DOCSLIB.ORG

C-Terminal HSP90 Inhibitors Block the HSP90:HIF-1Α Interaction and Inhibit the Cellular Hypoxic Response

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
C-Terminal HSP90 Inhibitors Block the HSP90:HIF-1Α Interaction and Inhibit the Cellular Hypoxic Response bioRxiv preprint doi: https://doi.org/10.1101/521989; this version posted January 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. C-terminal HSP90 Inhibitors Block the HSP90:HIF-1α Interaction and Inhibit the Cellular Hypoxic Response Nalin Katariaa, Bernadette Kerra, Samantha S. Zaiterb, Shelli McAlpineb and Kristina M Cooka† a. University of Sydney, Faculty of Medicine and Health, Charles Perkins Centre, Sydney, Australia. b. School of Chemistry, University of New South Wales, Sydney, Australia. † To whom correspondence should be addressed: Kristina M Cook, Charles Perkins Centre, University of Sydney, Sydney, NSW, Australia 2006; [email protected]; Tel: +61 286274858. Hypoxia Inducible Factor (HIF) is a transcription factor cancer cells known as a heat shock response (HSR)12. The activated by low oxygen, which is common in solid compounds also have poor selectivity for HSP9013,14. tumours. HIF controls the expression of genes involved in C-terminus inhibitors of HSP90 (SM molecules)11 act in a angiogenesis, chemotherapy resistance and metastasis. The selective manner, and in contrast to the N-terminus chaperone HSP90 (Heat Shock Protein 90) stabilizes the inhibitors, they do not induce a heat shock response13,15. subunit HIF-1α and prevents degradation. Previously Although the SM molecules block all co-chaperones that identified HSP90 inhibitors bind to the N-terminal pocket bind to the C-terminus of HSP90 and inhibit HSP90 of HSP90 which blocks binding to HIF-1α, and produces function16,17, there is no data discussing whether C- HIF-1α degradation. N-terminal inhibitors have failed in terminal inhibitors will impact the binding event between the clinic as single therapy treatments due in part because HSP90 and HIF-1α or the subsequent hypoxic response. they induce a heat shock response, which increases This is the first study examining the effect of C-terminal chemotherapy resistance. SM molecules are HSP90 HSP90 inhibitors on the activity of HIF-1α in hypoxia and inhibitors that bind to the C-terminus and do not activate downstream gene expression. the heat shock response. The effects of C-terminal HSP90 Herein we report that the SM compounds prevent inhibitors on HIF-1α are unreported. Herein we show that accumulation of HIF-1α in HCT116 colorectal cells in SM compounds block binding between HSP90 and HIF-1α, hypoxia (0.5% O2) (Fig 1). HIF-1α substantially increases leading to HIF-1α degradation through the proteasome in HCT116 cells under hypoxia. When hypoxic HCT116 using the PHD/pVHL pathway in hypoxic conditions. The cells are incubated in the presence of SM122, HIF-1α SM compounds decrease HIF-1α target gene expression at protein levels decreased to normoxia levels. SM253 and the mRNA and protein level under hypoxia in colorectal SM258 also decreased HIF-1α in hypoxia (Fig 1a and 1b). cancer cells, leading to cell death, without inducing a heat The HSP90 inhibitors were also compared to chetomin, a shock response. Our results suggest that targeting the C- potent HIF-1 inhibitor that blocks the interaction between terminus of HSP90 blocks the hypoxic response and may HIF-1α and p300 by a zinc ejection mechanism2. SM122 be an effective anti-cancer strategy. reduces HIF-1α levels similar to that seen with chetomin (Fig. 1a and b). Hypoxia is common in tumors and activates a transcription Cells treated with 17-AAG (10 µM, 100 fold over the factor known as Hypoxia Inducible Factor-1 (HIF-1). HIF- GI50), an N-terminal HSP90 inhibitor16,17 show similar 1 controls the expression of genes involved in glycolysis, degradation of HIF-1α when treated with SM122 (10 µM), angiogenesis