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1.
J Gen Virol ; 105(10)2024 Oct.
Article de Anglais | MEDLINE | ID: mdl-39373166

RÉSUMÉ

Feline calicivirus (FCV) icosahedral viral capsids are composed of dozens of structural subunits that rely on cellular chaperones to self-assemble in an orderly fashion. Here, we report that the heat shock protein 90 (Hsp90) inhibition significantly reduced FCV particle production, suggesting a role in the replicative cycle. We found that Hsp90 inhibition was not related to the synthesis or stability of the early proteins that translate from the gRNA nor to the minor capsid protein VP2 but with a reduction in the major capsid protein VP1 levels, both translated late in infection from the subgenomic RNAs. Reduction in VP1 levels was observed despite an augment of the leader of the capsid (LC)-VP1 precursor levels, from which the LC and VP1 proteins are produced after proteolytic processing by NS6/7. The direct interaction of VP1 with Hsp90 was observed in infected cells. These results suggest that upon release from the polyprotein precursor, VP1 becomes a client of Hsp90 and that this interaction is required for an efficient FCV replicative cycle.


Sujet(s)
Calicivirus félin , Protéines de capside , Protéines du choc thermique HSP90 , Réplication virale , Protéines du choc thermique HSP90/métabolisme , Protéines du choc thermique HSP90/génétique , Protéines de capside/métabolisme , Protéines de capside/génétique , Calicivirus félin/métabolisme , Calicivirus félin/physiologie , Calicivirus félin/génétique , Chats , Animaux , Lignée cellulaire , Infections à Caliciviridae/virologie , Infections à Caliciviridae/métabolisme
2.
Int J Mol Sci ; 25(16)2024 Aug 12.
Article de Anglais | MEDLINE | ID: mdl-39201468

RÉSUMÉ

Drug repositioning is an important therapeutic strategy for treating breast cancer. Hsp90ß chaperone is an attractive target for inhibiting cell progression. Its structure has a disordered and flexible linker region between the N-terminal and central domains. Geldanamycin was the first Hsp90ß inhibitor to interact specifically at the N-terminal site. Owing to the toxicity of geldanamycin, we investigated the repositioning of ritonavir as an Hsp90ß inhibitor, taking advantage of its proven efficacy against cancer. In this study, we used molecular modeling techniques to analyze the contribution of the Hsp90ß linker region to the flexibility and interaction between the ligands geldanamycin, ritonavir, and Hsp90ß. Our findings indicate that the linker region is responsible for the fluctuation and overall protein motion without disturbing the interaction between the inhibitors and the N-terminus. We also found that ritonavir established similar interactions with the substrate ATP triphosphate, filling the same pharmacophore zone.


Sujet(s)
Benzoquinones , Protéines du choc thermique HSP90 , Lactames macrocycliques , Ritonavir , Lactames macrocycliques/pharmacologie , Lactames macrocycliques/composition chimique , Ritonavir/composition chimique , Ritonavir/pharmacologie , Benzoquinones/composition chimique , Benzoquinones/pharmacologie , Benzoquinones/métabolisme , Protéines du choc thermique HSP90/composition chimique , Protéines du choc thermique HSP90/métabolisme , Protéines du choc thermique HSP90/antagonistes et inhibiteurs , Humains , Liaison aux protéines , Simulation de dynamique moléculaire , Simulation de docking moléculaire , Modèles moléculaires , Sites de fixation , Adénosine triphosphate/métabolisme , Adénosine triphosphate/composition chimique
3.
Biol Res ; 57(1): 28, 2024 May 15.
Article de Anglais | MEDLINE | ID: mdl-38750549

RÉSUMÉ

BACKGROUND: The activated microglia have been reported as pillar factors in neuropathic pain (NP) pathology, but the molecules driving pain-inducible microglial activation require further exploration. In this study, we investigated the effect of dorsal root ganglion (DRG)-derived exosomes (Exo) on microglial activation and the related mechanism. METHODS: A mouse model of NP was generated by spinal nerve ligation (SNL), and DRG-derived Exo were extracted. The effects of DRG-Exo on NP and microglial activation in SNL mice were evaluated using behavioral tests, HE staining, immunofluorescence, and western blot. Next, the differentially enriched microRNAs (miRNAs) in DRG-Exo-treated microglia were analyzed using microarrays. RT-qPCR, RNA pull-down, dual-luciferase reporter assay, and immunofluorescence were conducted to verify the binding relation between miR-16-5p and HECTD1. Finally, the effects of ubiquitination modification of HSP90 by HECTD1 on NP progression and microglial activation were investigated by Co-IP, western blot, immunofluorescence assays, and rescue experiments. RESULTS: DRG-Exo aggravated NP resulting from SNL in mice, promoted the activation of microglia in DRG, and increased neuroinflammation. miR-16-5p knockdown in DRG-Exo alleviated the stimulating effects of DRG-Exo on NP and microglial activation. DRG-Exo regulated the ubiquitination of HSP90 through the interaction between miR-16-5p and HECTD1. Ubiquitination alteration of HSP90 was involved in microglial activation during NP. CONCLUSIONS: miR-16-5p shuttled by DRG-Exo regulated the ubiquitination of HSP90 by interacting with HECTD1, thereby contributing to the microglial activation in NP.


