Changes in HSP gene and protein expression in natural scrapie with brain damage

Science.gov (United States)

2011-01-01

Heat shock proteins (Hsp) perform cytoprotective functions such as apoptosis regulation and inflammatory response control. These proteins can also be secreted to the extracellular medium, acting as inflammatory mediators, and their chaperone activity permits correct folding of proteins and avoids the aggregation of anomalous isoforms. Several studies have proposed the implication of Hsp in prion diseases. We analysed the gene expression and protein distribution of different members of the Hsp27, Hsp70, and Hsp90 families in the central nervous system of sheep naturally infected with scrapie. Different expression profiles were observed in the areas analysed. Whereas changes in transcript levels were not observed in the cerebellum or medulla oblongata, a significant decrease in HSP27 and HSP90 was detected in the prefrontal cortex. In contrast, HSP73 was over-expressed in diencephalons of scrapie animals. Western blotting did not reveal significant differences in Hsp90 and Hsp70 protein expression between scrapie and control animals. Expression rates identified by real-time RT-PCR and western blotting were compared with the extent of classical scrapie lesions using stepwise regression. Changes in Hsp gene and protein expression were associated with prion protein deposition, gliosis and spongiosis rather than with apoptosis. Finally, immunohistochemistry revealed intense Hsp70 and Hsp90 immunolabelling in Purkinje cells of scrapie sheep. In contrast, controls displayed little or no staining in these cells. The observed differences in gene expression and protein distribution suggest that the heat shock proteins analysed play a role in the natural form of the disease. PMID:21314976

Changes in HSP gene and protein expression in natural scrapie with brain damage

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Serrano Carmen

2011-01-01

Full Text Available Abstract Heat shock proteins (Hsp perform cytoprotective functions such as apoptosis regulation and inflammatory response control. These proteins can also be secreted to the extracellular medium, acting as inflammatory mediators, and their chaperone activity permits correct folding of proteins and avoids the aggregation of anomalous isoforms. Several studies have proposed the implication of Hsp in prion diseases. We analysed the gene expression and protein distribution of different members of the Hsp27, Hsp70, and Hsp90 families in the central nervous system of sheep naturally infected with scrapie. Different expression profiles were observed in the areas analysed. Whereas changes in transcript levels were not observed in the cerebellum or medulla oblongata, a significant decrease in HSP27 and HSP90 was detected in the prefrontal cortex. In contrast, HSP73 was over-expressed in diencephalons of scrapie animals. Western blotting did not reveal significant differences in Hsp90 and Hsp70 protein expression between scrapie and control animals. Expression rates identified by real-time RT-PCR and western blotting were compared with the extent of classical scrapie lesions using stepwise regression. Changes in Hsp gene and protein expression were associated with prion protein deposition, gliosis and spongiosis rather than with apoptosis. Finally, immunohistochemistry revealed intense Hsp70 and Hsp90 immunolabelling in Purkinje cells of scrapie sheep. In contrast, controls displayed little or no staining in these cells. The observed differences in gene expression and protein distribution suggest that the heat shock proteins analysed play a role in the natural form of the disease.

Genome-wide identification and analysis of biotic and abiotic stress regulation of small heat shock protein (HSP20) family genes in bread wheat.

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Muthusamy, Senthilkumar K; Dalal, Monika; Chinnusamy, Viswanathan; Bansal, Kailash C

2017-04-01

Small Heat Shock Proteins (sHSPs)/HSP20 are molecular chaperones that protect plants by preventing protein aggregation during abiotic stress conditions, especially heat stress. Due to global climate change, high temperature is emerging as a major threat to wheat productivity. Thus, the identification of HSP20 and analysis of HSP transcriptional regulation under different abiotic stresses in wheat would help in understanding the role of these proteins in abiotic stress tolerance. We used sequences of known rice and Arabidopsis HSP20 HMM profiles as queries against publicly available wheat genome and wheat full length cDNA databases (TriFLDB) to identify the respective orthologues from wheat. 163 TaHSP20 (including 109 sHSP and 54 ACD) genes were identified and classified according to the sub-cellular localization and phylogenetic relationship with sequenced grass genomes (Oryza sativa, Sorghum bicolor, Zea mays, Brachypodium distachyon and Setaria italica). Spatio-temporal, biotic and abiotic stress-specific expression patterns in normalized RNA seq and wheat array datasets revealed constitutive as well as inductive responses of HSP20 in different tissues and developmental stages of wheat. Promoter analysis of TaHSP20 genes showed the presence of tissue-specific, biotic, abiotic, light-responsive, circadian and cell cycle-responsive cis-regulatory elements. 14 TaHSP20 family genes were under the regulation of 8 TamiRNA genes. The expression levels of twelve HSP20 genes were studied under abiotic stress conditions in the drought- and heat-tolerant wheat genotype C306. Of the 13 TaHSP20 genes, TaHSP16.9H-CI showed high constitutive expression with upregulation only under salt stress. Both heat and salt stresses upregulated the expression of TaHSP17.4-CI, TaHSP17.7A-CI, TaHSP19.1-CIII, TaACD20.0B-CII and TaACD20.6C-CIV, while TaHSP23.7-MTI was specifically induced only under heat stress. Our results showed that the identified TaHSP20 genes play an important role under

RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells

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Gilmour, D.S.; Lis, J.T.

1986-01-01

By using a protein-DNA cross-linking method, we examined the in vivo distribution of RNA polymerase II on the hsp70 heat shock gene in Drosophila melanogaster Schneider line 2 cells. In heat shock-induced cells, a high level of RNA polymerase II was detected on the entire gene, while in noninduced cells, the RNA polymerase II was confined to the 5' end of the hsp70 gene, predominantly between nucleotides -12 and +65 relative to the start of transcription. This association of RNA polymerase II was apparent whether the cross-linking was performed by a 10-min UV irradiation of chilled cells with mercury vapor lamps or by a 40-microsecond irradiation of cells with a high-energy xenon flash lamp. We hypothesize that RNA polymerase II has access to, and a high affinity for, the promoter region of this gene before induction, and this poised RNA polymerase II may be critical in the mechanism of transcription activation

Identification and in silico analysis of the Citrus HSP70 molecular chaperone gene family

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Luciano G. Fietto

2007-01-01

Full Text Available The completion of the genome sequencing of the Arabidopsis thaliana model system provided a powerful molecular tool for comparative analysis of gene families present in the genome of economically relevant plant species. In this investigation, we used the sequences of the Arabidopsis Hsp70 gene family to identify and annotate the Citrus Hsp70 genes represented in the CitEST database. Based on sequence comparison analysis, we identified 18 clusters that were further divided into 5 subgroups encoding four mitochondrial mtHsp70s, three plastid csHsp70s, one ER luminal Hsp70 BiP, two HSP110/SSE-related proteins and eight cytosolic Hsp/Hsc70s. We also analyzed the expression profile by digital Northern of each Hsp70 transcript in different organs and in response to stress conditions. The EST database revealed a distinct population distribution of Hsp70 ESTs among isoforms and across the organs surveyed. The Hsp70-5 isoform was highly expressed in seeds, whereas BiP, mitochondrial and plastid HSp70 mRNAs displayed a similar expression profile in the organs analyzed, and were predominantly represented in flowers. Distinct Hsp70 mRNAs were also differentially expressed during Xylella infection and Citrus tristeza viral infection as well as during water deficit. This in silico study sets the groundwork for future investigations to fully characterize functionally the Citrus Hsp70 family and underscores the relevance of Hsp70s in response to abiotic and biotic stresses in Citrus.

Hsf1p and Msn2/4p cooperate in the expression of Saccharomyces cerevisiae genes HSP26 and HSP104 in a gene- and stress type-dependent manner.

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Amorós, M; Estruch, F

2001-03-01

Saccharomyces cerevisiae possesses several transcription factors involved in the transcriptional activation of stress-induced genes. Among them, the heat shock factor (Hsf1p) and the zinc finger proteins of the general stress response (Msn2p and Msn4p) have been shown to play a major role in stress protection. Some heat shock protein (HSP) genes contain both heat shock elements (HSEs) and stress response elements (STREs), suggesting the involvement of both transcription factors in their regulation. Analysis of the stress-induced expression of two of these genes, HSP26 and HSP104, reveals that the contribution of Hsf1p and Msn2/4p is different depending on the gene and the stress condition.

Concerted and nonconcerted evolution of the Hsp70 gene superfamily in two sibling species of nematodes.

