Molecular chaperones help other proteins to achieve and maintain their proper conformation. Chaperones bind to newly synthesized or unfolded polypeptide chains, actively modify their conformation and participate on their transport or degradation. Chaperones play an important role in cancer cell, where their increased activity enables stabilization of many mutant proteins and overcoming the stress generated by genetic instability. Hsp90 represents a key chaperone in cancer cells. Growth factor receptors, steroid hormone receptors and signal proctor teins are among its substrates, so-called client proteins; many of them being targets for anti-cancer therapy. Adverse conditions of the tumor microenvironment, such as hypoxia and nutrient deficiency, contribute to destabilization of proteins and further escalate dependence on chaperones. This is why molecular chaperones, in particular Hsp90, may represent a promising target for anticancer therapy. Importantly also, tumour-based Hsp90 has a significantly higher sensitivity to inhibitors than that in normal cells, and Hsp90 activity inhibition in tumours leads to a suppression of cellular signaling in many different oncogenic pathways. Several inhibitors of Hsp90 are currently undergoing clinical evaluation and new agents with different mechanisms of action are continually being identified.

• MeSH
• lidé MeSH
• molekulární chaperony antagonisté a inhibitory   fyziologie   MeSH
• nádory patofyziologie   MeSH
• proteiny tepelného šoku HSP90 antagonisté a inhibitory   fyziologie   MeSH
• protinádorové látky terapeutické užití   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• molekulární chaperony MeSH
• proteiny tepelného šoku HSP90 MeSH
• protinádorové látky MeSH

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.

• MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory prsu chemie   farmakoterapie   patologie   MeSH
• proteiny tepelného šoku HSP90 analýza   antagonisté a inhibitory   fyziologie   MeSH
• průtoková cytometrie MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny tepelného šoku HSP90 MeSH

Giardia lambliacauses giardiasis, one of the most common human infectious diseases globally. Previous studies from our lab have shown that hsp90 gene ofGiardia is split into two halves, namely hspN and hspC. The independent pre-mRNAs of these split genes join by trans-splicing, producing a full-length Hsp90 (FlHsp90) mRNA. Genetic manipulation of the participating genes is necessary to understand the mechanism and significance of such trans-splicing based expression of Hsp90. In this study, we have performed transfection based exogenous expression of hspN and/or hspC in G. lamblia. We electroporated a plasmid containing the Avi-tagged hspN component of Hsp90 and examined its fate in G. lamblia. We show that the exogenously expressed hspN RNA gets trans-spliced to endogenously expressed hspC RNA, giving rise to a hybrid-FlHsp90. We highlight the importance of cis-elements in this trans-splicing reaction through mutational analysis. The episomal plasmid carrying deletions in the intronic region of hspN, showed inhibition of the trans-splicing reaction.Additionally, exogenous hspC RNA also followed the same fate as of exogenous hspN, while upon co-transfection with episomal hspN, they underwent trans-splicing with each other. Using eGFP as a test protein, we have shown that intronic sequences of hsp90 gene can guide trans-splicing mediated repair of any associated exonic sequences. Our study provides in vivo validation of Hsp90 trans-splicing, showing crucial role of cis-elements and importantly highlights the potential of hsp90 intronic sequences to function as a minimal splicing tool.

• Klíčová slova
• Gene expression, Giardia lamblia, Hsp90, RNA splicing, Transfection,
• MeSH
• Giardia lamblia * genetika   MeSH
• introny genetika   MeSH
• prekurzory RNA genetika   MeSH
• proteiny tepelného šoku HSP90 * genetika   MeSH
• protozoální proteiny * genetika   MeSH
• trans-splicing * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• prekurzory RNA MeSH
• proteiny tepelného šoku HSP90 * MeSH
• protozoální proteiny * MeSH

BACKGROUND: Chaperone system inhibition is a recent promising strategy for cancer treatment that exploits increased metabolic needs required for rapid proliferation as well as higher level of proteotoxic stress in neoplastic cells. Chaperone HSP90 plays a key role in proper folding of many de novo synthesized proteins, so-called clients, including tumor suppressor p53 which is commonly mutated in majority of cancers. Aim of this work was therefore to understand the impact of HSP90 inhibition by NVP-AUY922 on breast cancer cell lines with wild-type and mutated p53. METHODS: Flow cytometry was used to analyze cell viability by fluorescein diacetate assay and changes in cell cycle. Western blotting was used to analyze expression of p53 and p21 proteins. RESULTS: Analysis of cell viability after HSP90 inhibition revealed higher sensitivity of cell line with wild-type p53. Cell cycle analysis then showed that both cell lines undergo increase in G2/M block of the cell cycle, but wild-type cell line had also substantial decrease in proliferative capacity of treated cells. We also observed increased expression of negative cell cycle regulator p21 in cell line with wild-type p53. CONCLUSIONS: Since p21 is directly regulated by p53, our results suggest that mutation status of p53 can be important factor in treatment of breast cancer cells by HSP90 chaperone inhibition and that wild-type p53 can increase sensitivity to HSP90 inhibition.Key words: breast cancer - cell cycle - chaperone - HSP90 - TP53 - p21 - p53 - NVP-AUY922This work was supported by the project 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: 6. 5. 2016Accepted: 17. 5. 2016.