and cell survival, and is associated with a or SM252 (25µM) or SM258 (25µM). The C-terminal poor cancer prognosis1. Given that HIF-1 is upregulated in majority of solid tumors, there are ongoing drug development efforts to inhibit its activity. Previous work has focused on inhibiting the HIF-1α subunit from binding to partners such as p3002-5 or HIF-1β6, or binding to the DNA itself7. HIF-1 is a heterodimer composed of α and β subunits. Activity of the HIF-1 transcriptional complex is regulated post-translationally through degradation of HIF-1α in an oxygen-dependent process1,8. HIF-1α stability and Fig. 1 SM compounds inhibit HIF-1α stabilization under hypoxia. a) Nuclear HIF-1α levels in HCT116 cells after 6 h of hypoxia and SM degradation is further regulated by the chaperone Heat treatment. b) Densitometry of HIF-1α western blots. mean± SEM of 4-5 Shock Protein 90 (HSP90)9,10. N-terminal HSP90 experiments (except 25 µM SM122 which is n = 2). **P < 0.01, ***P < inhibitors such as geldanamycin and related analogues 0.001, as compared to hypoxic control. bind in the ATP binding pocket on the amino terminus (N- terminus) of HSP9011, disassociating it from HIF-1α and inhibitors, SM122, SM253, and SM258, all have a GI50 = 16,17 inducing pVHL-independent degradation of HIF-1α9,10. ~5 µM , yet they are highly effective at inducing the While N-terminal HSP90 inhibitors effectively block the degradation of HIF-1α when cells are treated with 2-5 fold due to their ability to induce a survival mechanism in over their GI50 (Fig. 1a and b), compared to 17-AAG, bioRxiv preprint doi: https://doi.org/10.1101/521989; this version posted January 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. which is treated at 100 fold over its GI50. The considerable indicate that SM molecules functionally reduce the amount of HIF-1α degradation in the presence of SM glucose transporter and are more potent than the N- compounds indicates that targeting the C-terminus of terminal inhibitor 17-AAG. HSP90 is a more effective strategy for decreasing HIF-1α SM253 and SM258 reduced LDHA (lactate than targeting the ATP binding pocket in the N-terminus. dehydrogenase A) mRNA expression under hypoxia (Fig HCT116 cells treated with SM compounds showed 2a). SM122 had little effect on LDHA mRNA. SM253 and varying degrees of inhibition of HIF-1 target gene SM258 both decreased LDHA protein to similar levels as expression in hypoxia based on both mRNA (Fig. 2a) and chetomin (~33%) (Fig 2b and c). Protein levels of protein levels (Fig 2b). The expression of carbonic hexokinase 2 (HK2), another enzyme involved in anhydrase IX (CA9) is regulated by HIF-1 and increases glycolysis, were significantly reduced by SM122 and under hypoxia (Fig. 2a). Both the N-terminal (17-AAG) SM253 (~40%) (Fig. 2b and c). SM258 decreased HK2 by and C-terminal compounds (SM) significantly decreased ~60%. Thus, SM122 was highly effective at reducing HIF- the expression of CA9 mRNA (Fig. 2a), indicating that 1 target protein expression for Glut1, LDHA and HK2 HIF-1 target gene expression is inhibited by both series. compared to N-terminal HSP90 inhibitor 17-AAG, whilst We also examined the mRNA and protein expression of SM253 and SM258 were slightly more effective than 17- multiple glycolytic genes under HIF-1 control upon AAG at reducing Glut1. This indicates that C-terminal SM exposure to hypoxia and HSP90 inhibitors. At the mRNA compounds, and particularly SM122, are effective at level, 25 µM SM258 and 25 µM and 10µM 17-AAG reducing glycolytic enzymes that are commonly significantly inhibited glucose transporter SLC2A1 upregulated in cancers. (Glut1) mRNA expression (Fig. 2a). To test the cytotoxic effect of SM compounds, we Comparing the compounds’ impact on Glut1 protein measured HCT116 cell viability at 18 and 72 h and expression, SM122 (25 and 10 µM), produced a compared to 17-AAG. The majority of cells