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Nikolaidis, Nikolas; Nei, Masatoshi

2004-03-01

We have identified the Hsp70 gene superfamily of the nematode Caenorhabditis briggsae and investigated the evolution of these genes in comparison with Hsp70 genes from C. elegans, Drosophila, and yeast. The Hsp70 genes are classified into three monophyletic groups according to their subcellular localization, namely, cytoplasm (CYT), endoplasmic reticulum (ER), and mitochondria (MT). The Hsp110 genes can be classified into the polyphyletic CYT group and the monophyletic ER group. The different Hsp70 and Hsp110 groups appeared to evolve following the model of divergent evolution. This model can also explain the evolution of the ER and MT genes. On the other hand, the CYT genes are divided into heat-inducible and constitutively expressed genes. The constitutively expressed genes have evolved more or less following the birth-and-death process, and the rates of gene birth and gene death are different between the two nematode species. By contrast, some heat-inducible genes show an intraspecies phylogenetic clustering. This suggests that they are subject to sequence homogenization resulting from gene conversion-like events. In addition, the heat-inducible genes show high levels of sequence conservation in both intra-species and inter-species comparisons, and in most cases, amino acid sequence similarity is higher than nucleotide sequence similarity. This indicates that purifying selection also plays an important role in maintaining high sequence similarity among paralogous Hsp70 genes. Therefore, we suggest that the CYT heat-inducible genes have been subjected to a combination of purifying selection, birth-and-death process, and gene conversion-like events.

Genome-wide analysis of the potato Hsp20 gene family: identification, genomic organization and expression profiles in response to heat stress.

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Zhao, Peng; Wang, Dongdong; Wang, Ruoqiu; Kong, Nana; Zhang, Chao; Yang, Chenghui; Wu, Wentao; Ma, Haoli; Chen, Qin

2018-01-18

Heat shock proteins (Hsps) are essential components in plant tolerance mechanism under various abiotic stresses. Hsp20 is the major family of heat shock proteins, but little of Hsp20 family is known in potato (Solanum tuberosum), which is an important vegetable crop that is thermosensitive. To reveal the mechanisms of potato Hsp20s coping with abiotic stresses, analyses of the potato Hsp20 gene family were conducted using bioinformatics-based methods. In total, 48 putative potato Hsp20 genes (StHsp20s) were identified and named according to their chromosomal locations. A sequence analysis revealed that most StHsp20 genes (89.6%) possessed no, or only one, intron. A phylogenetic analysis indicated that all of the StHsp20 genes, except 10, were grouped into 12 subfamilies. The 48 StHsp20 genes were randomly distributed on 12 chromosomes. Nineteen tandem duplicated StHsp20s and one pair of segmental duplicated genes (StHsp20-15 and StHsp20-48) were identified. A cis-element analysis inferred that StHsp20s, except for StHsp20-41, possessed at least one stress response cis-element. A heatmap of the StHsp20 gene family showed that the genes, except for StHsp20-2 and StHsp20-45, were expressed in various tissues and organs. Real-time quantitative PCR was used to detect the expression level of StHsp20 genes and demonstrated that the genes responded to multiple abiotic stresses, such as heat, salt or drought stress. The relative expression levels of 14 StHsp20 genes (StHsp20-4, 6, 7, 9, 20, 21, 33, 34, 35, 37, 41, 43, 44 and 46) were significantly up-regulated (more than 100-fold) under heat stress. These results provide valuable information for clarifying the evolutionary relationship of the StHsp20 family and in aiding functional characterization of StHsp20 genes in further research.

Extracellular Hsp90 serves as a co-factor for MAPK activation and latent viral gene expression during de novo infection by KSHV

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Qin Zhiqiang; DeFee, Michael; Isaacs, Jennifer S.; Parsons, Chris

2010-01-01

The Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), an important cause of morbidity and mortality in immunocompromised patients. KSHV interaction with the cell membrane triggers activation of specific intracellular signal transduction pathways to facilitate virus entry, nuclear trafficking, and ultimately viral oncogene expression. Extracellular heat shock protein 90 localizes to the cell surface (csHsp90) and facilitates signal transduction in cancer cell lines, but whether csHsp90 assists in the coordination of KSHV gene expression through these or other mechanisms is unknown. Using a recently characterized non-permeable inhibitor specifically targeting csHsp90 and Hsp90-specific antibodies, we show that csHsp90 inhibition suppresses KSHV gene expression during de novo infection, and that this effect is mediated largely through the inhibition of mitogen-activated protein kinase (MAPK) activation by KSHV. Moreover, we show that targeting csHsp90 reduces constitutive MAPK expression and the release of infectious viral particles by patient-derived, KSHV-infected primary effusion lymphoma cells. These data suggest that csHsp90 serves as an important co-factor for KSHV-initiated MAPK activation and provide proof-of-concept for the potential benefit of targeting csHsp90 for the treatment or prevention of KSHV-associated illnesses.

Cloning of three heat shock protein genes (HSP70, HSP90α and HSP90β) and their expressions in response to thermal stress in loach (Misgurnus anguillicaudatus) fed with different levels of vitamin C.

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Yan, Jie; Liang, Xiao; Zhang, Yin; Li, Yang; Cao, Xiaojuan; Gao, Jian

2017-07-01

Heat shock protein 70 (HSP70) and 90 (HSP90) are the most broadly studied proteins in HSP families. They play key roles in cells as molecular chaperones, in response to stress conditions such as thermal stress. In this study, full-length cDNA sequences of HSP70, HSP90α and HSP90β from loach Misgurnus anguillicaudatus were cloned. The full-length cDNA of HSP70 in loach was 2332bp encoding 644 amino acids, while HSP90α and HSP90β were 2586bp and 2678bp in length, encoding 729 and 727 amino acids, respectively. The deduced amino acid sequences of HSP70 in loach shared the highest identity with those of Megalobrama amblycephala and Cyprinus carpio. The deduced amino acid sequences of HSP90α and HSP90β in loach both shared the highest identity with those of M. amblycephala. Their mRNA tissue expression results showed that the maximum expressions of HSP70, HSP90α and HSP90β were respectively present in the intestine, brain and kidney of loach. Quantitative real-time PCR was employed to analyze the temporal expressions of HSP70, HSP90α and HSP90β in livers of loaches fed with different levels of vitamin C under thermal stress. Expression levels of the three HSP genes in loach fed the diet without vitamin C supplemented at 0 h of thermal stress were significantly lower than those at 2 h, 6 h, 12 h and 24 h of thermal stress. It indicated that expressions of the three HSP genes were sensitive to thermal stress in loach. The three HSP genes in loaches fed with 1000 mg/kg vitamin C expressed significantly lower than other vitamin C groups at many time points of thermal stress, suggesting 1000 mg/kg dietary vitamin C might decrease the body damages caused by the thermal stress. This study will be of value for further studies into thermal stress tolerance in loach. Copyright © 2017 Elsevier Ltd. All rights reserved.

Obtaining a citric tristeza virus p65 protein antibody and preliminary results of p65 in vivo expression

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Yanneth Torres

2003-07-01

Full Text Available The citric tristeza virus (CTV belongs to the Closteroviridae family which indudes the only vegetal viruses possessing genes homologous to HSP70 thermal cellular shock proteins in their genome. Such is the case of the gene encoding for the CTV p65 protein which presents high homology with the HSP70 protein family. It has been shown recently that HSP70h viral proteins (such as CTV p65 are involved both in viral assembly, as a microtubule binding protein, and in cell-cell movement. Since CTV is the most deleterious citrus pathogen, understanding this protein's role in the pathogenesis process is important. Rabbits were immunised with four synthetic peptides (corresponding to CTV p65 thermal shock protein's carboxyl-terminal region to obtain polyclonal antibodies. All the peptides used were immunogenic, even though two of them showed greater response. Whilst none of the antibodies obtained reacted to non-infected plant extract, the p65 proteins was detected in extracts taken from citric plants infected with CTV Based on the antibody's reaction to two Colombian isolates having different serological characteristics, the p65 antibody's immunological behaviour appeared to be independent of the symptomatic severity of the CTV isolates. It was shown that the ORF encoded for the HSP70 homologue in CTV was expressed in vivo, even though the p65 antibody was only detected in concentrated protein extracts taken from infected plants, supporting reports from other studies that the concentration of this protein in plants infected with CTV is low. This is the first time that a polyclonal CTV antibody has been obtained in Colombia against p65 (a protein intervening in viral assembly and movement. Adapting a technique for obtaining p65 antibodies by using synthetic peptides as immunogens could be useful in the future for detecting or diagnosing p65 proteins present in different Colombian CTV isolates, especially in developing studies contributing towards greater

Genome-wide identification and expression profiling of tomato Hsp20 gene family in response to biotic and abiotic stresses

