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1.
Toxicol Lett ; 320: 19-27, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31778773

RESUMO

The deleterious effects of glucocorticoids on glucose homeostasis limit their clinical use. There is substantial evidence demonstrating that islet function impaired by long-term glucocorticoids exposure is a core defect in the progression of impaired glucose tolerance to diabetes. The activity of heat-shock protein (Hsp) 90 is required to maintain the hormone-binding activity and stability of glucocorticoid receptor (GR). In the present study, Hsp90 inhibition by 17-DMAG counteracted dexamethasone-mediated inhibition of glucose-stimulated insulin secretion in isolated rat islets as well as expressions of neuropeptide Y (NPY) and somatostatin receptor 3 (SSTR3), two negative regulators of insulin secretion. Like 17-DMAG, both the pan-histone deacetylase (HDAC) inhibitor TSA and HDAC6 inhibitor Tubacin exhibited a similar action in protecting islet function against dexamethasone-induced injury, along with the downregulation of NPY and SSTR3 expressions. The hyperacetylation of Hsp90 by TSA and Tubacin disrupted its binding ability to GR and blocked dexamethasone-elicited nuclear translocation of GR in INS-1 ß-cell lines. In addition, Tubacin treatment triggered the GR protein degradation through the ubiquitin-proteasome pathway. These findings suggest that Hsp90 acetylation by inhibiting HDAC6 activity may be a potential strategy to prevent the development of steroid diabetes mellitus via alleviating glucocorticoid-impaired islet function.


Assuntos
Anilidas/farmacologia , Benzoquinonas/farmacologia , Dexametasona/toxicidade , Glucocorticoides/toxicidade , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Lactamas Macrocíclicas/farmacologia , Acetilação , Animais , Linhagem Celular , Proteínas de Choque Térmico HSP90/metabolismo , Desacetilase 6 de Histona/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Ratos Sprague-Dawley , Via Secretória , Técnicas de Cultura de Tecidos
2.
Chemosphere ; 238: 124647, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31466007

RESUMO

Ground water arsenic contamination is a global menace. Since arsenic may affect the immune system, leading to immunesuppression, we investigated the effects of acute arsenic exposure on the thymus and spleen using Swiss albino mice, exposed to 5 ppm, 15 ppm and 300 ppm of sodium arsenite for 7 d. Effects on cytokine balance and cell survivability were subsequently analyzed. Our data showed that arsenic treatment induced debilitating alterations in the tissue architecture of thymus and spleen. A dose-dependent decrease in the ratio of CD4+-CD8+ T-cells was observed along with a pro-inflammatory response and redox imbalance. In addition, pioneering evidences established the ability of arsenic to induce an up regulation of Hsp90, eventually resulting in stabilization of its client protein Beclin-1, an important autophagy-initiating factor. This association initiated the autophagic process, confirmed by co-immunoprecipitation assay, acridine orange staining and Western blot, indicating the effort of cells trying to survive at lower doses. However, increased arsenic assault led to apoptotic cell death in the lymphoid organs, possibly by increased ROS generation. There are several instances of autophagy and apoptosis taking place either simultaneously or sequentially due to oxidative stress. Since arsenic is a potent environmental stress factor, exposure to arsenic led to a dose-dependent increase in both autophagy and apoptosis in the thymus and spleen, and cell death could therefore possibly be induced by autophagy. Therefore, exposure to arsenic leads to serious effects on the immune physiology in mice, which may further have dire consequences on the health of exposed animals.


Assuntos
Arsênico/farmacologia , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Animais , Apoptose/efeitos dos fármacos , Arsenitos/farmacologia , Relação CD4-CD8 , Inflamação/induzido quimicamente , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Compostos de Sódio/farmacologia , Baço/efeitos dos fármacos , Baço/patologia , Timo/efeitos dos fármacos , Timo/patologia
3.
Eur J Med Chem ; 183: 111699, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31561045