• MeSH
• inhibitor p21 cyklin-dependentní kinasy metabolismus   MeSH
• isoxazoly farmakologie   MeSH
• kontrolní body fáze G2 buněčného cyklu účinky léků   MeSH
• kontrolní body M fáze buněčného cyklu účinky léků   MeSH
• lidé MeSH
• mutace MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• nádory prsu farmakoterapie   genetika   MeSH
• proliferace buněk účinky léků   MeSH
• proteiny tepelného šoku HSP90 antagonisté a inhibitory   MeSH
• protinádorové látky farmakologie   MeSH
• resorcinoly farmakologie   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-morpholin-4-ylmethylphenyl)isoxazole-3-carboxylic acid ethylamide MeSH Prohlížeč
• CDKN1A protein, human MeSH Prohlížeč
• inhibitor p21 cyklin-dependentní kinasy MeSH
• isoxazoly MeSH
• nádorový supresorový protein p53 MeSH
• proteiny tepelného šoku HSP90 MeSH
• protinádorové látky MeSH
• resorcinoly MeSH

Auxin homeostasis and signaling affect a broad range of developmental processes in plants. The interplay between HSP90 and auxin signaling is channeled through the chaperoning capacity of the HSP90 on the TIR1 auxin receptor. The sophisticated buffering capacity of the HSP90 system through the interaction with diverse signaling protein components drastically shapes genetic circuitries regulating various developmental aspects. However, the elegant networking capacity of HSP90 in the global regulation of auxin response and homeostasis has not been appreciated. Arabidopsis hsp90 mutants were screened for gravity response. Phenotypic analysis of root meristems and cotyledon veins was performed. PIN1 localization in hsp90 mutants was determined. Our results showed that HSP90 affected the asymmetrical distribution of PIN1 in plasma membranes and influenced its expression in prompt cell niches. Depletion of HSP90 distorted polar distribution of auxin, as the acropetal auxin transport was highly affected, leading to impaired root gravitropism and lateral root formation. The essential role of the HSP90 in auxin homeostasis was profoundly evident from early development, as HSP90 depletion affected embryo development and the pattern formation of veins in cotyledons. Our data suggest that the HSP90-mediated distribution of PIN1 modulates auxin distribution and thereby auxin signaling to properly promote plant development.

• Klíčová slova
• PIN1, auxin transport, cell size, cotyledon veins, embryo development, gravitropism, lateral roots, root meristem,
• MeSH
• Arabidopsis * genetika   metabolismus   MeSH
• kořeny rostlin růst a vývoj   MeSH
• kyseliny indoloctové MeSH
• membránové transportní proteiny metabolismus   MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• proteiny tepelného šoku HSP90 * genetika   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyseliny indoloctové MeSH
• membránové transportní proteiny MeSH
• PIN1 protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku * MeSH
• proteiny tepelného šoku HSP90 * MeSH

Heat shock proteins 90 (HSP90) are essential and play critical roles in the adaptation of organisms to diverse stimuli. In plants, HSP90 are involved in auxin, jasmonate and brassinosteroid (BR) signalling pathways. The BR-promoted activation of the BES1 transcription factor regulates BR-responsive genes. Using genetic, physiological, fluorescence live cell imaging, molecular and biochemical approaches, such as phenotypic analysis, co-immunoprecipitation assay, yeast-two hybrid and Bimolecular fluorescence complementation (BiFC), we studied complex formation between BES1 and HSP90 under control conditions and active BR signalling. Further, we determined the effect of the pharmacological inhibition of HSP90 ATPase activity on hypocotyl elongation of bes1-D mutant. We determined that HSP90 interact with BES1 in the nucleus and in the cytoplasm. During active BR signalling, nuclear complexes were absent while cytoplasmic HSP90/BES1 complexes were prominent. Our results showed that the hypocotyl length of bes1-D mutants was highly reduced when HSP90 was challenged by the geldanamycin (GDA) inhibitor of the ATPase activity of HSP90. Active BR signalling could not rescue the GDA effect on the hypocotyl elongation of bes1-D. Our results reveal that the constitutively active BES1 in the bes1-D mutant is hypersensitive to GDA. The interaction of HSP90 with BES1 argues that HSP90 facilitate the nuclear metastable conformation of BES1 to regulate BR-dependent gene expression, and our data show that HSP90 assist in the compartmentalised cycle of BES1 during active BR signalling.