Sujet(s)
Exosomes , Ganglions sensitifs des nerfs spinaux , Protéines du choc thermique HSP90 , microARN , Microglie , Névralgie , Animaux , Mâle , Souris , Modèles animaux de maladie humaine , Exosomes/métabolisme , Ganglions sensitifs des nerfs spinaux/métabolisme , Protéines du choc thermique HSP90/métabolisme , Souris de lignée C57BL , Microglie/métabolisme , microARN/métabolisme , microARN/génétique , Névralgie/métabolisme , Névralgie/génétique , Ubiquitin-protein ligases/métabolisme , Ubiquitin-protein ligases/génétique
4.
Lett Appl Microbiol ; 77(5)2024 May 03.
Article de Anglais | MEDLINE | ID: mdl-38658187

RÉSUMÉ

Species from Candida parapsilosis complex are frequently found in neonatal candidemia. The antifungal agents to treat this infection are limited and the occurrence of low in vitro susceptibility to echinocandins such as micafungin has been observed. In this context, the chaperone Hsp90 could be a target to reduce resistance. Thus, the objective of this research was to identify isolates from the C. parapsilosis complex and verify the action of Hsp90 inhibitors associated with micafungin. The fungal identification was based on genetic sequencing and mass spectrometry. Minimal inhibitory concentrations were determined by broth microdilution method according to Clinical Laboratory and Standards Institute. The evaluation of the interaction between micafungin with Hsp90 inhibitors was realized using the checkerboard methodology. According to the polyphasic taxonomy, C. parapsilosis sensu stricto was the most frequently identified, followed by C. orthopsilosis and C. metapsilosis, and one isolate of Lodderomyces elongisporus was identified by genetic sequencing. The Hsp90 inhibitor geladanamycin associated with micafungin showed a synergic effect in 31.25% of the isolates, a better result was observed with radicicol, which shows synergic effect in 56.25% tested yeasts. The results obtained demonstrate that blocking Hsp90 could be effective to reduce antifungal resistance to echinocandins.


Sujet(s)
Antifongiques , Candida parapsilosis , Candidémie , Protéines du choc thermique HSP90 , Micafungine , Humains , Nouveau-né , Antifongiques/pharmacologie , Benzoquinones/pharmacologie , Candida parapsilosis/effets des médicaments et des substances chimiques , Candida parapsilosis/isolement et purification , Candida parapsilosis/génétique , Candidémie/microbiologie , Résistance des champignons aux médicaments , Synergie des médicaments , Échinocandines/pharmacologie , Protéines du choc thermique HSP90/antagonistes et inhibiteurs , Protéines du choc thermique HSP90/métabolisme , Protéines du choc thermique HSP90/génétique , Lactames macrocycliques/pharmacologie , Lipopeptides/pharmacologie , Micafungine/pharmacologie , Tests de sensibilité microbienne
5.
Cell Death Dis ; 14(12): 832, 2023 12 15.
Article de Anglais | MEDLINE | ID: mdl-38102125

RÉSUMÉ

In this work, we describe a novel ruthenium-xanthoxylin complex, [Ru(phen)2(xant)](PF6) (RXC), that can eliminate colorectal cancer (CRC) stem cells by targeting the chaperone Hsp90. RXC exhibits potent cytotoxicity in cancer cell lines and primary cancer cells, causing apoptosis in HCT116 CRC cells, as observed by cell morphology, YO-PRO-1/PI staining, internucleosomal DNA fragmentation, mitochondrial depolarization, and PARP cleavage (Asp214). Additionally, RXC can downregulate the HSP90AA1 and HSP90B1 genes and the expression of HSP90 protein, as well as the expression levels of its downstream/client elements Akt1, Akt (pS473), mTOR (pS2448), 4EBP1 (pT36/pT45), GSK-3ß (pS9), and NF-κB p65 (pS529), implying that these molecular chaperones can be molecular targets for RXC. Moreover, this compound inhibited clonogenic survival, the percentage of the CRC stem cell subpopulation, and colonosphere formation, indicating that RXC can eliminate CRC stem cells. RXC reduced cell migration and invasion, decreased vimentin and increased E-cadherin expression, and induced an autophagic process that appeared to be cytoprotective, as autophagy inhibitors enhanced RXC-induced cell death. In vivo studies showed that RXC inhibits tumor progression and experimental metastasis in mice with CRC HCT116 cell xenografts. Taken together, these results highlight the potential of the ruthenium complex RXC in CRC therapy with the ability to eliminate CRC stem cells by targeting the chaperone Hsp90.


Sujet(s)
Tumeurs colorectales , Ruthénium , Humains , Animaux , Souris , Transduction du signal , Glycogen synthase kinase 3 beta/métabolisme , Cellules HCT116 , Protéines du choc thermique HSP90/métabolisme , Tumeurs colorectales/traitement médicamenteux , Tumeurs colorectales/génétique , Tumeurs colorectales/métabolisme , Prolifération cellulaire , Lignée cellulaire tumorale
6.
Microbiol Spectr ; 11(6): e0296023, 2023 Dec 12.
Article de Anglais | MEDLINE | ID: mdl-37905935