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jiahong yu

2016-08-01

Full Text Available The Hsp20 genes are involved in the response of plants to environment stresses including heat shock and also play a vital role in plant growth and development. They represent the most abundant small heat shock proteins (sHsps in plants, but little is known about this family in tomato (Solanum lycopersicum, an important vegetable crop in the world. Here, we characterized heat shock protein 20 (SlHsp20 gene family in tomato through integration of gene structure, chromosome location, phylogenetic relationship and expression profile. Using bioinformatics-based methods, we identified at least 42 putative SlHsp20 genes in tomato. Sequence analysis revealed that most of SlHsp20 genes possessed no intron or a relatively short intron in length. Chromosome mapping indicated that inter-arm and intra-chromosome duplication events contributed remarkably to the expansion of SlHsp20 genes. Phylogentic tree of Hsp20 genes from tomato and other plant species revealed that SlHsp20 genes were grouped into 13 subfamilies, indicating that these genes may have a common ancestor that generated diverse subfamilies prior to the mono-dicot split. In addition, expression analysis using RNA-seq in various tissues and developmental stages of cultivated tomato and the wild relative Solanum pimpinellifolium revealed that most of these genes (83% were expressed in at least one stage from at least one genotype. Out of 42 genes, 4 genes were expressed constitutively in almost all the tissues analyzed, implying that these genes might have specific housekeeping function in tomato cell under normal growth conditions. Two SlHsp20 genes displayed differential expression levels between cultivated tomato and S. pimpinellifolium in vegetative (leaf and root and reproductive organs (floral bud and flower, suggesting inter-species diversification for functional specialization during the process of domestication. Based on genome-wide microarray analysis, we showed that the transcript

Genome-Wide Identification and Expression Profiling of Tomato Hsp20 Gene Family in Response to Biotic and Abiotic Stresses.

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Yu, Jiahong; Cheng, Yuan; Feng, Kun; Ruan, Meiying; Ye, Qingjing; Wang, Rongqing; Li, Zhimiao; Zhou, Guozhi; Yao, Zhuping; Yang, Yuejian; Wan, Hongjian

2016-01-01

The Hsp20 genes are involved in the response of plants to environment stresses including heat shock and also play a vital role in plant growth and development. They represent the most abundant small heat shock proteins (sHsps) in plants, but little is known about this family in tomato (Solanum lycopersicum), an important vegetable crop in the world. Here, we characterized heat shock protein 20 (SlHsp20) gene family in tomato through integration of gene structure, chromosome location, phylogenetic relationship, and expression profile. Using bioinformatics-based methods, we identified at least 42 putative SlHsp20 genes in tomato. Sequence analysis revealed that most of SlHsp20 genes possessed no intron or a relatively short intron in length. Chromosome mapping indicated that inter-arm and intra-chromosome duplication events contributed remarkably to the expansion of SlHsp20 genes. Phylogentic tree of Hsp20 genes from tomato and other plant species revealed that SlHsp20 genes were grouped into 13 subfamilies, indicating that these genes may have a common ancestor that generated diverse subfamilies prior to the mono-dicot split. In addition, expression analysis using RNA-seq in various tissues and developmental stages of cultivated tomato and the wild relative Solanum pimpinellifolium revealed that most of these genes (83%) were expressed in at least one stage from at least one genotype. Out of 42 genes, 4 genes were expressed constitutively in almost all the tissues analyzed, implying that these genes might have specific housekeeping function in tomato cell under normal growth conditions. Two SlHsp20 genes displayed differential expression levels between cultivated tomato and S. pimpinellifolium in vegetative (leaf and root) and reproductive organs (floral bud and flower), suggesting inter-species diversification for functional specialization during the process of domestication. Based on genome-wide microarray analysis, we showed that the transcript levels of SlHsp20

Characterization of six small HSP genes from Chironomus riparius (Diptera, Chironomidae): Differential expression under conditions of normal growth and heat-induced stress.

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Martín-Folgar, Raquel; de la Fuente, Mercedes; Morcillo, Gloria; Martínez-Guitarte, José-Luis

2015-10-01

Small heat shock proteins (sHSPs) comprise the most numerous, structurally diverse, and functionally uncharacterized family of heat shock proteins. Several Hsp genes (Hsp 90, 70, 40, and 27) from the insect Chironomus riparius are widely used in aquatic toxicology as biomarkers for environmental toxins. Here, we conducted a comparative study and characterized secondary structure of the six newly identified sHsp genes Hsp17, Hsp21, Hsp22, Hsp23, Hsp24, and Hsp34. A characteristic α-crystallin domain is predicted in all the new proteins. Phylogenetic analysis suggests a strong relation to other sHSPs from insects and interesting evidence regarding evolutionary origin and duplication events. Comparative analysis of transcription profiles for Hsp27, Hsp70, and the six newly identified genes revealed that Hsp17, Hsp21, and Hsp22 are constitutively expressed under normal conditions, while under two different heat shock conditions these genes are either not activated or are even repressed (Hsp22). In contrast, Hsp23, Hsp24, and Hsp34 are significantly activated along with Hsp27 and Hsp70 during heat stress. These results strongly suggest functional differentiation within the small HSP subfamily and provide new data to help understand the coping mechanisms induced by stressful environmental stimuli. Copyright © 2015 Elsevier Inc. All rights reserved.

Expression analysis of NOS family and HSP genes during thermal stress in goat ( Capra hircus)

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Yadav, Vijay Pratap; Dangi, Satyaveer Singh; Chouhan, Vikrant Singh; Gupta, Mahesh; Dangi, Saroj K.; Singh, Gyanendra; Maurya, Vijay Prakash; Kumar, Puneet; Sarkar, Mihir

2016-03-01

Approximately 50 genes other than heat shock protein (HSP) expression changes during thermal stress. These genes like nitric oxide synthase (NOS) need proper attention and investigation to find out their possible role in the adaptation to thermal stress in animals. So, the present study was undertaken to demonstrate the expressions of inducible form type II NOS (iNOS), endothelial type III NOS (eNOS), constitutively expressed enzyme NOS (cNOS), HSP70, and HSP90 in peripheral blood mononuclear cells (PBMCs) during different seasons in Barbari goats. Real-time polymerase chain reaction, western blot, and immunocytochemistry were applied to investigate messenger RNA (mRNA) expression, protein expression, and immunolocalization of examined factors. The mRNA and protein expressions of iNOS, eNOS, cNOS, HSP70, and HSP90 were significantly higher ( P goats.

The expression and function of hsp30-like small heat shock protein genes in amphibians, birds, fish, and reptiles.

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Heikkila, John J

2017-01-01

Small heat shock proteins (sHSPs) are a superfamily of molecular chaperones with important roles in protein homeostasis and other cellular functions. Amphibians, reptiles, fish and birds have a shsp gene called hsp30, which was also referred to as hspb11 or hsp25 in some fish and bird species. Hsp30 genes, which are not found in mammals, are transcribed in response to heat shock or other stresses by means of the heat shock factor that is activated in response to an accumulation of unfolded protein. Amino acid sequence analysis revealed that representative HSP30s from different classes of non-mammalian vertebrates were distinct from other sHSPs including HSPB1/HSP27. Studies with amphibian and fish recombinant HSP30 determined that they were molecular chaperones since they inhibited heat- or chemically-induced aggregation of unfolded protein. During non-mammalian vertebrate development, hsp30 genes were differentially expressed in selected tissues. Also, heat shock-induced stage-specific expression of hsp30 genes in frog embryos was regulated at the level of chromatin structure. In adults and/or tissue culture cells, hsp30 gene expression was induced by heat shock, arsenite, cadmium or proteasomal inhibitors, all of which enhanced the production of unfolded/damaged protein. Finally, immunocytochemical analysis of frog and chicken tissue culture cells revealed that proteotoxic stress-induced HSP30 accumulation co-localized with aggresome-like inclusion bodies. The congregation of damaged protein in aggresomes minimizes the toxic effect of aggregated protein dispersed throughout the cell. The current availability of probes to detect the presence of hsp30 mRNA or encoded protein has resulted in the increased use of hsp30 gene expression as a marker of proteotoxic stress in non-mammalian vertebrates. Copyright © 2016 Elsevier Inc. All rights reserved.

Ten tandem repeats of β-hCG 109-118 enhance immunogenicity and anti-tumor effects of β-hCG C-terminal peptide carried by mycobacterial heat-shock protein HSP65

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Zhang Yankai; Yan Rong; He Yi; Liu Wentao; Cao Rongyue; Yan Ming; Li Taiming; Liu Jingjing; Wu Jie

2006-01-01

The β-subunit of human chorionic gonadotropin (β-hCG) is secreted by many kinds of tumors and it has been used as an ideal target antigen to develop vaccines against tumors. In view of the low immunogenicity of this self-peptide,we designed a method based on isocaudamer technique to repeat tandemly the 10-residue sequence X of β-hCG (109-118), then 10 tandemly repeated copies of the 10-residue sequence combined with β-hCG C-terminal 37 peptides were fused to mycobacterial heat-shock protein 65 to construct a fusion protein HSP65-X10-βhCGCTP37 as an immunogen. In this study, we examined the effect of the tandem repeats of this 10-residue sequence in eliciting an immune by comparing the immunogenicity and anti-tumor effects of the two immunogens, HSP65-X10-βhCGCTP37 and HSP65-βhCGCTP37 (without the 10 tandem repeats). Immunization of mice with the fusion protein HSP65-X10-βhCGCTP37 elicited much higher levels of specific anti-β-hCG antibodies and more effectively inhibited the growth of Lewis lung carcinoma (LLC) in vivo than with HSP65-βhCGCTP37, which should suggest that HSP65-X10-βhCGCTP37 may be an effective protein vaccine for the treatment of β-hCG-dependent tumors and multiple tandem repeats of a certain epitope are an efficient method to overcome the low immunogenicity of self-peptide antigens

Integration of gene dosage and gene expression in non-small cell lung cancer, identification of HSP90 as potential target.