• Klíčová slova
• Arabidopsis, BES1, BR signalling, GDA inhibition, hypocotyl elongation, molecular chaperones,
• MeSH
• Arabidopsis * MeSH
• brassinosteroidy * metabolismus   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• proteiny huseníčku * metabolismus   MeSH
• proteiny tepelného šoku HSP90 * metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• signální transdukce * MeSH
• tabák metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• BES1 protein, Arabidopsis MeSH Prohlížeč
• brassinosteroidy * MeSH
• DNA vazebné proteiny MeSH
• proteiny huseníčku * MeSH
• proteiny tepelného šoku HSP90 * MeSH

Geldanamycin remains a driver in the medicinal chemistry of heat shock protein 90 (Hsp90) inhibition, even half a century after its original isolation from nature. This Perspective focuses on the properties of the benzoquinone ring of the natural product that enable a range of functionalization reactions to take place. Therefore, inherent reactivity at C-17, where the methoxy group serves as a vinylogous ester, and at C-19 that demonstrates nucleophilic, enamide-type character toward electrophiles, and also as a conjugate acceptor to react with nucleophiles, has facilitated the synthesis of semisynthetic derivatives. Thus, a range of C-17-substituted amine derivatives has been investigated in oncology applications, with a number of compounds in this series reaching clinical trials. In contrast, the 19-position of geldanamycin has received less attention, although 19-substituted derivatives offer promise with markedly reduced toxicity compared to geldanamycin itself, while retaining Hsp90 inhibitory activity albeit with diminished potency in cellular studies.

• MeSH
• benzochinony * chemie   farmakologie   chemická syntéza   MeSH
• farmaceutická chemie metody   MeSH
• lidé MeSH
• makrocyklické laktamy * chemie   farmakologie   chemická syntéza   MeSH
• proteiny tepelného šoku HSP90 * antagonisté a inhibitory   metabolismus   MeSH
• protinádorové látky farmakologie   chemie   chemická syntéza   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• benzochinony * MeSH
• geldanamycin MeSH Prohlížeč
• makrocyklické laktamy * MeSH
• proteiny tepelného šoku HSP90 * MeSH
• protinádorové látky MeSH

Maintenance of protein homeostasis by molecular chaperones Hsp70 and Hsp90 requires their spatial and functional coordination. The cooperation of Hsp70 and Hsp90 is influenced by their interaction with the network of co-chaperone proteins, some of which contain tetratricopeptide repeat (TPR) domains. Critical to these interactions are TPR domains that target co-chaperone binding to the EEVD-COOH motif that terminates Hsp70/Hsp90. Recently, the two-TPR domain-containing protein, Tomm34, was reported to bind both Hsp70 and Hsp90. Here we characterize the structural basis of Tomm34-Hsp70/Hsp90 interactions. Using multiple methods, including pull-down assays, fluorescence polarization, hydrogen/deuterium exchange, and site-directed mutagenesis, we defined the binding activities and specificities of Tomm34 TPR domains toward Hsp70 and Hsp90. We found that Tomm34 TPR1 domain specifically binds Hsp70. This interaction is partly mediated by a non-canonical TPR1 two-carboxylate clamp and is strengthened by so far unidentified additional intermolecular contacts. The two-carboxylate clamp of the isolated TPR2 domain has affinity for both chaperones, but as part of the full-length Tomm34 protein, the TPR2 domain binds specifically Hsp90. These binding properties of Tomm34 TPR domains thus enable simultaneous binding of Hsp70 and Hsp90. Importantly, we provide evidence for the existence of an Hsp70-Tomm34-Hsp90 tripartite complex. In addition, we defined the basic conformational demands of the Tomm34-Hsp90 interaction. These results suggest that Tomm34 represents a novel scaffolding co-chaperone of Hsp70 and Hsp90, which may facilitate Hsp70/Hsp90 cooperation during protein folding.