RÉSUMÉ

IMPORTANCE: In the unicellular parasites Leishmania spp., the etiological agents of leishmaniasis, a complex infectious disease that affects 98 countries in 5 continents, chemical inhibition of HSP90 protein leads to differentiation from promastigote to amastigote stage. Recent studies indicate potential role for protein phosphorylation in the life cycle control of Leishmania. Also, recent studies suggest a fundamentally important role of RNA-binding proteins (RBPs) in regulating the downstream effects of the HSP90 inhibition in Leishmania. Phosphorylation-dephosphorylation dynamics of RBPs in higher eukaryotes serves as an important on/off switch to regulate RNA processing and decay in response to extracellular signals and cell cycle check points. In the current study, using a combination of highly sensitive TMT labeling-based quantitative proteomic MS and robust phosphoproteome enrichment, we show for the first time that HSP90 inhibition distinctively modulates global protein phosphorylation landscapes in the different life cycle stages of Leishmania, shedding light into a crucial role of the posttranslational modification in the differentiation of the parasite under HSP90 inhibition stress. We measured changes in phosphorylation of many RBPs and signaling proteins including protein kinases upon HSP90 inhibition in the therapeutically relevant amastigote stage. This work provides insights into the importance of HSP90-mediated protein cross-talks and regulation of phosphorylation in Leishmania, thus significantly expanding our knowledge of the posttranslational modification in Leishmania biology.


Sujet(s)
Leishmania mexicana , Leishmania , Leishmania mexicana/métabolisme , Protéomique , Protéines de protozoaire/génétique , Protéines de protozoaire/métabolisme , Leishmania/métabolisme , Protéines du choc thermique HSP90/métabolisme , Protéome/métabolisme
7.
FEBS Lett ; 597(13): 1718-1732, 2023 07.
Article de Anglais | MEDLINE | ID: mdl-36932975

RÉSUMÉ

Systematic studies have revealed interactions between components of the Hsp90 chaperone system and Fe/S protein biogenesis or iron regulation. In addition, two chloroplast-localized DnaJ-like proteins, DJA5 and DJA6, function as specific iron donors in plastidial Fe/S protein biogenesis. Here, we used Saccharomyces cerevisiae to study the impact of both the Hsp90 chaperone and the yeast DJA5-DJA6 homologs, the essential cytosolic Ydj1, and the mitochondrial Mdj1, on cellular iron-related processes. Despite severe phenotypes induced upon depletion of these crucial proteins, there was no critical in vivo impact on Fe/S protein biogenesis or iron regulation. Importantly, unlike the plant DJA5-DJA6 iron chaperones, Ydj1 and Mdj1 did not bind iron in vivo, suggesting that these proteins use zinc for function under normal physiological conditions.


Sujet(s)
Ferrosulfoprotéines , Protéines de Saccharomyces cerevisiae , Saccharomyces cerevisiae/génétique , Saccharomyces cerevisiae/métabolisme , Ferrosulfoprotéines/génétique , Ferrosulfoprotéines/métabolisme , Protéines de Saccharomyces cerevisiae/génétique , Protéines de Saccharomyces cerevisiae/métabolisme , Fer/métabolisme , Protéines mitochondriales/métabolisme , Chaperons moléculaires/métabolisme , Protéines du choc thermique HSP90/génétique , Protéines du choc thermique HSP90/métabolisme
8.
Biochimie ; 200: 99-106, 2022 Sep.
Article de Anglais | MEDLINE | ID: mdl-35643212

RÉSUMÉ

The emergence of the COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a great threat to global health. ORF9b, an important accessory protein of SARS-CoV-2, plays a critical role in the viral host interaction, targeting TOM70, a member of the mitochondrial translocase of the outer membrane complex. The assembly between ORF9b and TOM70 is implicated in disrupting mitochondrial antiviral signaling, leading to immune evasion. We describe the expression, purification, and characterization of ORF9b alone or coexpressed with the cytosolic domain of human TOM70 in E. coli. ORF9b has 97 residues and was purified as a homodimer with an molecular mass of 22 kDa as determined by SEC-MALS. Circular dichroism experiments showed that Orf9b alone exhibits a random conformation. The ORF9b-TOM70 complex characterized by CD and differential scanning calorimetry showed that the complex is folded and more thermally stable than free TOM70, indicating strong binding. Importantly, protein-protein interaction assays demonstrated that full-length human Hsp90 is capable of binding to free TOM70 but not to the ORF9b-TOM70 complex. To narrow down the nature of this inhibition, the isolated C-terminal domain of Hsp90 was also tested. These results were used to build a model of the mechanism of inhibition, in which ORF9b efficiently targets two sites of interaction between TOM70 and Hsp90. The findings showed that ORF9b complexed with TOM70 prevents the interaction with Hsp90, and this is one major explanation for SARS-CoV-2 evasion of host innate immunity via the inhibition of the interferon activation pathway.


Sujet(s)
COVID-19 , SARS-CoV-2 , Protéines de transport/métabolisme , Escherichia coli/métabolisme , Protéines du choc thermique HSP90/métabolisme , Humains , Protéines de transport de la membrane mitochondriale/métabolisme , Protéines du complexe d'import des protéines précurseurs mitochondriales , Pandémies , Liaison aux protéines
9.
Electron. j. biotechnol ; Electron. j. biotechnol;53: 61-70, Sep.2021. ilus, tab
Article de Anglais | LILACS | ID: biblio-1451290

