scaffolding, and folding proteins and which are further diversified by the posttranslational modifications each carry, making their study through classic genetic and biochemical techniques quite a challenge. Chemical biology tools and techniques have been developed over the years to help decipher the complexities of the HSPs and this review provides an overview of such efforts with focus on HSP90 and HSP70., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: To investigate the expression and clinical significance of TRAP1 (tumor necrosis factor receptor-associated protein 1) in kidney cancer., Methods: TRAP1 expression was detected in kidney cancer and normal kidney tissues by qRT-PCR and immunohistochemistry (IHC), respectively. Then, the correlation of TRAP1 expression with clinicopathological characters and patients' prognosis was evaluated in kidney cancer., Results: IHC results revealed that the high-expression rates of TRAP1 in kidney cancer tissues and normal kidney tissues were 51.3% (41/80), 23.3% (7/30), and the difference was statistically significant (P=0.01). Also, TRAP1 mRNA level in kidney cancer was found to be significantly greater compared with those in normal kidney by qRT-PCR. In addition, TRAP1 expression in kidney cancer significantly correlated with lymph node metastasis and clinical stage (P<0.05). Kaplan-Meier survival analysis indicated that the mean survival time of patients with TRAP1 low-expression was significantly higher (56 months) than those patients with TRAP1 high-expression (47 months). Meanwhile, Kaplan-Meier and Cox survival analysis indicated that TRAP1, lymph node metastasis and clinical stage were correlated with patients' prognosis., Conclusion: TRAP1 is highly expressed in kidney cancer and correlates with patients prognosis, which may be served as a potential marker for the diagnosis and treatment of kidney cancer.

Aims: The aim of this study was to investigate the expression of Hsp90β and GRP94, and elucidate the clinical significance of their expression, in patients with resectable non-small-cell lung cancer (NSCLC)., Methods and Results: Surgical tissue specimens were obtained from 208 patients with NSCLC who underwent surgical resection. The expression levels of Hsp90β and GRP94 were assessed with tissue microarrays and immunohistochemistry. No correlations were observed between Hsp90β or GRP94 expression and several clinicopathological factors. The high-Hsp90β group [median overall survival (OS) 20.4 months; 95% confidence interval (CI) 0.000-40.864] showed a significant decrease in OS as compared with the low-Hsp90β group (median OS not reached; P = 0.003). In contrast to the Hsp90β analysis, the GRP94 analysis did not show a difference in OS. Moreover, in subgroup analyses of patients with squamous cell carcinoma histology, OS (P = 0.012) and relapse-free survival (P = 0.044) were significantly worse in the high-Hsp90β group than in the low-Hsp90β group. Multivariate analysis suggested that old age [hazard ratio (HR) 1.568; 95% CI 1.019-2.412; P = 0.041], advanced disease (HR 2.066; 95% CI 1.218-3.502; P = 0.007) and high Hsp90β expression (HR 1.802; 95% CI 1.061-3.060; P = 0.029) were independent poor prognostic factors for OS., Conclusions: Hsp90β expression might be a useful marker of poor OS, although further large prospective studies are warranted to validate our findings., (© 2015 John Wiley & Sons Ltd.)

Heat shock protein 90-beta (Hsp90-β) is associated with cell proliferation, differentiation and apoptosis and has been investigated as a prognostic factor in many cancers. However, Hsp90-β protein expression in lung adenocarcinoma (ADC) has not been thoroughly elucidated. The aim of this study was to determine the relationship between Hsp90-β expression, clinicopathological parameters and prognosis in lung adenocarcinomas. Seventy-five surgically resected lung adenocarcinomas and matched normal lung tissue samples were obtained to construct a tissue microarray (TMA), including 44 stage IA-IB cases. Then, Hsp90-β protein expression level in lung tissue was evaluated by immunohistochemistry. Kaplan-Meier survival analysis with a Log-rank significance test was used to estimate the survival differences among subgroups according to Hsp90-β expression in lung ADC tissues using SigmaPlot/SigmaStat v10 and 3.5, respectively. Hsp90-β protein expression was significantly upregulated in lung ADC tissues compared to that in the matched normal alveoli (P<0.001) and was associated with tumor differentiation (P<0.001). Furthermore, Hsp90-β over-expression was correlated with poor survival in stage I patients (P=0.026). Increased Hsp90-β expression was associated with reduced overall survival (HR, 2.440; 95% confidence interval, 1.076-5.530; P=0.033). To conclude, our data demonstrated that Hsp90-β protein was over-expressed in lung ADC tumor tissues and was associated with poor outcomes in early stage ADC patients and low pathological grade tumors. These data suggest that Hsp90-β could be a clinically useful biomarker for the prognosis of ADC and an effective anticancer target.