• Klíčová slova
• Co-chaperone, H/D Exchange, Hsp70, Hsp90, Molecular Chaperone, Protein Assembly, Protein Folding, Protein-Protein Interactions, TPR Domain,
• MeSH
• aminokyselinové motivy MeSH
• HEK293 buňky MeSH
• kvarterní struktura proteinů MeSH
• lidé MeSH
• missense mutace MeSH
• mitochondriální importní komplex MeSH
• multiproteinové komplexy * chemie   genetika   metabolismus   MeSH
• mutageneze cílená MeSH
• proteiny tepelného šoku HSP70 * chemie   genetika   metabolismus   MeSH
• proteiny tepelného šoku HSP90 * chemie   genetika   metabolismus   MeSH
• sbalování proteinů * MeSH
• substituce aminokyselin MeSH
• terciární struktura proteinů MeSH
• transportní proteiny mitochondriální membrány * chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HSP90AA1 protein, human MeSH Prohlížeč
• HSPA1A protein, human MeSH Prohlížeč
• mitochondriální importní komplex MeSH
• multiproteinové komplexy * MeSH
• proteiny tepelného šoku HSP70 * MeSH
• proteiny tepelného šoku HSP90 * MeSH
• TOMM34 protein, human MeSH Prohlížeč
• transportní proteiny mitochondriální membrány * MeSH

Heat shock proteins (Hsp) are important for the stability and function of cell proteins and thus for cell survival under physiological as well as stress conditions. Hsps were also reported to play an important role in tumorogenesis including leukemias. In this study we followed up Hsp70 and 90 protein levels in samples from patients with chronic myeloid leukemia (CML) to evaluate these Hsps with regard to their ability to characterize the disease status and disease prognosis. We analyzed 68 samples of total leukocytes of CML patients with different response to therapy with tyrosine kinase inhibitors. The results of Western blot analyses showed that the level of Hsp70 did not change in the course of the disease and did not correlate with response to therapy. In contrast, Hsp90 levels showed good correlation with the disease state. Patients with good response to therapy (major molecular response-MMR, molecular remission-CMR) had low expression levels of Hsp90, similar to those in healthy individuals. High Hsp90 levels were found in patients with resistance to therapy (hematological relapse-HR, accelerated phase or blast crisis), and in leukemic cell line K562. The results of the study suggested that not the kinetics but the particular level of Hsp90 at any time point since therapy start is of prognostic value: Hsp90 level above 0.27 significantly predicted poor response to TKI therapy (relapse, progression) and the level below 0.085 good response (MMR, CMR). In conclusion, Hsp90 level in total leukocytes could serve as a risk factor at diagnosis as well as during therapy and might help in clinical decision making especially in cases where BCR-ABL monitoring is of low predictive value. Our data suggest that high expression of HSP90 contributes to the aggressivity of the disease and should be considered as an important target for specialized CML therapy.

• MeSH
• bcr-abl fúzní proteiny genetika   MeSH
• buňky K562 MeSH
• chronická myeloidní leukemie farmakoterapie   genetika   metabolismus   MeSH
• dospělí MeSH
• genetická transkripce MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• nádorové biomarkery * MeSH
• nádorové buněčné linie MeSH
• prognóza MeSH
• progrese nemoci MeSH
• proteiny tepelného šoku HSP90 metabolismus   MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bcr-abl fúzní proteiny MeSH
• nádorové biomarkery * MeSH
• proteiny tepelného šoku HSP90 MeSH

Stomatal development is tightly connected with the overall plant growth, while changes in environmental conditions, like elevated temperature, affect negatively stomatal formation. Stomatal ontogenesis follows a well-defined series of cell developmental transitions in the cotyledon and leaf epidermis that finally lead to the production of mature stomata. YODA signaling cascade regulates stomatal development mainly through the phosphorylation and inactivation of SPEECHLESS (SPCH) transcription factor, while HSP90 chaperones have a central role in the regulation of YODA cascade. Here, we report that acute heat stress affects negatively stomatal differentiation, leads to high phosphorylation levels of MPK3 and MPK6, and alters the expression of SPCH and MUTE transcription factors. Genetic depletion of HSP90 leads to decreased stomatal differentiation rates. Thus, HSP90 chaperones safeguard the completion of distinct stomatal differentiation steps depending on these two transcription factors under normal and heat stress conditions.

• Klíčová slova
• Stomata, differentiation, heat shock proteins 90, mitogen-activated protein kinases,
• MeSH
• Arabidopsis genetika   metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• proteiny tepelného šoku HSP90 metabolismus   MeSH
• průduchy rostlin metabolismus   MeSH
• reakce na tepelný šok fyziologie   MeSH
• regulace genové exprese u rostlin MeSH
• signální transdukce genetika   fyziologie   MeSH
• transkripční faktory bHLH genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• MUTE protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH
• proteiny tepelného šoku HSP90 MeSH
• SPEECHLESS protein, Arabidopsis MeSH Prohlížeč
• transkripční faktory bHLH MeSH