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Mariëlle I Gallegos Ruiz

Full Text Available BACKGROUND: Lung cancer causes approximately 1.2 million deaths per year worldwide, and non-small cell lung cancer (NSCLC represents 85% of all lung cancers. Understanding the molecular events in non-small cell lung cancer (NSCLC is essential to improve early diagnosis and treatment for this disease. METHODOLOGY AND PRINCIPAL FINDINGS: In an attempt to identify novel NSCLC related genes, we performed a genome-wide screening of chromosomal copy number changes affecting gene expression using microarray based comparative genomic hybridization and gene expression arrays on 32 radically resected tumor samples from stage I and II NSCLC patients. An integrative analysis tool was applied to determine whether chromosomal copy number affects gene expression. We identified a deletion on 14q32.2-33 as a common alteration in NSCLC (44%, which significantly influenced gene expression for HSP90, residing on 14q32. This deletion was correlated with better overall survival (P = 0.008, survival was also longer in patients whose tumors had low expression levels of HSP90. We extended the analysis to three independent validation sets of NSCLC patients, and confirmed low HSP90 expression to be related with longer overall survival (P = 0.003, P = 0.07 and P = 0.04. Furthermore, in vitro treatment with an HSP90 inhibitor had potent antiproliferative activity in NSCLC cell lines. CONCLUSIONS: We suggest that targeting HSP90 will have clinical impact for NSCLC patients.

Expression profile of HSP genes during different seasons in goats (Capra hircus).

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Dangi, Satyaveer Singh; Gupta, Mahesh; Maurya, Divakar; Yadav, Vijay Prakash; Panda, Rudra Prasanna; Singh, Gyanendra; Mohan, Nitai Haridas; Bhure, Sanjeev Kumar; Das, Bikash Chandra; Bag, Sadhan; Mahapatra, Ramkrishna; Taru Sharma, Guttalu; Sarkar, Mihir

2012-12-01

The present study has demonstrated the expression of HSP60, HSP70, HSP90, and UBQ in peripheral blood mononuclear cells (PBMCs) during different seasons in three different age groups (Groups I, II, and III with age of 0-2, 2-5, and >5 years, respectively) of goats of tropical and temperate regions. Real-time polymerase chain reaction was applied to investigate mRNA expression of examined factors. Specificity of the desired products was documented using analysis of the melting temperature and high-resolution gel electrophoresis to verify that the transcripts are of the exact molecular size predicted. The mRNA expression of HSP60, HSP90, and UBQ was significantly higher (P tropical and temperate region goats. HSP70 mRNA expression was significantly higher (P tropical region goats. However, in the temperate region, in goats from all the three age groups studied, a non-significant difference of HSP70 expression between summer and winter seasons was noticed. In conclusion, results demonstrate that (1) HSP genes are expressed in caprine PBMCs and (2) higher expression of HSPs during thermal stress suggest possible involvement of them to ameliorate deleterious effect of thermal stress so as to maintain cellular integrity and homeostasis in goats.

Ammonia stress under high environmental ammonia induces Hsp70 and Hsp90 in the mud eel, Monopterus cuchia.

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Hangzo, Hnunlalliani; Banerjee, Bodhisattwa; Saha, Shrabani; Saha, Nirmalendu

2017-02-01

The obligatory air-breathing mud eel (Monopterus cuchia) is frequently being challenged with high environmental ammonia (HEA) exposure in its natural habitats. The present study investigated the possible induction of heat shock protein 70 and 90 (hsp70, hsc70, hsp90α and hsp90β) genes and more expression of Hsp70 and Hsp90 proteins under ammonia stress in different tissues of the mud eel after exposure to HEA (50 mM NH 4 Cl) for 14 days. HEA resulted in significant accumulation of toxic ammonia in different body tissues and plasma, which was accompanied with the stimulation of oxidative stress in the mud eel as evidenced by more accumulation of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) during exposure to HEA. Further, hyper-ammonia stress led to significant increase in the levels of mRNA transcripts for inducible hsp70 and hsp90α genes and also their translated proteins in different tissues probably as a consequence of induction of hsp70 and hsp90α genes in the mud eel. However, hyper-ammonia stress was neither associated with any significant alterations in the levels of mRNA transcripts for constitutive hsc70 and hsp90β genes nor their translated proteins in any of the tissues studied. More abundance of Hsp70 and Hsp90α proteins might be one of the strategies adopted by the mud eel to defend itself from the ammonia-induced cellular damages under ammonia stress. Further, this is the first report of ammonia-induced induction of hsp70 and hsp90α genes under hyper-ammonia stress in any freshwater air-breathing teleost.

Tah1 helix-swap dimerization prevents mixed Hsp90 co-chaperone complexes

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Morgan, Rhodri M. L.; Pal, Mohinder; Roe, S. Mark; Pearl, Laurence H.; Prodromou, Chrisostomos

2015-01-01

A helix swap involving the fifth helix between two adjacently bound Tah1 molecules restores the normal binding environment of the conserved MEEVD peptide of Hsp90. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes with Hsp90 and Tah1. Specific co-chaperone adaptors facilitate the recruitment of client proteins to the Hsp90 system. Tah1 binds the C-terminal conserved MEEVD motif of Hsp90, thus linking an eclectic set of client proteins to the R2TP complex for their assembly and regulation by Hsp90. Rather than the normal complement of seven α-helices seen in other tetratricopeptide repeat (TPR) domains, Tah1 unusually consists of the first five only. Consequently, the methionine of the MEEVD peptide remains exposed to solvent when bound by Tah1. In solution Tah1 appears to be predominantly monomeric, and recent structures have failed to explain how Tah1 appears to prevent the formation of mixed TPR domain-containing complexes such as Cpr6–(Hsp90) 2 –Tah1. To understand this further, the crystal structure of Tah1 in complex with the MEEVD peptide of Hsp90 was determined, which shows a helix swap involving the fifth α-helix between two adjacently bound Tah1 molecules. Dimerization of Tah1 restores the normal binding environment of the bound Hsp90 methionine residue by reconstituting a TPR binding site similar to that in seven-helix-containing TPR domain proteins. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes

Tah1 helix-swap dimerization prevents mixed Hsp90 co-chaperone complexes

Energy Technology Data Exchange (ETDEWEB)

Morgan, Rhodri M. L.; Pal, Mohinder; Roe, S. Mark; Pearl, Laurence H., E-mail: laurence.pearl@sussex.ac.uk; Prodromou, Chrisostomos, E-mail: laurence.pearl@sussex.ac.uk [University of Sussex, Falmer, Brighton BN1 9RQ (United Kingdom)

2015-05-01

A helix swap involving the fifth helix between two adjacently bound Tah1 molecules restores the normal binding environment of the conserved MEEVD peptide of Hsp90. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes with Hsp90 and Tah1. Specific co-chaperone adaptors facilitate the recruitment of client proteins to the Hsp90 system. Tah1 binds the C-terminal conserved MEEVD motif of Hsp90, thus linking an eclectic set of client proteins to the R2TP complex for their assembly and regulation by Hsp90. Rather than the normal complement of seven α-helices seen in other tetratricopeptide repeat (TPR) domains, Tah1 unusually consists of the first five only. Consequently, the methionine of the MEEVD peptide remains exposed to solvent when bound by Tah1. In solution Tah1 appears to be predominantly monomeric, and recent structures have failed to explain how Tah1 appears to prevent the formation of mixed TPR domain-containing complexes such as Cpr6–(Hsp90){sub 2}–Tah1. To understand this further, the crystal structure of Tah1 in complex with the MEEVD peptide of Hsp90 was determined, which shows a helix swap involving the fifth α-helix between two adjacently bound Tah1 molecules. Dimerization of Tah1 restores the normal binding environment of the bound Hsp90 methionine residue by reconstituting a TPR binding site similar to that in seven-helix-containing TPR domain proteins. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes.