were still significant reduction in the amount of Glut1, similar to viable at concentrations up to 25 µM at 18 h (see levels seen in normoxia (Fig. 2b and c). In contrast, 17- supplementary), where HIF-1 inhibition was documented AAG, slightly increased Glut-1 protein levels in hypoxia (Fig. 2). However, after 72 h, cell viability significantly decreased under normoxia and hypoxia for all drugs indicating HSP90 is essential for cell survival. A pitfall of N-terminal HSP90 inhibitors is that they induce a heat shock response (HSR). The HSR activates heat shock factor-1, a transcription factor that controls expression of HSP90, 70, 40 and 2718. The HSR therefore increases levels of HSP90, the protein targeted by N- terminal drugs, and activates mechanisms that block apoptosis and increase chemo-resistance18. SM HSP90 inhibitors do not induce the HSR in normoxia thus greatly increasing their cellular efficacy13. The HSR under hypoxic conditions is unknown, as is the impact of SM compounds on HSP protein levels. To test if a heat shock response is activated by N- and C- terminal HSP90 inhibitors in hypoxia, we measured HSP related proteins following an 18 h drug treatment. Treatments using 10 µM 17-AAG produced high levels of HSP70, and a slight increase in HSP90, while the SM compounds did not (Fig. 3a and b). We also compared the heat shock response from the HSP90 inhibitors to chetomin (Fig. 3a and b). At 1 µM chetomin, HSP70 significantly increases. This is not entirely unexpected given that the heat shock response can be induced by cell Fig 2 HIF-1 target gene expression is decreased by SM compounds in hypoxia. a) HIF-1 target mRNA expression. HCT116 cells were incubated in the presence or absence of compounds in hypoxia for 18 h. Total RNA was isolated, converted to cDNA and used for qPCR.
Load more

Recommended publications
  • Hsp90 Inhibitor As a Sensitizer of Cancer Cells to Different Therapies (Review)
    INTERNATIONAL JOURNAL OF ONCOLOGY 46: 907-926, 2015 Hsp90 inhibitor as a sensitizer of cancer cells to different therapies (Review) ZUZANA SOLÁROVÁ1, Ján MOJžiš1 and PETER SOLÁR2 1Department of Pharmacology, Faculty of Medicine, 2Laboratory of Cell Biology, Institute of Biology and Ecology, Faculty of Science, P.J. šafárik University, 040 01 Košice, Slovak Republic Received September 10, 2014; Accepted October 22, 2014 DOI: 10.3892/ijo.2014.2791 Abstract. Hsp90 is a molecular chaperone that maintains 1. Introduction the structural and functional integrity of various client proteins involved in signaling and many other functions of The cell responds to environmental stress by increasing cancer cells. The natural inhibitors, ansamycins influence the synthesis of several molecular chaperons known as heat shock Hsp90 chaperone function by preventing its binding to client proteins (Hsp). These proteins are cathegorized according to proteins and resulting in their proteasomal degradation. N- their molecular weight into five classes: small Hsp, Hsp60, and C-terminal inhibitors of Hsp90 and their analogues are Hsp70, Hsp90 and Hsp100. One of the most abundant widely tested as potential anticancer agents in vitro, in vivo molecular chaperones is Hsp90, which is a highly conserved as well as in clinical trials. It seems that Hsp90 competi- protein, whose association is required for the stability and tive inhibitors target different tumor types at nanomolar function of multiple-mutated, chimeric- and overexpressed- concentrations and might have therapeutic benefit. On the signaling proteins that promote the growth and/or survival contrary, some Hsp90 inhibitors increased toxicity and resis- of cancer cells (1). Hsp90 client proteins lack specific Hsp90 tance of cancer cells induced by heat shock response, and binding motifs and vary in terms of intracellular localization, through the interaction of survival signals, that occured as structure, and function.