RÉSUMÉ

BACKGROUND Heat shock proteins (HSPs) play important roles in the responses to different environmental stresses. In this study, the genomic and proteomic characteristics of three HSPs (HSP70, HSP90-a and HSP90-b) in five even-toed ungulates (sheep, goats, water buffalo, Zebu cattle and cattle) were analyzed using Multiple sequence alignment, SWISS modeling and phylogenetics analysis tools. RESULTS The bioinformatic analysis revealed that the HSP70 gene in cattle, Zebu cattle, and goat is located on chromosome 23, and is intronless, while in water buffalo and sheep it is located on chromosomes 2 and 20, respectively, and contains two exons linked by one intron. The HSP90-a gene is located on chromosome 21 in cattle, Zebu cattle, and goat, while in water buffalo and sheep it is located on chromosomes 20 and 18, respectively. The HSP90-b gene is located on the same chromosome as the HSP70 gene and contains 12 exons interspersed by 11 introns in all studied animals. In silico Expasy translate tool analysis revealed that HSP70, HSP90-a and HSP90-b encode 641, 733, and 724 amino acids, respectively. The data revealed that goat HSP70 protein has seven variable amino acid residues, while in both sheep and cattle only one such amino acid was detected. CONCLUSIONS This study will be supportive in providing new insights into HSPs for adaptive machinery in these studied animals and selection of target genes for molecular adaptation of livestock


Sujet(s)
Animaux , Protéines du choc thermique HSP90/génétique , Protéines du choc thermique HSP70/génétique , Buffles/génétique , Bovins/génétique , Capra/génétique , Ovis/génétique , Génome , Protéines du choc thermique HSP90/métabolisme , Protéines du choc thermique HSP70/métabolisme
10.
Clin Transl Oncol ; 23(8): 1688-1704, 2021 Aug.
Article de Anglais | MEDLINE | ID: mdl-33792840

RÉSUMÉ

Osteosarcoma, a common malignant tumor in orthopedics, often has a very poor prognosis after lung metastasis. Immunotherapy has not achieved much progress in the treatment because of the characteristics of solid tumors and immune environment of osteosarcoma. The tumor environment is rather essential for sarcoma treatment. Our previous study demonstrated that heat shock proteins could be used as antitumor vaccines by carrying tumor antigen peptides, and we hypothesize that an anti-osteosarcoma effect may be increased with an immune check point inhibitor (PD-L1 inhibitor) as a combination treatment strategy. The present study prepared a multisubtype mixed heat shock protein osteosarcoma vaccine (mHSP/peptide vaccine) and concluded that the mHSP/peptide vaccine was more effective than a single subtype heat shock protein, like Grp94. Therefore, we used the mHSP/peptide vaccine in combination with a PD-L1 inhibitor to treat osteosarcoma, and the deterioration of osteosarcoma was effectively hampered. The mechanism of combined therapy was investigated, and AKT expression participates with sarcoma lung metastasis. This study proposed an antisarcoma strategy via stimulation of the immune system as a further alternative approach for sarcoma treatment and elucidated the mechanism of combined therapy.


Sujet(s)
Tumeurs osseuses/thérapie , Vaccins anticancéreux/usage thérapeutique , Protéines du choc thermique/usage thérapeutique , Inhibiteurs de points de contrôle immunitaires/usage thérapeutique , Ostéosarcome/thérapie , Animaux , Tumeurs osseuses/immunologie , Tumeurs osseuses/anatomopathologie , Tumeurs osseuses/secondaire , Lignée cellulaire tumorale , Association thérapeutique/méthodes , Modèles animaux de maladie humaine , Femelle , Protéines du choc thermique HSP70/métabolisme , Protéines du choc thermique HSP90/métabolisme , Humains , Immunothérapie/méthodes , Interféron gamma/pharmacologie , Tumeurs du poumon/génétique , Tumeurs du poumon/secondaire , Glycoprotéines membranaires/métabolisme , Souris de lignée C57BL , Protéines tumorales/immunologie , Protéines tumorales/usage thérapeutique , Ostéosarcome/génétique , Ostéosarcome/immunologie , Ostéosarcome/secondaire , Protéines proto-oncogènes c-akt/métabolisme , Échappement de la tumeur à la surveillance immunitaire
11.
Int. j. morphol ; 39(1): 18-24, feb. 2021. ilus, tab
Article de Anglais | LILACS | ID: biblio-1385293

RÉSUMÉ

SUMMARY: Diabetes is a metabolic disorder characterized by high blood sugar levels and it causes complications in many systems, including the reproductive system. As a result of diabetic conditions, one of the mechanisms that can cause repression of reproductive activity is testicular oxidant stress. The identification of diabetes on the cell signaling molecules axis is still under discussion. The aim of this study was to determine the effect of Transforming Growth Factor (TGFβ), Nuclear Factor kappa B (NF-kB), Heat-schock 90β (HSP90β) signal pathways and E-cadherin cell adhesion molecule on infertility in diabetic rat testicular tissue. In our study, includes histological, molecular and biochemical analysis of testicular tissue removed at the end of the 2 weeks experiment period. A total of 14 adult male rats were divided as control and diabetes. No intervention was given to 7 male rats in the control group. For the diabetic group, 7 male rats were injected by intraperitoneal with a single dose of 55 mg/kg streptozotocin (STZ). TGFβ, NF-kB, HSP90β and E-cadherin proteins were immunohistochemically studied to investigate possible tissue damage, inflammatory process, cell stabilization and integrity due to diabetes. In order to determine oxidant stress, lipid peroxidation product malondialdehyde (MDA), glutathione (GSH) and glutathione peroxidase (GPx) analyzes were performed. Fibrosis, inflammatory changes and loss of spermatogenetic series are prominent findings in the diabetic group. On analysis of all the samples with immunostaining, in the diabetic group, TGFβ and NF-kB immunoexpression significantly increased, while Hsp90β and E-cadherin immunoexpression significantly decreased compared with control groups. Experimental diabetes was found to cause fibrosis, inflammation, disrupting cell adhesion and stabilization in testicular tissue. These results suggest that cellular therapy studies are needed for possible damage.