Investigating hsp Gene Expression in Liver of Channa striatus under Heat Stress for Understanding the Upper Thermal Acclimation

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Gopal Krishna Purohit

2014-01-01

Full Text Available Changes in hsp gene expression profiles in murrel Channa striatus experimentally exposed to temperature stress (36°C for 4, 15, and 30 days were investigated; fish collected from aquaculture ponds and maintained in laboratory at the pond temperature (25 ± 1°C served as control. Channa collected from a hot spring runoff (36°C was included in the study to examine the hsp profiles beyond 30 days of exposure. Gene expression analyses of a battery of hsps in liver tissues were carried out by quantitative RT-PCR and protein expressions were analyzed by immunoblotting. hsps could be grouped into three clusters based on similarity in response to heat stress: hsp70, hsp78, and hsp60, whose transcript level continued to increase with duration of exposure; hsp90 and hsp110 that increased to a much higher level and then decreased; hsp27 and hsp47 that did not significantly vary as compared to control. The results suggest that Hsp70, Hsp78, and Hsp60 are involved in thermal acclimation and long term survival at high temperature. Fish living in the hot spring runoff appears to continuously express hsps that can be approximated by long term induction of hsps in farmed fish if temperature of their environment is raised to 36°C.

Complexity of rice Hsp100 gene family: lessons from rice genome ...

Indian Academy of Sciences (India)

Madhu Sudhan

2007-03-29

Mar 29, 2007 ... Chaperonins are a class of molecular chaperones found in prokaryotes and in the ... Keywords. Chaperone, gene family, Hsp100, Oryza sativa ..... Sculpting the proteome with AAA+ proteases and disassembly machines; Cell ...

Effect of vibrational stress and spaceflight on regulation of heat shock proteins hsp70 and hsp27 in human lymphocytes (Jurkat)

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Cubano, L. A.; Lewis, M. L.

2001-01-01

Heat shock protein levels are increased in cells as a result of exposure to stress. To determine whether heat shock protein regulation could be used to evaluate stress in cells during spaceflight, the response of Jurkat cells to spaceflight and simulated space shuttle launch vibration was investigated by evaluating hsp70 and hsp27 gene expression. Gene expression was assessed by reverse transcription-polymerase chain reaction using mRNA extracted from vibrated, nonvibrated, space-flown, and ground control cells. Results indicate that mechanical stresses of vibration and low gravity do not up-regulate the mRNA for hsp70, although the gene encoding hsp27 is up-regulated by spaceflight but not by vibration. In ground controls, the mRNA for hsp70 and hsp27 increased with time in culture. We conclude that hsp70 gene expression is a useful indicator of stress related to culture density but is not an indicator of the stresses of launch vibration or microgravity. Up-regulation of hsp27 gene expression in microgravity is a new finding.

Single-nucleotide variations in the genes encoding the mitochondrial Hsp60/Hsp10 chaperone system and their disease-causing potential

DEFF Research Database (Denmark)

Bross, Peter; Li, Zhijie; Hansen, Jakob

2007-01-01

for variations in the HSPD1 and HSPE1 genes encoding the mitochondrial Hsp60/Hsp10 chaperone complex: two patients with multiple mitochondrial enzyme deficiency, 61 sudden infant death syndrome cases (MIM: #272120), and 60 patients presenting with ethylmalonic aciduria carrying non-synonymous susceptibility...... variations in the ACADS gene (MIM: *606885 and #201470). Besides previously reported variations we detected six novel variations: two in the bidirectional promoter region, and one synonymous and three non-synonymous variations in the HSPD1 coding region. One of the non-synonymous variations was polymorphic...... in patient and control samples, and the rare variations were each only found in single patients and absent in 100 control chromosomes. Functional investigation of the effects of the variations in the promoter region and the non-synonymous variations in the coding region indicated that none of them had...

Mitochondrial-type hsp70 genes of the amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and two microsporidians☆

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Arisue, Nobuko; Sánchez, Lidya B.; Weiss, Louis M.; Müller, Miklós; Hashimoto, Tetsuo

2011-01-01

Genes encoding putative mitochondrial-type heat shock protein 70 (mit-hsp70) were isolated and sequenced from amitochondriate protists, Giardia intestinalis, Entamoeba histolytica, and two microsporidians, Encephalitozoon hellem and Glugea plecoglossi. The deduced mit-hsp70 sequences were analyzed by sequence alignments and phylogenetic reconstructions. The mit-hsp70 sequence of these four amitochondriate protists were divergent from other mit-hsp70 sequences of mitochondriate eukaryotes. However, all of these sequences were clearly located within a eukaryotic mitochondrial clade in the tree including various type hsp70 sequences, supporting the emerging notion that none of these amitochondriate lineages are primitively amitochodrial, but lost their mitochondria secondarily in their evolutionary past. PMID:11880223

Very low amounts of glucose cause repression of the stress-responsive gene HSP12 in Saccharomyces cerevisiae.

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de Groot, E; Bebelman, J P; Mager, W H; Planta, R J

2000-02-01

Changing the growth mode of Saccharomyces cerevisiae by adding fermentable amounts of glucose to cells growing on a non-fermentable carbon source leads to rapid repression of general stress-responsive genes like HSP12. Remarkably, glucose repression of HSP12 appeared to occur even at very low glucose concentrations, down to 0.005%. Although these low levels of glucose do not induce fermentative growth, they do act as a growth signal, since upon addition of glucose to a concentration of 0.02%, growth rate increased and ribosomal protein gene transcription was up-regulated. In an attempt to elucidate how this type of glucose signalling may operate, several signalling mutants were examined. Consistent with the low amounts of glucose that elicit HSP12 repression, neither the main glucose-repression pathway nor cAMP-dependent activation of protein kinase A appeared to play a role in this regulation. Using mutants involved in glucose metabolism, evidence was obtained suggesting that glucose 6-phosphate serves as a signalling molecule. To identify the target for glucose repression on the promoter of the HSP12 gene, a promoter deletion series was used. The major transcription factors governing (stress-induced) transcriptional activation of HSP12 are Msn2p and Msn4p, binding to the general stress-responsive promoter elements (STREs). Surprisingly, glucose repression of HSP12 appeared to be independent of Msn2/4p: HSP12 transcription in glycerol-grown cells was unaffected in a deltamsn2deltamsn4 strain. Nevertheless, evidence was obtained that STRE-mediated transcription is the target of repression by low amounts of glucose. These data suggest that an as yet unidentified factor is involved in STRE-mediated transcriptional regulation of HSP12.

UV Radiation and Visible Light Induce hsp70 Gene Expression in the Antarctic Psychrophilic Ciliate Euplotes focardii.

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Fulgentini, Lorenzo; Passini, Valerio; Colombetti, Giuliano; Miceli, Cristina; La Terza, Antonietta; Marangoni, Roberto

2015-08-01

The psychrophilic ciliate Euplotes focardii inhabits the shallow marine coastal sediments of Antarctica, where, over millions of years of evolution, it has reached a strict molecular adaptation to such a constant-temperature environment (about -2 °C). This long evolution at sub-zero temperatures has made E. focardii unable to respond to heat stress with the activation of its heat shock protein (hsp) 70 genes. These genes can, however, be expressed in response to other stresses, like the oxidative one, thus indicating that the molecular adaptation has exclusively altered the heat stress signaling pathways, while it has preserved hsp70 gene activation in response to other environmental stressors. Since radiative stress has proved to be affine to oxidative stress in several organisms, we investigated the capability of UV radiation to induce hsp70 transcription. E. focardii cell cultures were exposed to several different irradiation regimes, ranging from visible only to a mixture of visible, UV-A and UV-B. The irradiation values of each spectral band have been set to be comparable with those recorded in a typical Antarctic spring. Using Northern blot analysis, we measured the expression level of hsp70 immediately after irradiation (0-h-labeled samples), 1 h, and 2 h from the end of the irradiation. Surprisingly, our results showed that besides UV radiation, the visible light was also able to induce hsp70 expression in E. focardii. Moreover, spectrophotometric measurements have revealed no detectable endogenous pigments in E. focardii, making it difficult to propose a possible explanation for the visible light induction of its hsp70 genes. Further research is needed to conclusively clarify this point.

Plasmodium falciparum Hep1 Is Required to Prevent the Self Aggregation of PfHsp70-3.

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David O Nyakundi

Full Text Available The majority of mitochondrial proteins are encoded in the nucleus and need to be imported from the cytosol into the mitochondria, and molecular chaperones play a key role in the efficient translocation and proper folding of these proteins in the matrix. One such molecular chaperone is the eukaryotic mitochondrial heat shock protein 70 (Hsp70; however, it is prone to self-aggregation and requires the presence of an essential zinc-finger protein, Hsp70-escort protein 1 (Hep1, to maintain its structure and function. PfHsp70-3, the only Hsp70 predicted to localize in the mitochondria of P. falciparum, may also rely on a Hep1 orthologue to prevent self-aggregation. In this study, we identified a putative Hep1 orthologue in P. falciparum and co-expression of PfHsp70-3 and PfHep1 enhanced the solubility of PfHsp70-3. PfHep1 suppressed the thermally induced aggregation of PfHsp70-3 but not the aggregation of malate dehydrogenase or citrate synthase, thus showing specificity for PfHsp70-3. Zinc ions were indeed essential for maintaining the function of PfHep1, as EDTA chelation abrogated its abilities to suppress the aggregation of PfHsp70-3. Soluble and functional PfHsp70-3, acquired by co-expression with PfHep-1, will facilitate the biochemical characterisation of this particular Hsp70 protein and its evaluation as a drug target for the treatment of malaria.