    [Show full text]
  • Combination of Anti-Cancer Drugs with Molecular Chaperone Inhibitors
    International Journal of Molecular Sciences Review Combination of Anti-Cancer Drugs with Molecular Chaperone Inhibitors Maxim Shevtsov 1,2,3,4,5,6,* , Gabriele Multhoff 1 , Elena Mikhaylova 2, Atsushi Shibata 7 , Irina Guzhova 2 and Boris Margulis 2,* 1 Center for Translational Cancer Research, TUM (TranslaTUM), Technische Universität München (TUM), Klinikum rechts der Isar, Radiation Immuno Oncology, Einsteinstr. 25, 81675 Munich, Germany; gabriele.multhoff@tum.de 2 Institute of Cytology of the Russian Academy of Sciences (RAS), Tikhoretsky Ave., 4, St. Petersburg 194064, Russia; [email protected] (E.M.); [email protected] (I.G.) 3 Department of Biotechnology Pavlov First Saint Petersburg State Medical University, L’va Tolstogo str., 6/8, St. Petersburg 197022, Russia 4 Almazov National Medical Research Centre, Polenov Russian Scientific Research Institute of Neurosurgery, Mayakovskogo str., 12, St. Petersburg 191014, Russia 5 Department of Biomedical Cell Technologies Far Eastern Federal University, Russky Island, Vladivostok 690000, Russia 6 National Center for Neurosurgery, Turan Ave., 34/1, Nur-Sultan 010000, Kazakhstan 7 Signal Transduction Program, Gunma University Initiative for Advanced Research (GIAR), Gunma University, Maebashi, Gunma 371-8511, Japan; [email protected] * Correspondence: [email protected] (M.S.); [email protected] (B.M.) Received: 31 August 2019; Accepted: 22 October 2019; Published: 24 October 2019 Abstract: Most molecular chaperones belonging to heat shock protein (HSP) families are known to protect cancer cells from pathologic, environmental and pharmacological stress factors and thereby can hamper anti-cancer therapies. In this review, we present data on inhibitors of the heat shock response (particularly mediated by the chaperones HSP90, HSP70, and HSP27) either as a single treatment or in combination with currently available anti-cancer therapeutic approaches.
    [Show full text]
  • Holdase Activity of Secreted Hsp70 Masks Amyloid-Β42 Neurotoxicity in Drosophila
    Holdase activity of secreted Hsp70 masks amyloid-β42 neurotoxicity in Drosophila Pedro Fernandez-Funeza,b,c,1, Jonatan Sanchez-Garciaa, Lorena de Menaa, Yan Zhanga, Yona Levitesb, Swati Kharea, Todd E. Goldea,b, and Diego E. Rincon-Limasa,b,c,1 aDepartment of Neurology, McKnight Brain Institute, University of Florida, Gainesville, FL 32611; bDepartment of Neuroscience, Center for Translational Research on Neurodegenerative Diseases, University of Florida, Gainesville, FL 32611; and cGenetics Institute, University of Florida, Gainesville, FL 32611 Edited by Nancy M. Bonini, University of Pennsylvania, Philadelphia, PA, and approved July 11, 2016 (received for review May 25, 2016) Alzheimer’s disease (AD) is the most prevalent of a large group of cell-free systems by dissociating preformed oligomers but not fi- related proteinopathies for which there is currently no cure. Here, we brils, suggesting that Hsp70 targets oligomeric intermediates (18). used Drosophila to explore a strategy to block Aβ42 neurotoxicity More recent in vitro studies show that Hsp70 and other chaperones through engineering of the Heat shock protein 70 (Hsp70), a chap- promote the aggregation of oligomers into less toxic species (19). erone that has demonstrated neuroprotective activity against several Also, Hsp70 demonstrates neuroprotection against intracellular intracellular amyloids. To target its protective activity against extra- Aβ42 in primary cultures (20), whereas down-regulation of Hsp70 cellular Aβ42, we added a signal peptide to Hsp70. This secreted form leads to increased protein aggregation in transgenic worms of Hsp70 (secHsp70) suppresses Aβ42 neurotoxicity in adult eyes, expressing intracellular Aβ42 (21). A recent study in a transgenic reduces cell death, protects the structural integrity of adult neurons, mouse model of AD overexpressing the Amyloid precursor pro- alleviates locomotor dysfunction, and extends lifespan.