Background: The rate of progression of the disease depends on various factors and the tumor microenvironment takes not the last place among them. One part of researchers argues that the presence of tumor-infiltrating leukocytes serves as a favorable marker of the disease. There exists a completely different point of view on the matter. The investigation of the effects of the inflammatory infiltration on the course of breast cancer process., Methods: We found a pronounced inflammatory infiltration in the tumor microenvironment in 24 cases. Nineteen cases of IDC without inflammatory infiltration were used as a control group. Immunohistochemical reaction showed expression of ERα, PR, HER2/neu, E-cadherin, Hsp90α, Bcl-2, CD3, CD79α, S100 and Myeloperoxidase receptors. Mathematical calculations were done using Microsoft Excel 2010 with 12.0.5 Attestat option., Results: We have determined five variants of immune microenvironment: interstitial, trabecular, nodular, diffuse and mixed. We have established a direct correlation between the expression of ERα and PR and indirect correlation between the receptors of steroid hormones and HER2/neo in both groups of breast cancer. HER2/neo positive tumors in 100% of cases were accompanied by the presence of heat shock proteins. There was a combination of Bcl-2 presence with the steroid receptors expression in 90 % of cases. There was found the indirect correlation between the presence of B lymphocytes and expression of steroid receptors., Conclusions: The presence of B lymphocytes in an inflammatory infiltrate leads to the disappearance of estrogen receptors and progesterone receptors. It provokes the accumulation of Hsp90 in a cell. It contributes to the stabilization of HER2/neu receptors and most proteins that promote tumor progression., Virtual Slides: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1362330168161694.

Liver fibrosis occurring as an outcome of non-alcoholic steatohepatitis (NASH) can precede the development of cirrhosis. We investigated the effects of sorafenib in preventing liver fibrosis in a rodent model of NASH. Adult Sprague-Dawley rats were fed a choline-deficient high-fat diet and exposed to diethylnitrosamine for 6 weeks. The NASH group (n=10) received vehicle and the sorafenib group (n=10) received 2.5 mg·kg(-1)·day(-1) by gavage. A control group (n=4) received only standard diet and vehicle. Following treatment, animals were sacrificed and liver tissue was collected for histologic examination, mRNA isolation, and analysis of mitochondrial function. Genes related to fibrosis (MMP9, TIMP1, TIMP2), oxidative stress (HSP60, HSP90, GST), and mitochondrial biogenesis (PGC1α) were evaluated by real-time quantitative polymerase chain reaction (RT-qPCR). Liver mitochondrial oxidation activity was measured by a polarographic method, and cytokines by enzyme-linked immunosorbent assay (ELISA). Sorafenib treatment restored mitochondrial function and reduced collagen deposition by nearly 63% compared to the NASH group. Sorafenib upregulated PGC1α and MMP9 and reduced TIMP1 and TIMP2 mRNA and IL-6 and IL-10 protein expression. There were no differences in HSP60, HSP90 and GST expression. Sorafenib modulated PGC1α expression, improved mitochondrial respiration and prevented collagen deposition. It may, therefore, be useful in the treatment of liver fibrosis in NASH.

Diminished glucocorticoid signaling is associated with an age-related decline in hippocampal functioning. In this study we demonstrate the effect of intermittent, every other day (EOD) feeding on the glucocorticoid hormone/glucocorticoid receptor (GR) system in the hippocampus of middle-aged (18-month-old) and aged (24-month-old) Wistar rats. In aged ad libitum-fed rats, a decrease in the level of total GR and GR phosphorylated at Ser(232) (pGR) was detected. Conversely, aged rats subjected to EOD feeding, starting from 6 months of age, showed an increase in GR and pGR levels and a higher content of hippocampal corticosterone. Furthermore, prominent nuclear staining of pGR was observed in CA1 pyramidal and DG granule neurons of aged EOD-fed rats. These changes were accompanied by increased Sgk-1 and decreased GFAP transcription, pointing to upregulated transcriptional activity of GR. EOD feeding also induced an increase in the expression of the mineralocorticoid receptor. Our results reveal that intermittent feeding restores impaired GR signaling in the hippocampus of aged animals by inducing rather than by stabilizing GR signaling during aging., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Microfluidic systems with monolithic columns have been developed for preconcentration and on-chip labeling of model proteins. Monoliths were prepared in microchannels by photopolymerization, and their properties were optimized by varying the composition and concentration of the monomers to improve flow and extraction. On-chip labeling of proteins was achieved by driving solutions through the monolith by use of voltage then incubating fluorescent dye with protein retained on the monolith. Subsequently, the labeled proteins were eluted, by applying voltages to reservoirs on the microdevice, and then detected, by monitoring laser-induced fluorescence. Monoliths prepared from octyl methacrylate combine the best protein retention with the possibility of separate elution of unattached fluorescent label with 50% acetonitrile. Finally, automated on-chip extraction and fluorescence labeling of a model protein were successfully demonstrated. This method involves facile sample pretreatment, and therefore has potential for production of integrated bioanalysis microchips.