RESUMO

The recent burst of explorations on heat shock protein 90 (HSP90) in virus research supports its emergence as a promising target to overcome the drawbacks of current antiviral therapeutic regimen. In continuation of our efforts towards the discovery of novel anti-retroviral molecules, we designed, synthesized fifteen novels 2-isoxazol-3-yl-acetamide based compounds (2a-o) followed by analysis of their anti-HIV activity and cytotoxicity studies. 2a-b, 2e, 2j, and 2l-m were found to be active with inhibitory potentials >80% at their highest non-cytotoxic concentration (HNC). Further characterization of anti-HIV activity of these molecules suggests that 2l has ∼3.5 fold better therapeutic index than AUY922, the second generation HSP90 inhibitor. The anti-HIV activity of 2l is a cell type, virus isolate and viral load independent phenomena. Interestingly, 2l does not significantly modulate viral enzymes like Reverse Transcriptase (RT), Integrase (IN) and Protease (PR) as compared to their known inhibitors in a cell free in vitro assay system at its HNC. Further, 2l mediated inhibition of HSP90 attenuates HIV-1 LTR driven gene expression. Taken together, structural rationale, modeling studies and characterization of biological activities suggest that this novel scaffold can attenuate HIV-1 replication significantly within the host and thus opens a new horizon to develop novel anti-HIV therapeutic candidates.


Assuntos
Acetamidas/farmacologia , Androstenóis/farmacologia , Fármacos Anti-HIV/farmacologia , Descoberta de Drogas , HIV-1/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Acetamidas/síntese química , Acetamidas/química , Androstenóis/síntese química , Androstenóis/química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Relação Dose-Resposta a Droga , HIV-1/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos
4.
J Chem Theory Comput ; 15(11): 6368-6381, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31538783

RESUMO

The molecular chaperone HSP90 oversees the functional activation of a large number of client proteins. Because of its role in multiple pathways linked to cancer and neurodegeneration, drug discovery targeting HSP90 has been actively pursued. Yet, a number of inhibitors failed to meet expectations due to induced toxicity problems. In this context, allosteric perturbation has emerged as an alternative strategy for the pharmacological modulation of HSP90 functions. Specifically, novel allosteric stimulators showed the interesting capability of accelerating HSP90 closure dynamics and ATPase activities while inducing tumor cell death. Here, we gain atomistic insight into the mechanisms of allosteric ligand recognition and their consequences on the functional dynamics of HSP90, starting from the fully unbound state. We integrate advanced computational sampling methods based on FunnelMetadynamics, with the analysis of internal dynamics of the structural ensembles visited during the simulations. We observe several binding/unbinding events, and from these, we derive an accurate estimation of the absolute binding free energy. Importantly, we show that different binding poses induce different dynamics states. Our work for the first time explicitly correlates HSP90 responses to binding/unbinding of an allosteric ligand to the modulation of functionally oriented protein motions.


Assuntos
Proteínas de Choque Térmico HSP90/química , Ligantes , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Sítios de Ligação , Proteínas de Choque Térmico HSP90/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Termodinâmica
5.
Plant Sci ; 287: 110170, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31481192

RESUMO

Protein ubiquitination is a major post-translational modification important for diverse biological processes. In wheat (Triticum aestivum) and Arabidopsis thaliana, STRESS-ASSOCIATED PROTEIN 5 (SAP5) is involved in drought tolerance, acting as an E3 ubiquitin ligase to target DRIP and MBP-1 for degradation. To identify further target proteins of SAP5, we implemented two independent approaches in this work. We used ubiquitylome capture with a di-Gly-Lys antibody-based peptide enrichment and affinity purification with a polyubiquitin antibody coupled with mass spectrometry to elucidate the SAP5-dependent ubiquitylation of its target proteins in response to osmotic stress. Wild type or TaSAP5-overexpressing Arabidopsis line, which was more tolerant to osmotic stress according to our previous study, were used here. We identified HSP90C (chloroplast heat shock protein 90) as a substrate of TaSAP5. Further biochemical experiments indicated that TaSAP5 interacts with HSP90C and mediates its degradation by the 26S proteasome. Our work also demonstrates that ubiquitylome profiling is an effective approach to search for substrates of the TaSAP5 E3 ubiquitin ligase when heterologously expressed in Arabidopsis.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Plantas/metabolismo , Saporinas/metabolismo , Triticum/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Arabidopsis , Eletroforese em Gel Bidimensional , Metabolômica , Plantas Geneticamente Modificadas , Tabaco , Triticum/enzimologia , Ubiquitinação
6.
Cell Physiol Biochem ; 53(3): 480-495, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31486323