    [Show full text]
  • The Hsp90 Chaperone Network Modulates Candida Virulence Traits
    HHS Public Access Author manuscript Author ManuscriptAuthor Manuscript Author Trends Microbiol Manuscript Author . Author Manuscript Author manuscript; available in PMC 2018 October 01. Published in final edited form as: Trends Microbiol. 2017 October ; 25(10): 809–819. doi:10.1016/j.tim.2017.05.003. The Hsp90 Chaperone Network Modulates Candida Virulence Traits Teresa R. O'Meara, Nicole Robbins, and Leah E. Cowen* Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5G 1M1, Canada Abstract Hsp90 is a conserved molecular chaperone that facilitates the folding and function of client proteins. Hsp90 function is dynamically regulated by interactions with co-chaperones and by post- translational modifications. In the fungal pathogen Candida albicans, Hsp90 enables drug resistance and virulence by stabilizing diverse signal transducers. Here, we review studies that have unveiled regulators of Hsp90 function, as well as downstream effectors that govern the key virulence traits of morphogenesis and drug resistance. We highlight recent work mapping the Hsp90 genetic network in C. albicans under diverse environmental conditions, and how these interactions provide insight into circuitry important for drug resistance, morphogenesis, and virulence. Ultimately, elucidating the Hsp90 chaperone network will aid in the development of therapeutics to treat fungal disease. Keywords Candida albicans; stress response; Hsp90; virulence; development; drug resistance Hsp90 enables temperature-dependent morphogenesis, drug resistance and virulence in fungal pathogens For fungal pathogens, the ability to grow at human physiological temperatures is a requirement for successful colonization and infection [1-4]. Of the estimated 1.5 million fungal species, only approximately 300 can cause disease in humans and only a handful are common human pathogens [5].
    [Show full text]
  • Hsp90 Inhibitors for the Treatment of Chronic Myeloid Leukemia
    Hindawi Publishing Corporation Leukemia Research and Treatment Volume 2015, Article ID 757694, 16 pages http://dx.doi.org/10.1155/2015/757694 Review Article Hsp90 Inhibitors for the Treatment of Chronic Myeloid Leukemia Kalubai Vari Khajapeer and Rajasekaran Baskaran Department of Biochemistry and Molecular Biology, School of Life Sciences, Pondicherry University, Pondicherry 605014, India Correspondence should be addressed to Rajasekaran Baskaran; [email protected] Received 26 August 2015; Revised 11 November 2015; Accepted 12 November 2015 Academic Editor: Massimo Breccia Copyright © 2015 K. V. Khajapeer and R. Baskaran. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Chronic myeloid leukemia (CML) is a hematological malignancy that arises due to reciprocal translocation of 3 sequences from c-Abelson (ABL) protooncogene of chromosome 9 with 5 sequence of truncated break point cluster region (BCR) on chromosome 22. BCR-ABL is a functional oncoprotein p210 that exhibits constitutively activated tyrosine kinase causing genomic alteration of hematopoietic stem cells. BCR-ABL specific tyrosine kinase inhibitors (TKIs) successfully block CML progression. However, drug resistance owing to BCR-ABL mutations and overexpression is still an issue. Heat-shock proteins (Hsps) function as molecular chaperones facilitating proper folding of nascent polypeptides. Their increased expression under stressful conditions protects cells by stabilizing unfolded or misfolded peptides. Hsp90 is the major mammalian protein and is required by BCR-ABL for stabilization and maturation. Hsp90 inhibitors destabilize the binding of BCR-ABL protein thus leading to the formation of heteroprotein complex that is eventually degraded by the ubiquitin-proteasome pathway.