Robust heat-inducible gene expression by two endogenous hsp70-derived promoters in transgenic Aedes aegypti

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Carpenetti, Tiffany L. G.; Aryan, Azadeh; Myles, Kevin M.; Adelman, Zach N.

2011-01-01

Aedes aegypti is an important vector of the viruses that cause dengue fever, dengue hemorrhagic fever, and yellow fever. Reverse genetic approaches to the study of gene function in this mosquito have been limited by the lack of a robust inducible promoter to allow precise temporal control over a protein-encoding or hairpin RNA transgene. Likewise, investigations into the molecular and biochemical basis of vector competence would benefit from the ability to activate an anti-pathogen molecule at specific times during infection. We have characterized the ability of genomic sequences derived from two Ae. aegypti hsp70 genes to drive heat-inducible expression of a reporter in both transient and germline transformation contexts. AaHsp70-luciferase transcripts accumulated specifically after heat shock, and displayed a pattern of rapid induction and decay similar to endogenous AaHsp70 genes. Luciferase expression in transgenic Ae. aegypti increased by ∼25-50 fold in whole adults by four hours after heat-shock, with significant activity (∼20 fold) remaining at 24 hr. Heat-induced expression was even more dramatic in midgut tissues, with one strain showing a ∼2500-fold increase in luciferase activity. The AaHsp70 promoters described could be valuable for gene function studies as well as for the precise timing of the expression of anti-pathogen molecules. PMID:22142225

Protective Role of Hsp27 Protein Against Gamma Radiation-Induced Apoptosis and Radiosensitization Effects of Hsp27 Gene Silencing in Different Human Tumor Cells

International Nuclear Information System (INIS)

Aloy, Marie-Therese; Hadchity, Elie; Bionda, Clara; Diaz-Latoud, Chantal; Claude, Line; Rousson, Robert; Arrigo, Andre-Patrick; Rodriguez-Lafrasse, Claire

2008-01-01

Purpose: The ability of heat shock protein 27 (Hsp27) to protect cells from stressful stimuli and its increased levels in tumors resistant to anticancer therapeutics suggest that it may represent a target for sensitization to radiotherapy. In this study, we investigate the protective role of Hsp27 against radiation-induced apoptosis and the effect of its attenuation in highly expressing radioresistant cancer cell lines. Methods and Materials: We examined clonogenic death and the kinetics of apoptotic events in different tumor cell lines overexpressing or underexpressing Hsp27 protein irradiated with photons. The radiosensitive Jurkat cell line, which does not express Hsp27 constitutively or in response to γ-rays, was stably transfected with Hsp27 complementary DNA. Attenuation of Hsp27 expression was accomplished by antisense or RNAi (interfering RNA) strategies in SQ20B head-and-neck squamous carcinoma, PC3 prostate cancer, and U87 glioblastoma radioresistant cells. Results: We measured concentration-dependent protection against the cytotoxic effects of radiation in Jurkat-Hsp27 cells, which led to a 50% decrease in apoptotic cells at 48 hours in the highest expressing cells. Underlying mechanisms leading to radiation resistance involved a significant increase in glutathione levels associated with detoxification of reactive oxygen species, a delay in mitochondrial collapse, and caspase activation. Conversely, attenuation of Hsp27 in SQ20B cells, characterized by their resistance to apoptosis, sensitizes cells to irradiation. This was emphasized by increased apoptosis, decreased glutathione basal level, and clonogenic cell death. Sensitization to irradiation was confirmed in PC3 and U87 radioresistant cells. Conclusion: Hsp27 gene therapy offers a potential adjuvant to radiation-based therapy of resistant tumors

Association of HSP70-2 Gene 1267A/G Polymorphism With Cataract Incidence Among Guilan Population

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Zivar Salehi

2017-01-01

Full Text Available Background: Cataract is a visible opacity of the eye lens and it is the main reason of reversible blindness in the world. Oxidative stress is known as a major factor in cataract formation HSP70-2 protein is a molecular chaperone which is essential for cell survival in stress conditions. HSP70-2 gene is located in the human leukocyte antigen class ΙΙΙ region. This gene encodes an inducible protein. One of the common polymorphism of HSP70-2 is 1267A/G which is located in coding region. The aim of this study was to analysis of 1267A/G polymorphism of hsp70-2 gene in cataract patients. Material and Methods: This case-control study included 118 cataract patients and 122 healthy people as a control groups. Genomic DNA was extracted from peripheral blood leukocytes and genotyping determination was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP method. Statistical analysis was performed by using the MedCalc software (Version 12.1. Results: The frequency of G allele was significantly higher in patients than the controls, (0.36 and 0.24, respectively. A higher frequency of the GG genotype of the HSP70-2 1267A/G polymorphism was found in the patients compared with controls (21.19% and 8.20%, respectively. The patients carrying the GG genotype were 3.2-fold at a higher risk of cataract compared with AA genotype (P=0.005. Conclusion: The finding of this study suggested that the HSP70-2 1267A/G may affect the susceptibility to cataract in the studied population. Anyway the randomized multicenter studies with greater sample size still need to confirmed our results.

Hsp72 (HSPA1A Prevents Human Islet Amyloid Polypeptide Aggregation and Toxicity: A New Approach for Type 2 Diabetes Treatment.

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Paola C Rosas

Full Text Available Type 2 diabetes is a growing public health concern and accounts for approximately 90% of all the cases of diabetes. Besides insulin resistance, type 2 diabetes is characterized by a deficit in β-cell mass as a result of misfolded human islet amyloid polypeptide (h-IAPP which forms toxic aggregates that destroy pancreatic β-cells. Heat shock proteins (HSP play an important role in combating the unwanted self-association of unfolded proteins. We hypothesized that Hsp72 (HSPA1A prevents h-IAPP aggregation and toxicity. In this study, we demonstrated that thermal stress significantly up-regulates the intracellular expression of Hsp72, and prevents h-IAPP toxicity against pancreatic β-cells. Moreover, Hsp72 (HSPA1A overexpression in pancreatic β-cells ameliorates h-IAPP toxicity. To test the hypothesis that Hsp72 (HSPA1A prevents aggregation and fibril formation, we established a novel C. elegans model that expresses the highly amyloidogenic human pro-IAPP (h-proIAPP that is implicated in amyloid formation and β-cell toxicity. We demonstrated that h-proIAPP expression in body-wall muscles, pharynx and neurons adversely affects C. elegans development. In addition, we demonstrated that h-proIAPP forms insoluble aggregates and that the co-expression of h-Hsp72 in our h-proIAPP C. elegans model, increases h-proIAPP solubility. Furthermore, treatment of transgenic h-proIAPP C. elegans with ADAPT-232, known to induce the expression and release of Hsp72 (HSPA1A, significantly improved the growth retardation phenotype of transgenic worms. Taken together, this study identifies Hsp72 (HSPA1A as a potential treatment to prevent β-cell mass decline in type 2 diabetic patients and establishes for the first time a novel in vivo model that can be used to select compounds that attenuate h-proIAPP aggregation and toxicity.

Introgression of heat shock protein (Hsp70 and sHsp) genes into the Malaysian elite chilli variety Kulai (Capsicum annuum L.) through the application of marker-assisted backcrossing (MAB).