RESUMEN: La diabetes es una enfermedad metabólica caracterizada por niveles altos de azúcar en sangre y causa complicaciones en muchos sistemas, incluido el sistema reproductivo. Como resultado de las condiciones diabéticas, uno de los mecanismos que puede causar alteraciones en la actividad reproductiva es el estrés oxidativo testicular. La identificación de la diabetes en el eje de las moléculas de señalización celular aún está en discusión. El objetivo de este estudio fue determinar el efecto del factor de crecimiento transformante (TGFβ), el factor nuclear kappa B (NF-kB), las vías de señalización de Heat-Schock 90b (HSP90β) y la molécula de adhesión celular de E-cadherina sobre la infertilidad en testículo de rata diabética. Al término de dos semanas se realizaron análisis histológico, molecular y bioquímico del tejido testicular extraído. Las 7 ratas macho del grupo control no fueron intervenidas. Para el grupo de diabéticos, 7 ratas macho fueron inyectadas por vía intraperitoneal con una dosis única de 55 mg / kg de estreptozotocina (STZ). Se estudiaron inmunohistoquímicamente las proteínas TGFβ, NF-kB, HSP90β y E-cadherina para investigar el posible daño tisular, el proceso inflamatorio, la estabilización celular y la integridad debido a la diabetes. Para determinar el estrés oxidativo, se realizaron análisis del producto de peroxidación lipídica malondialdehído (MDA), glutatión (GSH) y glutatión peroxidasa (GPx). La fibrosis, los cambios inflamatorios y la pérdida de series espermatogenéticas son hallazgos destacados en el grupo de ratas diabéticas. En el análisis de todas las muestras con inmunotinción, en el grupo diabético, la inmunoexpresión de TGFβ y NF-kB aumentó significativamente, mientras que la inmunoexpresión de Hsp90β y e-cadherina disminuyó significativamente en comparación con los grupos control. Se encontró que la diabetes experimental causa fibrosis, inflamación, alteración de la adhesión celular y estabilización en el tejido testicular. Estos resultados sugieren que son necesarios estudios de terapia celular para verificar posibles daños.


Sujet(s)
Animaux , Mâle , Rats , Testicule/anatomopathologie , Diabète expérimental/métabolisme , Testicule/métabolisme , Immunohistochimie , Facteurs de croissance transformants/métabolisme , Cadhérines/métabolisme , Facteur de transcription NF-kappa B/métabolisme , Protéines du choc thermique HSP90/métabolisme
12.
Cell Microbiol ; 23(2): e13273, 2021 02.
Article de Anglais | MEDLINE | ID: mdl-33010083

RÉSUMÉ

The initiation of Aspergillus fumigatus infection occurs via dormant conidia deposition into the airways. Therefore, conidial germination and subsequent hyphal extension and growth occur in a sustained heat shock (HS) environment promoted by the host. The cell wall integrity pathway (CWIP) and the essential eukaryotic chaperone Hsp90 are critical for fungi to survive HS. Although A. fumigatus is a thermophilic fungus, the mechanisms underpinning the HS response are not thoroughly described and important to define its role in pathogenesis, virulence and antifungal drug responses. Here, we investigate the contribution of the CWIP in A. fumigatus thermotolerance. We observed that the CWIP components PkcA, MpkA and RlmA are Hsp90 clients and that a PkcAG579R mutation abolishes this interaction. PkcAG579R also abolishes MpkA activation in the short-term response to HS. Biochemical and biophysical analyses indicated that Hsp90 is a dimeric functional ATPase, which has a higher affinity for ADP than ATP and prevents MpkA aggregation in vitro. Our data suggest that the CWIP is constitutively required for A. fumigatus to cope with the temperature increase found in the mammalian lung environment, emphasising the importance of this pathway in supporting thermotolerance and cell wall integrity.


Sujet(s)
Adaptation physiologique , Aspergillus fumigatus/physiologie , Paroi cellulaire/physiologie , Protéines du choc thermique HSP90/métabolisme , Réaction de choc thermique , Aspergillose/microbiologie , Protéines fongiques/métabolisme , Régulation de l'expression des gènes fongiques , Interactions hôte-microbes , Mutation , Protéine kinase C/métabolisme , Transduction du signal , Spores fongiques/croissance et développement , Virulence
13.
Anat Histol Embryol ; 50(1): 50-57, 2021 Jan.
Article de Anglais | MEDLINE | ID: mdl-32776605

RÉSUMÉ

Heat shock proteins play a crucial role in cellular development, proliferation, differentiation and apoptosis. Heat shock protein 90 (HSP90) has been localised in the human endometrium, where its immunoexpression changes during the menstrual cycle. Similar studies have not been done for the equid species, so the present study aimed to describe endometrial HSP90 immunoexpression in mare endometrium. Endometrial biopsies were formalin-fixed and paraffin-embedded, and sections were stained with haematoxylin-eosin in preparation for HSP90 immunohistochemistry. Immunostaining and morphometric analyses were performed on the epithelial lining, endometrial glands and connective stroma during oestrus, dioestrus phase and anoestrus period (n = 7 per phase or period). Immunoexpression was localised in the basal region of the epithelial cells lining the lumen. Immunoexpression was greater during oestrus than during either dioestrus or anoestrus. During anoestrus, there was little immunostaining in the endometrium, suggesting that HSP90 is involved in the functional modulation of sex steroid receptors in cyclic mares. Indeed, the function of HSP90 as a chaperone in the folding of proteins, such as steroid receptors, might explain the greater intensity of immunostaining during the oestrus and dioestrus phases, compared the anoestrus period. We conclude that, in the mare, HSP90 plays a role in endometrial function and that further studies are needed to test whether it is important in pathological conditions as endometritis.