    [Show full text]
  • Associated 16P11.2 Deletion in Drosophila Melanogaster
    ARTICLE DOI: 10.1038/s41467-018-04882-6 OPEN Pervasive genetic interactions modulate neurodevelopmental defects of the autism- associated 16p11.2 deletion in Drosophila melanogaster Janani Iyer1, Mayanglambam Dhruba Singh1, Matthew Jensen1,2, Payal Patel 1, Lucilla Pizzo1, Emily Huber1, Haley Koerselman3, Alexis T. Weiner 1, Paola Lepanto4, Komal Vadodaria1, Alexis Kubina1, Qingyu Wang 1,2, Abigail Talbert1, Sneha Yennawar1, Jose Badano 4, J. Robert Manak3,5, Melissa M. Rolls1, Arjun Krishnan6,7 & 1234567890():,; Santhosh Girirajan 1,2,8 As opposed to syndromic CNVs caused by single genes, extensive phenotypic heterogeneity in variably-expressive CNVs complicates disease gene discovery and functional evaluation. Here, we propose a complex interaction model for pathogenicity of the autism-associated 16p11.2 deletion, where CNV genes interact with each other in conserved pathways to modulate expression of the phenotype. Using multiple quantitative methods in Drosophila RNAi lines, we identify a range of neurodevelopmental phenotypes for knockdown of indi- vidual 16p11.2 homologs in different tissues. We test 565 pairwise knockdowns in the developing eye, and identify 24 interactions between pairs of 16p11.2 homologs and 46 interactions between 16p11.2 homologs and neurodevelopmental genes that suppress or enhance cell proliferation phenotypes compared to one-hit knockdowns. These interac- tions within cell proliferation pathways are also enriched in a human brain-specific network, providing translational relevance in humans. Our study indicates a role for pervasive genetic interactions within CNVs towards cellular and developmental phenotypes. 1 Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA. 2 Bioinformatics and Genomics Program, The Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802, USA.
    [Show full text]
  • Celastrol Increases Glucocerebrosidase Activity in Gaucher Disease by Modulating Molecular Chaperones
    Celastrol increases glucocerebrosidase activity in Gaucher disease by modulating molecular chaperones Chunzhang Yanga,1, Cody L. Swallowsa, Chao Zhanga, Jie Lua, Hongbin Xiaob, Roscoe O. Bradya,1, and Zhengping Zhuanga,1 aSurgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892-1260; and bInstitute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China Contributed by Roscoe O. Brady, November 19, 2013 (sent for review October 21, 2013) Gaucher disease is caused by mutations in the glucosidase, beta, acid increased the catalytic activity of mutant GCase. Celastrol interfered gene that encodes glucocerebrosidase (GCase). Glucosidase, beta, with the recruitment of Cdc37 to Hsp90 halting the assembly of the acid mutations often cause protein misfolding and quantitative loss requisite chaperone complex. Inhibition of Hsp90 reduced its rec- of GCase. In the present study, we found that celastrol, an herb de- ognition of mutant GCase and therefore limited the proteasomal rivative with known anticancer, anti-inflammatory, and antioxidant degradation of the mutant protein. Additionally, celastrol triggered activity, significantly increased the quantity and catalytic activity of a reorganization of the gene expression pattern of molecular chap- GCase. Celastrol interfered with the establishment of the heat-shock erones such as DnaJ homolog subfamily B members 1 and 9 protein 90/Hsp90 cochaperone Cdc37/Hsp90-Hsp70-organizing pro- (DNAJB1/9), heat shock 70kDa proteins 1A and 1B (HSPA1A/B), tein chaperone complex with mutant GCase and reduced heat-shock and Bcl2-associated athanogene 3 (BAG3). The presence of BAG protein 90-associated protein degradation. In addition, celastrol mod- family molecular chaperone regulator 3 (BAG3) further stabi- ulated the expression of molecular chaperones.