RESUMO

BACKGROUND/AIMS: Hypoxia Inducible Factor-1α (HIF-1α) is involved in cancer progression and is stabilized by the chaperone HSP90 (Heat Shock Protein 90), preventing degradation. Previously identified HSP90 inhibitors bind to the N-terminal pocket of HSP90, which blocks binding to HIF-1α and induces HIF-1α degradation. N-terminal inhibitors have failed in the clinic as single therapy treatments partially because they induce a heat shock response. SM molecules are HSP90 inhibitors that bind to the C-terminus of HSP90 and do not induce a heat shock response. The effects of these C-terminal inhibitors on HIF-1α are unreported. METHODS: HCT116, MDA-MB-231, PC3, and HEK293T cells were treated with HSP90 inhibitors. qRT-PCR and western blotting was performed to assess mRNA and protein levels of HIF-1α, HSP- and RACK1-related genes. siRNA was used to knockdown RACK1, while MG262 was used to inhibit proteasome activity. Dimethyloxalylglycine (DMOG) was used to inhibit activity of the prolyl hydroxylases (PHDs). Anti-angiogenic activity of HSP90 inhibitors was assessed using a HUVEC tubule formation assay. RESULTS: We show that SM compounds decrease HIF-1α target expression at the mRNA and protein level under hypoxia in colorectal, breast and prostate cancer cells, leading to cell death, without inducing a heat shock response. Surprisingly, we found that when the C-terminal of HSP90 is inhibited, HIF-1α degradation occurs through the proteasome and prolyl hydroxylases in an oxygen-dependent manner even in very low levels of oxygen (tumor hypoxia levels). RACK1 was not required for proteasomal degradation of HIF-1α. CONCLUSION: Our results suggest that by targeting the C-terminus of HSP90 we can exploit the prolyl hydroxylase and proteasome pathway to induce HIF-1α degradation in hypoxic tumors.


Assuntos
Hipóxia Celular/fisiologia , Proteínas de Choque Térmico HSP90/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Aminoácidos Dicarboxílicos/metabolismo , Western Blotting , Hipóxia Celular/genética , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Células HCT116 , Células HEK293 , Proteínas de Choque Térmico HSP90/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Células PC-3 , Prolil Hidroxilases/genética , Prolil Hidroxilases/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
7.
Comput Biol Chem ; 83: 107113, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31493740

RESUMO

Breast cancer is one of the major impediments affecting women globally. The ATP-dependant heat shock protein 90 (Hsp90) forms the central component of molecular chaperone machinery that predominantly governs the folding of newly synthesized peptides and their conformational maturation. It regulates the stability and function of numerous client proteins that are frequently upregulated and/or mutated in cancer cells, therefore, making Hsp90 inhibition a promising therapeutic strategy for the development of new efficacious drugs to treat breast cancer. In the present in silico investigation, a structure-based pharmacophore model was generated with hydrogen bond donor, hydrogen bond acceptor and hydrophobic features complementary to crucial residues Ala55, Lys58, Asp93, Ile96, Met98 and Thr184 directed at inhibiting the ATP-binding activity of Hsp90. Subsequently, the phytochemical dataset of 3210 natural compounds was screened to retrieve the prospective inhibitors after rigorous validation of the model pharmacophore. The retrieved 135 phytocompounds were further filtered by drug-likeness parameters including Lipinski's rule of five and ADMET properties, then investigated via molecular docking-based scoring. Molecular interactions were assessed using Genetic Optimisation for Ligand Docking program for 95 drug-like natural compounds against Hsp90 along with two clinical drugs as reference compounds - Geldanamycin and Radicicol. Docking studies revealed three phytochemicals are better than the investigated clinical drugs. The reference and hit compounds with dock scores of 48.27 (Geldanamycin), 40.90 (Radicicol), 73.04 (Hit1), 72.92 (Hit2) and 68.12 (Hit3) were further validated for their binding stability through molecular dynamics simulations. We propose that the non-macrocyclic scaffolds of three identified phytochemicals might aid in the development of novel therapeutic candidates against Hsp90-driven cancers.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Produtos Biológicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Modelos Moleculares , Antineoplásicos Fitogênicos/síntese química , Antineoplásicos Fitogênicos/química , Produtos Biológicos/síntese química , Produtos Biológicos/química , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Feminino , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Relação Estrutura-Atividade
8.
Biomed Res ; 40(4): 169-178, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31413238