Sujet(s)
Anoestrus/physiologie , Dioestrus/physiologie , Endomètre/métabolisme , Oestrus/physiologie , Protéines du choc thermique HSP90/métabolisme , Equus caballus/physiologie , Animaux , Femelle , Régulation de l'expression des gènes/effets des médicaments et des substances chimiques , Protéines du choc thermique HSP90/génétique , Immunohistochimie/médecine vétérinaire
14.
J Cell Sci ; 133(12)2020 06 16.
Article de Anglais | MEDLINE | ID: mdl-32467326

RÉSUMÉ

It has been demonstrated that tetratricopeptide-repeat (TPR) domain proteins regulate the subcellular localization of glucocorticoid receptor (GR). This study analyses the influence of the TPR domain of high molecular weight immunophilins in the retrograde transport and nuclear retention of GR. Overexpression of the TPR peptide prevented efficient nuclear accumulation of the GR by disrupting the formation of complexes with the dynein-associated immunophilin FKBP52 (also known as FKBP4), the adaptor transporter importin-ß1 (KPNB1), the nuclear pore-associated glycoprotein Nup62 and nuclear matrix-associated structures. We also show that nuclear import of GR was impaired, whereas GR nuclear export was enhanced. Interestingly, the CRM1 (exportin-1) inhibitor leptomycin-B abolished the effects of TPR peptide overexpression, although the drug did not inhibit GR nuclear export itself. This indicates the existence of a TPR-domain-dependent mechanism for the export of nuclear proteins. The expression balance of those TPR domain proteins bound to the GR-Hsp90 complex may determine the subcellular localization and nucleocytoplasmic properties of the receptor, and thereby its pleiotropic biological properties in different tissues and cell types.


Sujet(s)
Récepteurs aux glucocorticoïdes , Répétition tétratricopeptide , Transport nucléaire actif , Noyau de la cellule/métabolisme , Protéines du choc thermique HSP90/métabolisme , Pore nucléaire/métabolisme , Récepteurs aux glucocorticoïdes/génétique , Récepteurs aux glucocorticoïdes/métabolisme , Protéines de liaison au tacrolimus/génétique , Protéines de liaison au tacrolimus/métabolisme
15.
Int J Biol Macromol ; 156: 522-530, 2020 Aug 01.
Article de Anglais | MEDLINE | ID: mdl-32302629

RÉSUMÉ

Cellular proteostasis is maintained by a system consisting of molecular chaperones, heat shock proteins (Hsps) and proteins involved with degradation. Among the proteins that play important roles in the function of this system is Hsp90, which acts as a node of this network, interacting with at least 10% of the proteome. Hsp90 is ATP-dependent, participates in critical cell events and protein maturation and interacts with large numbers of co-chaperones. The study of Hsp90 orthologs is justified by their differences in ATPase activity levels and conformational changes caused by Hsp90 interaction with nucleotides. This study reports the characterization of Hsp90 from Aedes aegypti, a vector of several diseases in many regions of the planet. Aedes aegypti Hsp90, AaHsp90, was cloned, purified and characterized for its ATPase and chaperone activities and structural conformation. These parameters indicate that it has the characteristics of eukaryotic Hsp90s and resembles orthologs from yeast rather than from human. Finally, constitutive and increased stress expression in Aedes cells was confirmed. Taken together, the results presented here help to understand the relationship between structure and function in the Hsp90 family and have strong potential to form the basis for studies on the network of chaperone and Hsps in Aedes.


Sujet(s)
Aedes , Protéines du choc thermique HSP90/composition chimique , Réaction de choc thermique , Protéines d'insecte/composition chimique , Conformation des protéines , Adenosine triphosphatases/composition chimique , Adenosine triphosphatases/métabolisme , Aedes/métabolisme , Séquence d'acides aminés , Animaux , Clonage moléculaire , Expression des gènes , Protéines du choc thermique HSP90/génétique , Protéines du choc thermique HSP90/métabolisme , Hydrodynamique , Protéines d'insecte/métabolisme
16.
Molecules ; 25(4)2020 Feb 20.
Article de Anglais | MEDLINE | ID: mdl-32093392

RÉSUMÉ

A series of benzo[g]benzothiazolo[2,3-b]quinazoline-7,12-quinones were prepared from 2-acylnaphthohydroquinones and 2-aminobenzothiazoles and were evaluated for their in vitro antiproliferative activity. After screening using the MTT reduction assay, their IC50 values were calculated on a panel of cancer cells (T24, DU-145, MCF-7). Current standard anticancer drugs were included as control, and their calculated IC50 values were 7.8 and 23.5 µM for 5-fluorouracil and tamoxifen, respectively. Non-cancer cells (AG1523) were included to assess cancer cell sensitivity and drug selectivity. Four members of the series, with IC50 values from 0.11 to 2.98 µM, were chosen for further assays. The selected quinones were evaluated regarding their effects on cancer cell proliferation (clonogenic assay) and on Hsp90 and poly(ADPribose)polymerase (PARP) protein integrity. The most active compound (i.e., 15) substantially inhibited colony forming unit (CFU) formation at 0.25 µM. In the presence of ascorbate, it induced an oxidative cleavage of Hsp90 but had no effect on PARP protein integrity. In an in vivo animal model, it discreetly increased the mean survival time (m.s.t.) of tumor-bearing mice. In light of these results, compound 15 represents a potential lead-molecule to be further developed.