    [Show full text]
  • Human/Mouse/Rat HSP70/HSPA1A Antibody
    Human/Mouse/Rat HSP70/HSPA1A Antibody Monoclonal Mouse IgG2A Clone # 242707 Catalog Number: A-400 DESCRIPTION Species Reactivity Human/Mouse/Rat Specificity Detects the induced form of human and mouse HSP70/HSPA1A in Western blots. No cross reactivity with HSC70 (HSP73) is observed. Source Monoclonal Mouse IgG2A Clone # 242707 Purification Protein A or G purified from hybridoma culture supernatant Immunogen E. coli­derived recombinant human HSP70/HSPA1A Met1­Asp641 Formulation Supplied as a solution in PBS containing Glycerol and Sodium Azide. See Certificate of Analysis for details. APPLICATIONS Please Note: Optimal dilutions should be determined by each laboratory for each application. General Protocols are available in the Technical Information section on our website. Recommended Sample Concentration Western Blot 0.5 µg/mL See Below Immunohistochemistry 8­25 µg/mL No Sample Info DATA Western Blot Detection of Human HSP70/HSPA1A by Western Blot. Western blot shows lysates of HEK293 human embryonic kidney cell line. PVDF membrane was probed with 0.5 µg/mL of Mouse Anti­Human/Mouse/Rat HSP70/HSPA1A Monoclonal Antibody (Catalog # A­400) followed by HRP­ conjugated Anti­Mouse IgG Secondary Antibody (Catalog # HAF007). A specific band was detected for HSP70/HSPA1A at approximately 70­75 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1. PREPARATION AND STORAGE Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. Stability & Storage Use a manual defrost freezer and avoid repeated freeze­thaw cycles. l 12 months from date of receipt, ­20 to ­70 °C as supplied.
    [Show full text]
  • A Novel Hsp90 Inhibitor Activates Compensatory Heat Shock Protein Responses and Autophagy and Alleviates Mutant A53T A-Synuclein Toxicity S
    Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2015/09/24/mol.115.101451.DC1 1521-0111/88/6/1045–1054$25.00 http://dx.doi.org/10.1124/mol.115.101451 MOLECULAR PHARMACOLOGY Mol Pharmacol 88:1045–1054, December 2015 Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics A Novel Hsp90 Inhibitor Activates Compensatory Heat Shock Protein Responses and Autophagy and Alleviates Mutant A53T a-Synuclein Toxicity s Rui Xiong, Wenbo Zhou, David Siegel, Russell R. A. Kitson, Curt R. Freed, Christopher J. Moody, and David Ross Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences (R.X., D.S., D.R.), and Department of Medicine, Division of Clinical Pharmacology and Toxicology (W.Z., C.R.F.), University of Colorado Anschutz Medical Campus, Aurora, Colorado; and School of Chemistry, University of Nottingham, Nottingham, United Kingdom (R.R.A.K., Downloaded from C.J.M.) Received August 20, 2015; accepted September 16, 2015 ABSTRACT A potential cause of neurodegenerative diseases, including 2,5-diphenyl-tetrazolium bromide (MTT), and apoptosis assays. molpharm.aspetjournals.org Parkinson’s disease (PD), is protein misfolding and aggregation Meanwhile, 19-phenyl-GA retained the ability to induce autophagy that in turn leads to neurotoxicity. Targeting Hsp90 is an and potentially protective heat shock proteins (HSPs) such as attractive strategy to halt neurodegenerative diseases, and Hsp70 and Hsp27. We found that transduction of A53T, but not benzoquinone ansamycin (BQA) Hsp90 inhibitors such as gelda- wild type (WT) a-synuclein, induced toxicity in SH-SY5Y cells.
    [Show full text]
  • The HSP70 Chaperone Machinery: J Proteins As Drivers of Functional Specificity
    REVIEWS The HSP70 chaperone machinery: J proteins as drivers of functional specificity Harm H. Kampinga* and Elizabeth A. Craig‡ Abstract | Heat shock 70 kDa proteins (HSP70s) are ubiquitous molecular chaperones that function in a myriad of biological processes, modulating polypeptide folding, degradation and translocation across membranes, and protein–protein interactions. This multitude of roles is not easily reconciled with the universality of the activity of HSP70s in ATP-dependent client protein-binding and release cycles. Much of the functional diversity of the HSP70s is driven by a diverse class of cofactors: J proteins. Often, multiple J proteins function with a single HSP70. Some target HSP70 activity to clients at precise locations in cells and others bind client proteins directly, thereby delivering specific clients to HSP70 and directly determining their fate. In their native cellular environment, polypeptides are participates in such diverse cellular functions. Their constantly at risk of attaining conformations that pre- functional diversity is remarkable considering that vent them from functioning properly and/or cause them within and across species, HSP70s have high sequence to aggregate into large, potentially cytotoxic complexes. identity. They share a single biochemical activity: an Molecular chaperones guide the conformation of proteins ATP-dependent client-binding and release cycle com- throughout their lifetime, preventing their aggregation bined with client protein recognition, which is typi- by protecting interactive surfaces against non-productive cally rather promiscuous. This apparent conundrum interactions. Through such inter actions, molecular chap- is resolved by the fact that HSP70s do not work alone, erones aid in the folding of nascent proteins as they are but rather as ‘HSP70 machines’, collaborating with synthesized by ribosomes, drive protein transport across and being regulated by several cofactors.