RESUMO

Migration of osteoblasts to the sites resorbed by osteoclasts is an essential step in bone remodeling. However, the exact mechanism of osteoblast migration is still not known. We have shown that platelet-derived growth factor (PDGF)-BB induces the migration of osteoblast-like MC3T3-E1 cells through the activation of p38 mitogen-activated protein (MAP) kinase, c-Jun N-terminal kinase (JNK) and p44/p42 MAP kinase. Evidence is accumulating that heat shock protein 90 (HSP90) acts as a central regulator of proteostasis under stress conditions and physiological cell functions. In the present study, using transwell cell migration assay and wound-healing assay, we investigated the involvement of HSP90 in the PDGF-BB-stimulated migration of MC3T3-E1 cells, and the underlying signaling mechanism estimated by Western blot analyses. Onalespib, an HSP90 inhibitor, significantly reduced the PDGF-BB-stimulated migration evaluated by the two types of migration assays. The cell migration was also suppressed by geldanamycin, another type of HSP90 inhibitor. Onalespib markedly attenuated the PDGF-BB-elicited phosphorylation of p44/p42 MAP kinase without affecting that of p38 MAP kinase or JNK. In addition, the phosphorylation of p44/p42 MAP kinase by PDGF-BB was reduced by geldanamycin. Taken together, these results strongly suggest that HSP90 inhibitors suppress the PDGF-BB-induced osteoblast migration through the attenuation of p44/p42 MAP kinase activity.


Assuntos
Becaplermina/farmacologia , Benzamidas/farmacologia , Benzoquinonas/farmacologia , Movimento Celular/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Isoindóis/farmacologia , Lactamas Macrocíclicas/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Osteoblastos/metabolismo , Animais , Linhagem Celular , Proteínas de Choque Térmico HSP90/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos
9.
Nat Commun ; 10(1): 3626, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399574

RESUMO

The molecular chaperone Hsp90 is an important regulator of proteostasis. It has remained unclear why S. cerevisiae possesses two Hsp90 isoforms, the constitutively expressed Hsc82 and the stress-inducible Hsp82. Here, we report distinct differences despite a sequence identity of 97%. Consistent with its function under stress conditions, Hsp82 is more stable and refolds more efficiently than Hsc82. The two isoforms also differ in their ATPases and conformational cycles. Hsc82 is more processive and populates closed states to a greater extent. Variations in the N-terminal ATP-binding domain modulate its dynamics and conformational cycle. Despite these differences, the client interactomes are largely identical, but isoform-specific interactors exist both under physiological and heat shock conditions. Taken together, changes mainly in the N-domain create a stress-specific, more resilient protein with a shifted activity profile. Thus, the precise tuning of the Hsp90 isoforms preserves the basic mechanism but adapts it to specific needs.


Assuntos
Proteínas de Choque Térmico HSP90/química , Chaperonas Moleculares/química , Isoformas de Proteínas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico HSP90/fisiologia , Resposta ao Choque Térmico/fisiologia , Ligantes , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Estresse Fisiológico
10.
Nat Commun ; 10(1): 3613, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399576

RESUMO

Small-molecule inhibitors for the 90-kDa heat shock protein (HSP90) have been extensively exploited in preclinical studies for the therapeutic interventions of human diseases accompanied with proteotoxic stress. By using an unbiased quantitative proteomic method, we uncover that treatment with three HSP90 inhibitors results in elevated expression of a large number of heat shock proteins. We also demonstrate that the HSP90 inhibitor-mediated increase in expression of DNAJB4 protein occurs partly through an epitranscriptomic mechanism, and is substantially modulated by the writer, eraser, and reader proteins of N6-methyladenosine (m6A). Furthermore, exposure to ganetespib leads to elevated modification levels at m6A motif sites in the 5'-UTR of DNAJB4 mRNA, and the methylation at adenosine 114 site in the 5'-UTR promotes the translation of the reporter gene mRNA. This m6A-mediated mechanism is also at play upon heat shock treatment. Cumulatively, we unveil that HSP90 inhibitors stimulate the translation of DNAJB4 through an epitranscriptomic mechanism.