Science.gov (United States)

Usman, Magaji G; Rafii, Mohd Y; Martini, Mohammad Y; Yusuff, Oladosu A; Ismail, Mohd R; Miah, Gous

2018-03-01

Backcrossing together with simple sequence repeat marker strategy was adopted to improve popular Malaysian chilli Kulai (Capsicum annuum L.) for heat tolerance. The use of molecular markers in backcross breeding and selection contributes significantly to overcoming the main drawbacks such as increase linkage drag and time consumption, in the ancient manual breeding approach (conventional), and speeds up the genome recovery of the recurrent parent. The strategy was adopted to introgress heat shock protein gene(s) from AVPP0702 (C. annuum L.), which are heat-tolerant, into the genetic profile of Kulai, a popular high-yielding chilli but which is heat sensitive. The parents were grown on seed trays, and parental screening was carried out with 252 simple sequence repeat markers. The selected parents were crossed and backcrossed to generate F 1 hybrids and backcross generations. Sixty-eight markers appeared to be polymorphic and were used to assess the backcross generation; BC 1 F 1 , BC 2 F 1 and BC 3 F 1 . The average recipient allele of the selected four BC 1 F 1 plants was 80.75% which were used to produce the BC 2 F 1 generation. BC 1 -P 7 was the best BC 1 F 1 plant because it had the highest recovery at 83.40% and was positive to Hsp-linked markers (Hsp70-u2 and AGi42). After three successive generations of backcrossing, the average genome recovery of the recurrent parent in the selected plants in BC 3 F 1 was 95.37%. Hsp gene expression analysis was carried out on BC 1 F 1 , BC 2 F 1 and BC 3 F 1 selected lines. The Hsp genes were found to be up-regulated when exposed to heat treatment. The pattern of Hsp expression in the backcross generations was similar to that of the donor parent. This confirms the successful introgression of a stress-responsive gene (Hsp) into a Kulai chilli pepper variety. Furthermore, the yield performance viz. plant height, number of fruits, fruit length and weight and total yield of the improved plant were similar with the recurrent

Oral administration of Lactococcus lactis-expressing heat shock protein 65 and tandemly repeated IA2P2 prevents type 1 diabetes in NOD mice.

Science.gov (United States)

Liu, Kun-Feng; Liu, Xiao-Rui; Li, Guo-Liang; Lu, Shi-Ping; Jin, Liang; Wu, Jie

2016-06-01

Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease characterized by the destruction of insulin-secreting β cells upon autoreactive T cell attack. Oral administration of autoantigens is an attractive approach to treating T1DM, but an effective carrier should be used in order to protect antigens. Lactococcus lactis, a safe engineering strain, was used for this task in the present study. Two recombinant L. lactis expressing protein HSP65-6IA2P2 were used and be investigated the effects and mechanisms against T1DM in NOD mice. Our findings demonstrate that recombinant L. lactis strains can successfully both deliver antigens to intestinal mucosa and maintain the epitopes for a long time in NOD mice. Oral administration of recombinant L. lactis could prevent hyperglycemia, improve glucose tolerance, and reduce insulitis by inhibiting antigen-specific proliferation of T cells, augmenting regulatory immune reactions, and balancing ratios of Th17/Tregs and Th1/Th2. These results prove that orally administrated L. lactis expressing HSP65-6IA2P2 is an effective approach for the prevention of T1DM in NOD mice. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

B-chromosome effects on Hsp70 gene expression does not occur at transcriptional level in the grasshopper Eyprepocnemis plorans.

Science.gov (United States)

Navarro-Domínguez, Beatriz; Cabrero, Josefa; Camacho, Juan Pedro M; López-León, María Dolores

2016-10-01

As intragenomic parasites, B chromosomes can elicit stress in the host genome, thus inducing a response for host adaptation to this kind of continuous parasitism. In the grasshopper Eyprepocnemis plorans, B-chromosome presence has been previously associated with a decrease in the amount of the heat-shock protein 70 (HSP70). To investigate whether this effect is already apparent at transcriptional level, we analyze the expression levels of the Hsp70 gene in gonads and somatic tissues of males and females with and without B chromosomes from two populations, where the predominant B chromosome variants (B2 and B24) exhibit different levels of parasitism, by means of quantitative real-time PCR (qPCR) on complementary DNA (cDNA). The results revealed the absence of significant differences for Hsp70 transcripts associated with B-chromosome presence in virtually all samples. This indicates that the decrease in HSP70 protein levels, formerly reported in this species, may not be a consequence of transcriptional down-regulation of Hsp70 genes, but the result of post-transcriptional regulation. These results will help to design future studies oriented to identifying factors modulating Hsp70 expression, and will also contribute to uncover the biological role of B chromosomes in eukaryotic genomes.

Stressing Out Hsp90 in Neurotoxic Proteinopathies.

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Inda, Carmen; Bolaender, Alexander; Wang, Tai; Gandu, Srinivasa R; Koren, John

2016-01-01

A toxic accumulation of proteins is the hallmark pathology of several neurodegenerative disorders. Protein accumulation is regularly prevented by the network of molecular chaperone proteins, including and especially Hsp90. For reasons not yet elucidated, Hsp90 and the molecular chaperones interact with, but do not degrade, these toxic proteins resulting in the pathogenic accumulation of proteins such as tau, in Alzheimer's Disease, and α-synuclein, in Parkinson's Disease. In this review, we describe the associations between Hsp90 and the pathogenic and driver proteins of several neurodegenerative disorders. We additionally describe how the inhibition of Hsp90 promotes the degradation of both mutant and pathogenic protein species in models of neurodegenerative diseases. We also examine the current state of Hsp90 inhibitors capable of crossing the blood-brain barrier; compounds which may be capable of slowing, preventing, and possible reversing neurodegenerative diseases.

Molecular cloning of a Candida albicans gene (SSB1) coding for a protein related to the Hsp70 family.

Science.gov (United States)

Maneu, V; Cervera, A M; Martinez, J P; Gozalbo, D

1997-06-15

We have cloned and sequenced a Candida albicans gene (SSB1) encoding a potential member of the heat-shock protein seventy (hsp70) family. The protein encoded by this gene contains 613 amino acids and shows a high degree (85%) of sequence identity to the ssb subfamily (ssb1 and ssb2) of the Saccharomyces cerevisiae hsp70 family. The transcribed mRNA (2.1 kb) is present in similar amounts both in yeast and germ tube cells of C. albicans.

Expression of Helicobacter pylori hspA Gene in Lactococcus lactis NICE System and Experimental Study on Its Immunoreactivity

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Xiao-Juan Zhang

2015-01-01

Full Text Available Aim. The aim of this study was to develop an oral Lactococcus lactis (L. lactis vaccine against Helicobacter pylori (H. pylori. Methods. After L. lactis NZ3900/pNZ8110-hspA was constructed, growth curves were plotted to study whether the growth of recombinant L. lactis was affected after hspA was cloned into L. lactis and whether the growth of empty bacteria, empty plasmid bacteria, and recombinant L. lactis was affected by different concentrations of Nisin; SDS-PAGE and Western blot were adopted, respectively, to detect the HspA expressed by recombinant L. lactis and its immunoreactivity. Results. There was no effect observed from the growth curve after exogenous gene hspA was cloned into L. lactis NZ3900; different concentrations of Nisin did not affect the growth of NZ3900 and NZ3900/pNZ8110, while different concentrations of Nisin inhibited the growth of NZ3900/pNZ8110-hspA except 10 ng/mL Nisin. No HspA strip was observed from SDS-PAGE. Western blot analysis showed that HspA expressed by recombinant bacteria had favorable immunoreactivity. Conclusion. The growth of recombinant L. lactis was suppressed even though a small amount of HspA had been induced to express. Therefore recombinant L. lactis only express HspA which was not suitable to be oral vaccine against Helicobacter pylori.

Hsp40 gene therapy exerts therapeutic effects on polyglutamine disease mice via a non-cell autonomous mechanism.

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H Akiko Popiel

Full Text Available The polyglutamine (polyQ diseases such as Huntington's disease (HD, are neurodegenerative diseases caused by proteins with an expanded polyQ stretch, which misfold and aggregate, and eventually accumulate as inclusion bodies within neurons. Molecules that inhibit polyQ protein misfolding/aggregation, such as Polyglutamine Binding Peptide 1 (QBP1 and molecular chaperones, have been shown to exert therapeutic effects in vivo by crossing of transgenic animals. Towards developing a therapy using these aggregation inhibitors, we here investigated the effect of viral vector-mediated gene therapy using QBP1 and molecular chaperones on polyQ disease model mice. We found that injection of adeno-associated virus type 5 (AAV5 expressing QBP1 or Hsp40 into the striatum both dramatically suppresses inclusion body formation in the HD mouse R6/2. AAV5-Hsp40 injection also ameliorated the motor impairment and extended the lifespan of R6/2 mice. Unexpectedly, we found even in virus non-infected cells that AAV5-Hsp40 appreciably suppresses inclusion body formation, suggesting a non-cell autonomous therapeutic effect. We further show that Hsp40 inhibits secretion of the polyQ protein from cultured cells, implying that it inhibits the recently suggested cell-cell transmission of the polyQ protein. Our results demonstrate for the first time the therapeutic effect of Hsp40 gene therapy on the neurological phenotypes of polyQ disease mice.

Mycobacterium tuberculosis HspX/EsxS Fusion Protein: Gene Cloning, Protein Expression, and Purification in Escherichia coli.