Sujet(s)
Antinéoplasiques , Prolifération cellulaire/effets des médicaments et des substances chimiques , Protéines du choc thermique HSP90 , Protéines tumorales , Tumeurs expérimentales , Quinazolines , Animaux , Antinéoplasiques/synthèse chimique , Antinéoplasiques/composition chimique , Antinéoplasiques/pharmacologie , Acide ascorbique , Protéines du choc thermique HSP90/antagonistes et inhibiteurs , Protéines du choc thermique HSP90/métabolisme , Humains , Cellules MCF-7 , Souris , Protéines tumorales/antagonistes et inhibiteurs , Protéines tumorales/métabolisme , Tumeurs expérimentales/traitement médicamenteux , Tumeurs expérimentales/métabolisme , Tumeurs expérimentales/anatomopathologie , Quinazolines/synthèse chimique , Quinazolines/composition chimique , Quinazolines/pharmacologie , Tests d'activité antitumorale sur modèle de xénogreffe
17.
J Enzyme Inhib Med Chem ; 35(1): 639-649, 2020 Dec.
Article de Anglais | MEDLINE | ID: mdl-32048531

RÉSUMÉ

Leishmaniasis is a neglected disease caused by the protozoa Leishmania ssp. Environmental differences found by the parasites in the vector and the host are translated into cellular stress, leading to the production of heat shock proteins (Hsp). These are molecular chaperones involved in the folding of nascent proteins as well as in the regulation of gene expression, signalling events and proteostasis. Since Leishmania spp. use Hsp90 to trigger important transitions between their different stages of the life cycle, this protein family becomes a profitable target in anti-parasite drug discovery. In this work, we implemented a multidisciplinary strategy coupling molecular modelling with in vitro assays to identify small molecules able to inhibit Hsp90 from L. braziliensis (LbHsp90). Overall, we identified some compounds able to kill the promastigote form of the L. braziliensis, and to inhibit LbHsp90 ATPase activity.


Sujet(s)
Antiprotozoaires/pharmacologie , Antienzymes/pharmacologie , Protéines du choc thermique HSP90/antagonistes et inhibiteurs , Leishmania brasiliensis/effets des médicaments et des substances chimiques , Chaperons moléculaires/pharmacologie , Bibliothèques de petites molécules/pharmacologie , Adenosine triphosphatases/antagonistes et inhibiteurs , Adenosine triphosphatases/métabolisme , Antiprotozoaires/synthèse chimique , Antiprotozoaires/composition chimique , Relation dose-effet des médicaments , Découverte de médicament , Antienzymes/synthèse chimique , Antienzymes/composition chimique , Protéines du choc thermique HSP90/métabolisme , Leishmania brasiliensis/composition chimique , Modèles moléculaires , Chaperons moléculaires/synthèse chimique , Chaperons moléculaires/composition chimique , Structure moléculaire , Tests de sensibilité parasitaire , Bibliothèques de petites molécules/synthèse chimique , Bibliothèques de petites molécules/composition chimique , Relation structure-activité
18.
Mycotoxin Res ; 36(1): 23-30, 2020 Feb.
Article de Anglais | MEDLINE | ID: mdl-31264166

RÉSUMÉ

The mycotoxin enniatin B1 (ENN B1) is widely present in grain-based feed and food products. In the present study, we have investigated how this lipophilic and ionophoric molecule can affect the lysosomal stability and chaperone-mediated autophagy (CMA) in wild-type (WT) and in lysosome-associated membrane proteins (LAMP)-1/2 double-deficient (DD) mouse embryonic fibroblasts (MEF). The cell viability and lysosomal pH were assessed using the Neutral Red (NR) cytotoxicity assay and the LysoSensor® Yellow/Blue DND-160, respectively. Changes in the expression of the CMA-related components LAMP-2 and the chaperones heat shock cognate (hsc) 70 and heat shock protein (hsp) 90 were determined in cytosolic extracts by immunoblotting. In the NR assay, LAMP-1/2 DD MEF cells were significantly less sensitive to ENN B1 than WT MEF cells after 24 h exposure to ENN B1 at levels of 2.5-10 µmol/L. Exposure to ENN B1 at concentrations below the half maximal effective concentration (EC50) (1.5-1.7 µmol/L) increased the lysosomal pH in WT MEF, but not in LAMP-1/2 DD cells, suggesting that lysosomal LAMP-2 is an early target of ENN B1-induced lysosomal alkalization and cytotoxicity in MEF cells. Additionally, cytosolic hsp90 and LAMP-2 levels slightly increased after exposure for 4 h, indicating lysosomal membrane permeabilization (LMP). In summary, it appeared that ENN B1 can destabilize the LAMP-2 complex in the lysosomal membrane at concentrations close to the EC50, resulting in the alkalinization of lysosomes, partial LMP, and thereby leakage of CMA-associated components into the cytosol.