Assuntos
Adenosina/análogos & derivados , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteômica , Regiões 5' não Traduzidas , Adenosina/metabolismo , Linhagem Celular Tumoral , Genes Reporter , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP90/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Metilação , RNA Mensageiro/metabolismo , Triazóis
11.
Chemphyschem ; 20(21): 2759-2766, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31460692

RESUMO

This paper describes the excellent performance of a newly developed scoring function (SF), based on the semiempirical QM (SQM) PM6-D3H4X method combined with the conductor-like screening implicit solvent model (COSMO). The SQM/COSMO, Amber/GB and nine widely used SFs have been evaluated in terms of ranking power on the HSP90 protein with 72 biologically active compounds and 4469 structurally similar decoys. Among conventional SFs, the highest early and overall enrichment measured by EF1 and AUC% obtained using single-scoring-function ranking has been found for Glide SP and Gold-ASP SFs, respectively (7, 75 % and 3, 76 %). The performance of other standard SFs has not been satisfactory, mostly even decreasing below random values. The SQM/COSMO SF, where P-L structures were optimised at the advanced Amber level, has resulted in a dramatic enrichment increase (47, 98 %), almost reaching the best possible receiver operator characteristic (ROC) curve. The best SQM frame thus inserts about seven times more active compounds into the selected dataset than the best standard SF.


Assuntos
Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Teoria Quântica , Proteínas de Choque Térmico HSP90/metabolismo , Ligantes , Modelos Moleculares , Curva ROC , Termodinâmica
12.
Biochim Biophys Acta Mol Cell Res ; 1866(10): 1544-1555, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31326539

RESUMO

Plasma membrane transporter SLC6A14 transports all neutral and basic amino acids in a Na/Cl - dependent way and it is up-regulated in many types of cancer. Mass spectrometry analysis of overexpressed SLC6A14-associated proteins identified, among others, the presence of cytosolic heat shock proteins (HSPs) and co-chaperones. We detected co-localization of overexpressed and native SLC6A14 with HSP90-beta and HSP70 (HSPA14). Proximity ligation assay confirmed a direct interaction of overexpressed SLC6A14 with both HSPs. Treatment with radicicol and VER155008, specific inhibitors of HSP90 and HSP70, respectively, attenuated these interactions and strongly reduced transporter presence at the cell surface, what resulted from the diminished level of the total transporter protein. Distortion of SLC6A14 proper folding by both HSPs inhibitors directed the transporter towards endoplasmic reticulum-associated degradation pathway, a process reversed by the proteasome inhibitor - bortezomib. As demonstrated in an in vitro ATPase assay of recombinant purified HSP90-beta, the peptides corresponding to C-terminal amino acid sequence following the last transmembrane domain of SLC6A14 affected the HSP90-beta activity. These results indicate that a plasma membrane protein folding can be controlled not only by chaperones in the endoplasmic reticulum, but also those localized in the cytosol.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Membrana Celular/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Transporte Proteico/fisiologia , Adenosina Trifosfatases/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Biotinilação , Bortezomib/farmacologia , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Humanos , Células MCF-7 , Macrolídeos/farmacologia , Chaperonas Moleculares/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Dobramento de Proteína , Transporte Proteico/efeitos dos fármacos , Nucleosídeos de Purina/farmacologia
13.
Cancer Sci ; 110(9): 2941-2959, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31343810

RESUMO

A sensitive and specific diagnosis biomarker, in principle scalable to most cancer types, is needed to reduce the prevalent cancer mortality. Meanwhile, the investigation of diagnosis determinants of a biomarker will facilitate the interpretation of its screening results in clinic. Here we design a large-scale (1558 enrollments), multicenter (multiple hospitals), and cross-validation (two datasets) clinic study to validate plasma Hsp90α quantified by ELISA as a pan-cancer biomarker. ROC curve shows the optimum diagnostic cutoff is 69.19 ng/mL in discriminating various cancer patients from all controls (AUC 0.895, sensitivity 81.33% and specificity 81.65% in test cohort; AUC 0.893, sensitivity 81.72% and specificity 81.03% in validation cohort). Similar results are noted in detecting early-stage cancer patients. Plasma Hsp90α maintains also broad-spectrum for cancer subtypes, especially with 91.78% sensitivity and 91.96% specificity in patients with AFP-limited liver cancer. In addition, we demonstrate levels of plasma Hsp90α are determined by ADAM10 expression, which will affect Hsp90α content in exosomes. Furthermore, Western blotting and PRM-based quantitative proteomics identify that partial false ELISA-negative patients secret high levels of plasma Hsp90α. Mechanism analysis reveal that TGFß-PKCγ gene signature defines a distinct pool of hyperphosphorylated Hsp90α at Theronine residue. In clinic, a mechanistically relevant population of false ELISA-negative patients express also higher levels of PKCγ. In sum, plasma Hsp90α is a novel pan-cancer diagnosis biomarker, and cancer diagnosis with plasma Hsp90α is particularly effective in those patients with high expression of ADAM10, but may be insufficient to detect the patients with low ADAM10 and those with hyperphosphorylated Hsp90α.