    [Show full text]
  • Mosaic Origin of the Eukaryotic Kinetochore 2 Jolien J.E
    bioRxiv preprint doi: https://doi.org/10.1101/514885; this version posted January 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Mosaic origin of the eukaryotic kinetochore 2 Jolien J.E. van Hooff12*, Eelco Tromer123*#, Geert J.P.L. Kops2+ and Berend Snel1+# 3 4 1 Theoretical Biology and Bioinformatics, Biology, Science Faculty, Utrecht University, the Netherlands 5 2 Oncode Institute, Hubrecht Institute – KNAW (Royal Netherlands Academy of Arts and Sciences) and 6 University Medical Centre Utrecht, Utrecht, The Netherlands 7 3 Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom 8 9 * equal contribution as first author 10 + equal contribution as senior author 11 # corresponding authors ([email protected]; [email protected]) 1 bioRxiv preprint doi: https://doi.org/10.1101/514885; this version posted January 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 12 Abstract 13 The emergence of eukaryotes from ancient prokaryotic lineages was accompanied by a remarkable increase in 14 cellular complexity. While prokaryotes use simple systems to connect DNA to the segregation machinery during 15 cell division, eukaryotes use a highly complex protein assembly known as the kinetochore. Although conceptually 16 similar, prokaryotic segregation systems and eukaryotic kinetochore proteins share no homology, raising the 17 question of the origins of the latter.
    [Show full text]
  • HSP90 Inhibitor 17-AAG Selectively Eradicates Lymphoma Stem Cells
    Published OnlineFirst June 29, 2012; DOI: 10.1158/0008-5472.CAN-11-3600 Cancer Tumor and Stem Cell Biology Research HSP90 Inhibitor 17-AAG Selectively Eradicates Lymphoma Stem Cells Bryan Newman2, Yan Liu1, Hsiu-Fang Lee2, Duxin Sun2, and Yin Wang1 Abstract Cancer stem cells (CSC; also called tumor-initiating cells) comprise tumor cell subpopulations that preserve the properties of quiescence, self-renewal, and differentiation of normal stem cells. In addition, CSCs are therapeutically important because of their key contributions toward drug resistance. The hypoxia- inducible transcription factor HIF1a is critical for CSC maintenance in mouse lymphoma. In this study, we showed that low concentrations of the HSP90 inhibitor 17-AAG eliminate lymphoma CSCs in vitro and in vivo by disrupting the transcriptional function of HIF1a, a client protein of HSP90. 17-AAG preferentially induced apoptosis and eliminated the colony formation capacity of mouse lymphoma CSCs and human acute myeloid leukemia (AML) CSCs. However, low concentrations of 17-AAG failed to eliminate highly proliferative lymphoma and AML cells (non-CSCs), in which the AKT-GSK3 signaling pathway is constitutively active. The heat shock transcription factor HSF1 is highly expressed in non-CSCs, but it was weakly expressed in lymphoma CSCs. However, siRNA-mediated attenuation of HSF1 abrogated the colony formation ability of both lymphoma and AML CSCs. This study supports the use of 17-AAG as a CSC targeting agent and, in addition, shows that HSF1 is an important target for elimination of both CSCs and non-CSCs in cancer. Cancer Res; 72(17); 1–11. Ó2012 AACR. Introduction HSP90 inhibitors is a derivative of the geldanamycin antibiotic, Molecular chaperone proteins function to ensure the proper 17-N-allylamino-17-demethoxy geldanamycin(17-AAG;ref.12).
    [Show full text]