Objective: Heat shock protein (Hsp90) resides exclusively in the cytosol in normal cells, but is activated and then removes to the cell surface in tumor cells. The detecting upregulation or activation of Hsp90 is an early indicator of malignant behavior of cancer cells. Hsp90 has emerged as an important target for diagnosis or therapy of prostate cancer. In this study, we labeled Hsp90α specific monoclonal antibody (Hsp90α-mAb) with radioiodine Na131I and investigated its potential usage in diagnostic imaging of prostate tumor in xenograft mice model., Methods: Hsp90α-mAb was radioiodinated by using chloramine-T. The radiolabeling efficiency and radiochemical purity were assessed in vitro. 131I-Hsp90α-mAb was then injected into the nude mice bearing human prostate carcinoma. The planar gamma Imaging was performed at 3, 6, 9 and 12 h after injection., Results: The radiochemical purity of 131I-Hsp90α-mAb exceeded 95% after purification. This radiolabeled mAb was stable in human blood serum. In planar gamma imaging study, the prostate tumors in mice model were imaged clearly at 3h after injection of 131I-Hsp90α-mAb., Conclusions: The results suggest that 131I-HSP90α-mAb could be a new promising molecular probe for diagnostic imaging of prostate tumors.

Heat shock protein 90 plays critical roles in client protein maturation, signal transduction, protein folding and degradation, and morphological evolution; however, its function in human sperm is not fully understood. Therefore, our objective in this study was to elucidate the mechanism by which heat shock protein 90 exerts its effects on human sperm function. By performing indirect immunofluorescence staining, we found that heat shock protein 90 was localized primarily in the neck, midpiece, and tail regions of human sperm, and that its expression increased with increasing incubation time under capacitation conditions. Geldanamycin, a specific inhibitor of heat shock protein 90, was shown to inhibit this increase in heat shock protein 90 expression in western blotting analyses. Using a multifunctional microplate reader to examine Fluo-3 AM-loaded sperm, we observed for the first time that inhibition of heat shock protein 90 by using geldanamycin significantly decreased intracellular calcium concentrations during capacitation. Moreover, western blot analysis showed that geldanamycin enhanced tyrosine phosphorylation of several proteins, including heat shock protein 90, in a dose-dependent manner. The effects of geldanamycin on human sperm function in the absence or presence of progesterone was evaluated by performing chlortetracycline staining and by using a computer-assisted sperm analyzer. We found that geldanamycin alone did not affect sperm capacitation, hyperactivation, and motility, but did so in the presence of progesterone. Taken together, these data suggest that heat shock protein 90, which increases in expression in human sperm during capacitation, has roles in intracellular calcium homeostasis, protein tyrosine phosphorylation regulation, and progesterone-stimulated sperm function. In this study, we provide new insights into the roles of heat shock protein 90 in sperm function.

The role and diagnostic efficacy of gene and protein products RB1, CDK4, CHMP2B, HSP90, and cPLA2G4A, all previously shown to be involved in tumor genesis and cell proliferation, were examined by immunohistochemical techniques in 32 cases of myxofibrosarcomas and 29 myxoid liposarcomas (all diagnosis had been confirmed by fluorescence in situ hybridization). HSP90 demonstrated strong nuclear and cytoplasmic positivity in all myxoid liposarcoma cases, while only 4 myxofibrosarcomas showed scattered HSP90 positivity. All but 4 cases of myxofibrosarcoma displayed strong positivity for cPLA2G4A, while only 2 myxoid liposarcoma cases were cPLA2G4A positive and both were CHMP2B negative. Overexpression of both cPLA2G4A and CHMP2B also suggested higher tumor grade. In conclusion, HSP90 and cPLA2G4A immunohistochemical stains are useful markers to distinguish myxofibrosarcoma from myxoid liposarcoma., (© The Author(s) 2014.)

The objective of this study is to investigate the expression of HSP90 and the effect of HSP90 inhibitor AUY922 in ACC. The expression of HSP90 was measured in tissue samples from 36 human sporadic adrenocortical tumors by immunohistochemistry, Western blotting, and real-time PCR. The effect of AUY922 was tested on SW13 and H295R cells by evaluating cell viability and apoptosis in vitro. Transwell assay was performed to evaluate the migration of SW13 cells after different concentrations of AUY922. Western blot, real-time PCR, and immunohistochemistry revealed that both HSP90 mRNA and protein were obviously expressed in a higher degree in ACC tissues than ACA tissues and normal adrenal tissues (P < 0.01). Positive staining for HSP90 was found in 15 of 20 ACCs (75.00 %) and in 3 of 16 (18.75 %) ACAs. There existed the significant statistical difference (P < 0.001). AUY922 inhibited the proliferation of ACC cells in a time- and concentration-dependent manner, and increasing apoptosis was observed in tumor cells treated with the HSP90 inhibitor. Finally, migration of SW13 cells was distinctly suppressed after undergoing treatment with AUY922. Our data suggest that the specific HSP90 inhibitor AUY922 can play a therapeutic role in treatment of ACC and, thus, HSP90 could qualify as a promising new target in ACC.