Assuntos
Biomarcadores Tumorais/sangue , Proteínas de Choque Térmico HSP90/sangue , Neoplasias/diagnóstico , Proteína ADAM10/genética , Proteína ADAM10/metabolismo , Adolescente , Adulto , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Criança , Pré-Escolar , Conjuntos de Dados como Assunto , Ensaio de Imunoadsorção Enzimática , Exossomos/metabolismo , Reações Falso-Negativas , Feminino , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactente , Recém-Nascido , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Pessoa de Meia-Idade , Neoplasias/sangue , Neoplasias/patologia , Fosforilação , Estudos Prospectivos , Curva ROC , Treonina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
14.
Life Sci ; 232: 116672, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31336120

RESUMO

AIMS: Obesity is not only associated with metabolic diseases but is also a symptom of menopause in women. To date, there are no effective drugs for the management of obesity, and it is important to find new agents with fewer side effects, for the treatment of obesity. This study aimed to determine the anti-obesity effect of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), a heat shock protein 90 (Hsp90) inhibitor, and its underlying mechanism in rats with ovariectomy-induced obesity. MAIN METHODS: Ovariectomy (Ovx) rats were treated with 17-DMAG (1 mg kg-1, intraperitoneally) for eight weeks from one week after surgery. The body weight, food intake, locomotor activity, adipogenic- and autophagy-related protein expression in white adipose tissue (WAT) and plasma triglyceride (TG) levels were measured in sham and Ovx rats. KEY FINDINGS: Compared with sham rats, Ovx rats showed increased weight gain, food intake, WAT mass, TG levels, adipogenic protein expression, and decreased locomotor activity. Furthermore, autophagy-related proteins and Foxo3a of WAT were significantly increased in Ovx rats. However, with the exclusion of increased food intake, the changes induced by Ovx were all reversed in 17-DMAG-treated Ovx rats. In addition, the expression of Hsp70 and phosphorylation of Akt increased in 17-DMAG-treated Ovx rats. SIGNIFICANCE: These results suggest that 17-DMAG significantly ameliorated obesity induced by Ovx, and this phenomenon is accompanied by the downregulation of adipogenic-related and autophagy-related proteins as well as the upregulation of Akt-phosphorylation and Hsp70 expression. Therefore, 17-DMAG may be a potential agent for preventing or treating obesity in postmenopausal women.


Assuntos
Benzoquinonas/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , Obesidade/etiologia , Obesidade/prevenção & controle , Ovariectomia/efeitos adversos , Adipogenia , Tecido Adiposo/metabolismo , Animais , Autofagia , Ingestão de Alimentos/efeitos dos fármacos , Feminino , Proteínas de Choque Térmico HSP90/metabolismo , Locomoção/efeitos dos fármacos , Ratos
15.
BMC Neurosci ; 20(1): 34, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307382

RESUMO

BACKGROUND: Emerging data suggests that volatile anesthetic agents may have organ protection properties in the setting of critical illness. The purpose of this study was to better understand the effect of inflammation on cerebral subcellular energetics in animals exposed to two different anesthetic agents-a GABA agonist (propofol) and a volatile agent (isoflurane). RESULTS: Forty-eight Sprague-Dawley rats were anesthetized with isoflurane or propofol. In each group, rats were randomized to celiotomy and closure (sham) or cecal ligation and puncture (inflammation [sepsis model]) for 8 h. Brain tissue oxygen saturation and the oxidation state of cytochrome aa3 were measured. Brain tissue was extracted using the freeze-blow technique. All rats experienced progressive increases in tissue oxygenation and cytochrome aa3 reduction over time. Inflammation had no impact on cytochrome aa3, but isoflurane caused significant cytochrome aa3 reduction. During isoflurane (not propofol) anesthesia, inflammation led to an increase in lactate (+ 0.64 vs. - 0.80 mEq/L, p = 0.0061). There were no differences in ADP:ATP ratios between groups. In the isoflurane (not propofol) group, inflammation increased the expression of hypoxia-inducible factor-1α (62%, p = 0.0012), heme oxygenase-1 (67%, p = 0.0011), and inducible nitric oxide synthase (31%, p = 0.023) in the brain. Animals exposed to inflammation and isoflurane (but not propofol) exhibited increased expression of protein carbonyls (9.2 vs. 7.0 nM/mg protein, p = 0.0050) and S-nitrosylation (49%, p = 0.045) in the brain. RNA sequencing identified an increase in heat shock protein 90 and NF-κß inhibitor mRNA in the inflammation/isoflurane group. CONCLUSIONS: In the setting of inflammation, rats exposed to isoflurane show increased hypoxia-inducible factor-1α expression despite a lack of hypoxia, increased oxidative stress in the brain, and increased serum lactate, all of which suggest a relative increase in anaerobic metabolism compared to propofol. Differences in oxidative stress as well as heat shock protein 90 and NF-κß inhibitor may account for the differential expression of cerebral hypoxia-inducible factor-1α during inflammation.