Sujet(s)
Depsipeptides/toxicité , Membranes intracellulaires/effets des médicaments et des substances chimiques , Lysosomes/anatomopathologie , Mycotoxines/toxicité , Perméabilité/effets des médicaments et des substances chimiques , Animaux , Autophagie médiée par les chaperonnes/effets des médicaments et des substances chimiques , Fibroblastes , Délétion de gène , Protéines du choc thermique HSC70/effets des médicaments et des substances chimiques , Protéines du choc thermique HSC70/métabolisme , Protéines du choc thermique HSP90/effets des médicaments et des substances chimiques , Protéines du choc thermique HSP90/métabolisme , Concentration en ions d'hydrogène/effets des médicaments et des substances chimiques , Protéine de membrane-2 associée au lysosome/effets des médicaments et des substances chimiques , Protéine de membrane-2 associée au lysosome/génétique , Protéine de membrane-2 associée au lysosome/métabolisme , Souris , Chaperons moléculaires/effets des médicaments et des substances chimiques , Chaperons moléculaires/métabolisme
19.
Sci Rep ; 9(1): 14756, 2019 10 14.
Article de Anglais | MEDLINE | ID: mdl-31611575

RÉSUMÉ

Leishmaniasis is a neglected disease that affects millions of individuals around the world. Regardless of clinical form, treatment is based primarily on the use of pentavalent antimonials. However, such treatments are prolonged and present intense side effects, which lead to patient abandonment in many cases. The search for chemotherapeutic alternatives has become a priority. Heat Shock Protein 90 (Hsp90) inhibitors have recently come under investigation due to antiparasitic activity in Plasmodium sp., Trypanosoma sp. and Leishmania sp. Some of these inhibitors, such as geldanamycin and its analogs, 17-AAG and 17-DMAG, bind directly to Hsp90, thereby inhibiting its activity. Previous studies have demonstrated that different parasite species are more susceptible to some of these inhibitors than host cells. We hypothesized that this increased susceptibility may be due to differences in binding of Hsp90 inhibitors to Leishmania protein compared to host protein. Based on the results of the in silico approach used in the present study, we propose that geldanamycin, 17-AAG and 17-DMAG present an increased tendency to bind to the N-terminal domain of Leishmania amazonensis Hsp83 in comparison to human Hsp90. This could be partially explained by differences in intermolecular interactions between each of these inhibitors and Hsp83 or Hsp90. The present findings demonstrate potential for the use of these inhibitors in the context of anti-Leishmania therapy.


Sujet(s)
Benzoquinones/pharmacologie , Protéines du choc thermique HSP90/antagonistes et inhibiteurs , Lactames macrocycliques/pharmacologie , Leishmania/effets des médicaments et des substances chimiques , Protéines de protozoaire/antagonistes et inhibiteurs , Trypanocides/pharmacologie , Benzoquinones/composition chimique , Protéines du choc thermique HSP90/métabolisme , Humains , Lactames macrocycliques/composition chimique , Leishmania/métabolisme , Leishmaniose/traitement médicamenteux , Leishmaniose/parasitologie , Simulation de docking moléculaire , Protéines de protozoaire/métabolisme , Trypanocides/composition chimique
20.
PLoS One ; 14(7): e0220393, 2019.
Article de Anglais | MEDLINE | ID: mdl-31361762

RÉSUMÉ

The wild type huntingtin protein (Htt), supports the production of brain-derived neurotrophic factor (BDNF), a survival factor for striatal neurons, through cytoplasmic sequestering of RE-1silencing transcription factor (REST). In Huntington´s Disease an inherited degenerative disease, caused by a CAG expansion in the 5´coding region of the gene, the mutant huntingtin protein (mHtt), causes that REST enters pathologically into the nucleus of cells, resulting in the repression of neuronal genes including BDNF, resulting in the progressive neuronal death. It has been reported that Htt associates with Hsp90 and this interaction is involved in regulation of huntingtin aggregation. Discovering mechanisms to reduce the cellular levels of mutant huntingtin and REST provide promising strategies for treating Huntington disease. Here, we use the yeast two-hybrid system to show that N-terminus or REST interacts with the heat shock protein 90 (Hsp90) and identifies REST as an Hsp90 Client Protein. To assess the effects of Hsp90 we used antisense oligonucleotide, and evaluated the levels mHtt and REST levels. Our results show that direct knockdown of endogenous Hsp90 significantly reduces the levels of REST and mutant Huntingtin, decreased the percentage of cells with mHtt in nucleus and rescued cells from mHtt-induced cellular cytotoxicity. Additionally Hsp90-specific inhibitors geldanamicyn and PUH71 dramatically reduced mHtt and REST levels, thereby providing neuroprotective activity. Our data show that Hsp90 is necessary to maintain the levels of REST and mHtt, which suggests that the interactions between Hsp90-REST and Hsp90-Huntingtin could be potential therapeutic targets in Huntington's disease.


Sujet(s)
Protéines du choc thermique HSP90/métabolisme , Protéine huntingtine/métabolisme , Maladie de Huntington/métabolisme , Protéines de répression/composition chimique , Protéines de répression/métabolisme , Benzoquinones/pharmacologie , Sites de fixation , Lignée cellulaire , Noyau de la cellule/métabolisme , Cytoplasme/métabolisme , Techniques de knock-down de gènes , Protéines du choc thermique HSP90/génétique , Humains , Protéine huntingtine/génétique , Maladie de Huntington/génétique , Lactames macrocycliques/pharmacologie , Modèles biologiques , Mutation , Liaison aux protéines/effets des médicaments et des substances chimiques
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