Assuntos
Encéfalo/metabolismo , Inflamação/metabolismo , Isoflurano/administração & dosagem , Propofol/administração & dosagem , Tiflite/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Anestésicos Inalatórios , Animais , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isoflurano/farmacologia , Ácido Láctico/metabolismo , Masculino , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Oxirredução , Oxigênio/metabolismo , Propofol/farmacologia , Carbonilação Proteica/efeitos dos fármacos , Ratos
16.
Genes Cells ; 24(8): 524-533, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31273901

RESUMO

The outcome of epigenetic responses to stress depends strictly on genetic background, suggesting that altered phenotypes, when induced, are created by a combination of induced epigenetic factors and pre-existing allelic ones. When individuals with altered phenotypes are selected and subjected to successive breeding, alleles that potentiate epigenetic responses could accumulate in offspring populations. It is reasonable to suppose that many, if not all, of these allelic genes could also be involved in creating new phenotypes under nonstressful conditions. In this review, I discuss the possibility that the accumulation of such alleles in selected individuals with an epigenetic phenotype could give rise to individuals that exhibit the same phenotype even in the absence of stress.


Assuntos
Adaptação Biológica/genética , Evolução Biológica , Epigênese Genética , Alelos , Animais , Cromatina/genética , Cromatina/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Padrões de Herança , Modelos Genéticos , Mutação , Fenótipo , Seleção Genética , Estresse Fisiológico
17.
PLoS Biol ; 17(7): e3000358, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31283755

RESUMO

Hsp90 is a conserved molecular chaperone that assists in the folding and function of diverse cellular regulators, with a profound impact on biology, disease, and evolution. As a central hub of protein interaction networks, Hsp90 engages with hundreds of protein-protein interactions within eukaryotic cells. These interactions include client proteins, which physically interact with Hsp90 and depend on the chaperone for stability or function, as well as co-chaperones and partner proteins that modulate chaperone function. Currently, there are no methods to accurately predict Hsp90 interactors and there has been considerable network rewiring over evolutionary time, necessitating experimental approaches to define the Hsp90 network in the species of interest. This is a pressing challenge for fungal pathogens, for which Hsp90 is a key regulator of stress tolerance, drug resistance, and virulence traits. To address this challenge, we applied a novel biochemical fractionation and quantitative proteomic approach to examine alterations to the proteome upon perturbation of Hsp90 in a leading human fungal pathogen, Candida albicans. In parallel, we performed affinity purification coupled to mass spectrometry to define physical interacting partners for Hsp90 and the Hsp90 co-chaperones and identified 164 Hsp90-interacting proteins, including 111 that are specific to the pathogen. We performed the first analysis of the Hsp90 interactome upon antifungal drug stress and demonstrated that Hsp90 stabilizes processing body (P-body) and stress granule proteins that contribute to drug tolerance. We also describe novel roles for Hsp90 in regulating posttranslational modification of the Rvb1-Rvb2-Tah1-Pih1 (R2TP) complex and the formation of protein aggregates in response to thermal stress. This study provides a global view of the Hsp90 interactome in a fungal pathogen, demonstrates the dynamic role of Hsp90 in response to environmental perturbations, and highlights a novel connection between Hsp90 and the regulation of mRNA-associated protein granules.


Assuntos
Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Proteômica/métodos , Candida albicans/genética , Candida albicans/patogenicidade , Candidíase/microbiologia , Proteínas Fúngicas/genética , Redes Reguladoras de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico HSP90/genética , Humanos , Microscopia Confocal , Chaperonas Moleculares/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ligação Proteica , Proteoma/genética , Proteoma/metabolismo , Virulência/genética
18.
Eur J Med Chem ; 178: 48-63, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31176095

RESUMO

The ninety kilo Dalton molecular weight heat shock protein (Hsp90) is an attractive target for the discovery of novel anticancer agents. Several strategies have been employed for the development of inhibitors against this polypeptide. The most successful strategy is targeting the N-terminal ATP binding region of the chaperone. However, till date not a single molecule reached Phase-IV of clinical trials from this class of Hsp90 inhibitors. The other approach is to target the Cterminal region of the protein. The success with this approach has been limited due to lack of well-defined ligand binding pocket in this terminal. The other promising strategy is to prevent the interaction of client proteins/co-chaperones with Hsp90 protein, i.e., protein-protein interaction inhibitors of Hsp90. The review focuses on advantage of this approach along with the recent advances in the discovery of inhibitors by following this strategy. Additionally, the biology of the client protein/co-chaperone binding site of Hsp90 is also discussed.


Assuntos
Antineoplásicos/uso terapêutico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Neoplasias/tratamento farmacológico , Ligação Proteica/efeitos dos fármacos , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Proteínas de Choque Térmico HSP90/química , Humanos
19.
Molecules ; 24(11)2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31163701

RESUMO

Hsp90 (Heat shock protein 90) is involved in various processes in cancer occurrence and development, and therefore represents a promising drug target for cancer therapy. In this work, a virtual screening strategy was employed, leading to the identification of a series of compounds bearing a scaffold of 1,3-dibenzyl-2-aryl imidazolidine as novel Hsp90 inhibitors. Compound 4a showed the highest binding affinity to Hsp90α (IC50 = 12 nM) in fluorescence polarization (FP) competition assay and the strongest anti-proliferative activity against human breast adenocarcinoma cell line (MCF-7) and human lung epithelial cell line (A549) with IC50 values of 21.58 µM and 31.22 µM, respectively. Western blotting assays revealed that these novel Hsp90 inhibitors significantly down-regulated the expression level of Her2, a client protein of Hsp90, resulting in the cytotoxicity of these novel Hsp90 inhibitors. The molecular docking study showed that these novel Hsp90 inhibitors bound to the adenosine triphosphate (ATP) binding site at the N-terminus of Hsp90. Furthermore, structure-activity relationship studies indicated that the N-benzyl group is important for the anti-cancer activity of 1,3-dibenzyl-2-aryl imidazolidines.


Assuntos
Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Imidazolidinas/química , Imidazolidinas/farmacologia , Células A549 , Proliferação de Células/efeitos dos fármacos , Ensaios Clínicos como Assunto , Avaliação Pré-Clínica de Medicamentos , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Concentração Inibidora 50 , Células MCF-7 , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Interface Usuário-Computador
20.
Cells ; 8(6)2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31163702

RESUMO

Heat shock protein 90 (HSP90) molecular chaperones are a family of ubiquitous proteins participating in several cellular functions through the regulation of folding and/or assembly of large multiprotein complexes and client proteins. Thus, HSP90s chaperones are, directly or indirectly, master regulators of a variety of cellular processes, such as adaptation to stress, cell proliferation, motility, angiogenesis, and signal transduction. In recent years, it has been proposed that HSP90s play a crucial role in carcinogenesis as regulators of genotype-to-phenotype interplay. Indeed, HSP90 chaperones control metabolic rewiring, a hallmark of cancer cells, and influence the transcription of several of the key-genes responsible for tumorigenesis and cancer progression, through either direct binding to chromatin or through the quality control of transcription factors and epigenetic effectors. In this review, we will revise evidence suggesting how this interplay between epigenetics and metabolism may affect oncogenesis. We will examine the effect of metabolic rewiring on the accumulation of specific metabolites, and the changes in the availability of epigenetic co-factors and how this process can be controlled by HSP90 molecular chaperones. Understanding deeply the relationship between epigenetic and metabolism could disclose novel therapeutic scenarios that may lead to improvements in cancer treatment.


Assuntos
Antineoplásicos/uso terapêutico , Progressão da Doença , Epigênese Genética , Proteínas de Choque Térmico HSP90/metabolismo , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Epigênese Genética/efeitos dos fármacos , Humanos , Neoplasias/genética , Fenótipo
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