Science.gov (United States)

Khademi, Farzad; Yousefi-Avarvand, Arshid; Derakhshan, Mohammad; Meshkat, Zahra; Tafaghodi, Mohsen; Ghazvini, Kiarash; Aryan, Ehsan; Sankian, Mojtaba

2017-10-01

The purpose of this study was to clone, express, and purify a novel multidomain fusion protein of Micobacterium tuberculosis (Mtb) in a prokaryotic system. An hspX/esxS gene construct was synthesized and ligated into a pGH plasmid, E. coli TOP10 cells were transformed, and the vector was purified. The vector containing the construct and pET-21b (+) plasmid were digested with the same enzymes and the construct was ligated into pET-21b (+). The accuracy of cloning was confirmed by colony PCR and sequencing. E. coli BL21 cells were transformed with the pET-21b (+)/hspX/esxS expression vector and protein expression was evaluated. Finally, the expressed fusion protein was purified on a Ni-IDA column and verified by SDS-PAGE and western blotting. The hspX/esxS gene construct was inserted into pET-21b (+) and recombinant protein expression was induced with IPTG in E. coli BL21 cells. Various concentrations of IPTG were tested to determine the optimum concentration for expression induction. The recombinant protein was expressed in insoluble inclusion bodies. Three molar guanidine HCl was used to solubilize the insoluble protein. An HspX/EsxS Mtb fusion protein was expressed in E. coli and the recombinant protein was purified. After immunological analysis, the HspX/EsxS fusion protein might be an anti-tuberculosis vaccine candidate in future clinical trial studies.

Hsp12p and PAU genes are involved in ecological interactions between natural yeast strains.

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Rivero, Damaríz; Berná, Luisa; Stefanini, Irene; Baruffini, Enrico; Bergerat, Agnes; Csikász-Nagy, Attila; De Filippo, Carlotta; Cavalieri, Duccio

2015-08-01

The coexistence of different yeasts in a single vineyard raises the question on how they communicate and why slow growers are not competed out. Genetically modified laboratory strains of Saccharomyces cerevisiae are extensively used to investigate ecological interactions, but little is known about the genes regulating cooperation and competition in ecologically relevant settings. Here, we present evidences of Hsp12p-dependent altruistic and contact-dependent competitive interactions between two natural yeast isolates. Hsp12p is released during cell death for public benefit by a fast-growing strain that also produces a killer toxin to inhibit growth of a slow grower that can enjoy the benefits of released Hsp12p. We also show that the protein Pau5p is essential in the defense against the killer effect. Our results demonstrate that the combined action of Hsp12p, Pau5p and a killer toxin is sufficient to steer a yeast community. © 2015 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

IL-10 down-regulates the expression of survival associated gene hspX of Mycobacterium tuberculosis in murine macrophage

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Babban Jee

2017-07-01

Full Text Available Mycobacterium tuberculosis (MTB adopts a special survival strategy to overcome the killing mechanism(s of host immune system. Amongst the many known factors, small heat shock protein 16.3 (sHSP16.3 of MTB encoded by gene hspX has been reported to be critical for the survival of MTB. In the present study, the effect of recombinant murine interferon-gamma (rmIFN-γ and recombinant murine interleukin-10 (rmIL-10 on the expression of gene hspX of MTB in murine macrophage RAW264.7 has been investigated. By real-time RT-PCR, it was observed that three increasing concentrations (5, 25 and 50 ng/ml of rmIFN-γ significantly up-regulated the expression of hspX whereas similar concentrations of rmIL-10 (5, 25 and 50 ng/ml significantly down-regulated the hspX expression. This effect was not only dependent on the concentration of the stimulus but this was time-dependent as well. A contrasting pattern of hspX expression was observed against combinations of two different concentrations of rmIFN-γ and rmIL-10. The study results suggest that rIL-10 mediated down-regulation of hspX expression, in the presence of low concentration of rIFN-γ, could be used as an important strategy to decrease the dormancy of MTB in its host and thus making MTB susceptible to the standard anti-mycobacterial therapy used for treating tuberculosis. However, as these are only preliminary results in the murine cell line model, this hypothesis needs to be first validated in human cell lines and subsequently in animal models mimicking the latent infection using clinical isolates of MTB before considering the development of modified regimens for humans.

Genetic Diversity and Phylogenetic Analysis of the Iranian Leishmania Parasites Based on HSP70 Gene PCR-RFLP and Sequence Analysis.

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Nemati, Sara; Fazaeli, Asghar; Hajjaran, Homa; Khamesipour, Ali; Anbaran, Mohsen Falahati; Bozorgomid, Arezoo; Zarei, Fatah

2017-08-01

Despite the broad distribution of leishmaniasis among Iranians and animals across the country, little is known about the genetic characteristics of the causative agents. Applying both HSP70 PCR-RFLP and sequence analyses, this study aimed to evaluate the genetic diversity and phylogenetic relationships among Leishmania spp. isolated from Iranian endemic foci and available reference strains. A total of 36 Leishmania isolates from almost all districts across the country were genetically analyzed for the HSP70 gene using both PCR-RFLP and sequence analysis. The original HSP70 gene sequences were aligned along with homologous Leishmania sequences retrieved from NCBI, and subjected to the phylogenetic analysis. Basic parameters of genetic diversity were also estimated. The HSP70 PCR-RFLP presented 3 different electrophoretic patterns, with no further intraspecific variation, corresponding to 3 Leishmania species available in the country, L. tropica, L. major, and L. infantum. Phylogenetic analyses presented 5 major clades, corresponding to 5 species complexes. Iranian lineages, including L. major, L. tropica, and L. infantum, were distributed among 3 complexes L. major, L. tropica, and L. donovani. However, within the L. major and L. donovani species complexes, the HSP70 phylogeny was not able to distinguish clearly between the L. major and L. turanica isolates, and between the L. infantum, L. donovani, and L. chagasi isolates, respectively. Our results indicated that both HSP70 PCR-RFLP and sequence analyses are medically applicable tools for identification of Leishmania species in Iranian patients. However, the reduced genetic diversity of the target gene makes it inevitable that its phylogeny only resolves the major groups, namely, the species complexes.

Association of eNOS and HSP70 gene polymorphisms with glaucoma in Pakistani cohorts.

NARCIS (Netherlands)

Ayub, H.; Khan, M.I.; Micheal, S.; Akhtar, F.; Ajmal, M.; Shafique, S.; Ali, S.H.; Hollander, A.I. den; Ahmed, A.; Qamar, R.

2010-01-01

PURPOSE: To investigate the involvement of stress-regulating genes, endothelial nitric oxide synthase (eNOS) and heat shock protein 70 (HSP70) with primary open angle glaucoma (POAG) and primary closed angle glaucoma (PCAG). METHODS: POAG and PCAG patients recruited from different areas of Pakistan

Comparison of inhibitory effects of 17-AAG nanoparticles and free 17-AAG on HSP90 gene expression in breast cancer.

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Ghalhar, Masoud Gandomkar; Akbarzadeh, Abolfazl; Rahmati, Mohammad; Mellatyar, Hassan; Dariushnejad, Hassan; Zarghami, Nosratallah; Barkhordari, Amin

2014-01-01

HSP90 may be overexpressed in cancer cells which are greatly dependent on Hsp90 function. Geldanamycin derivative 17 allylamino-17-demethoxygeldanamycin (17-AAG) inhibits the function and expression of HSP90. 17-AAG has poor water-solubility which is a potential problem for clinical practice. In this study for improving the stability and solubility of molecules in drug delivery systems we used a β-cyclodextrin- 17AAG complex. To assess cytotoxic effects of β-cyclodextrin-17AAG complexes and free 17AAG, colorimetric cell viability (MTT) assays were performed. Cells were treated with equal concentrations of β-cyclodextrin- 17AAG complex and free 17AAG and Hsp90 gene expression levels in the two groups was compared by real-time PCR. MTT assay confirmed that β-cyclodextrin- 17AAG complex enhanced 17AAG cytotoxicity and drug delivery in T47D breast cancer cells. The level of Hsp90 gene expression in cells treated with β-cyclodextrin- 17AAG complex was lower than that of cells treated with free 17AAG (P=0.001). The results demonstrated that β-cyclodextrin- 17AAG complexes are more effective than free 17AAG in down-regulating HSP90 expression due to enhanced β-cyclodextrin-17AAG uptake by cells. Therefore, β-cyclodextrin could be superior carrier for this kind of hydrophobic agent.

The association of SNPs in Hsp90β gene 5' flanking region with thermo tolerance traits and tissue mRNA expression in two chicken breeds.

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Chen, Zhuo-Yu; Gan, Jian-Kang; Xiao, Xiong; Jiang, Li-Yan; Zhang, Xi-Quan; Luo, Qing-Bin

2013-09-01

Thermo stress induces heat shock proteins (HSPs) expression and HSP90 family is one of them that has been reported to involve in cellular protection against heat stress. But whether there is any association of genetic variation in the Hsp90β gene in chicken with thermo tolerance is still unknown. Direct sequencing was used to detect possible SNPs in Hsp90β gene 5' flanking region in 3 chicken breeds (n = 663). Six mutations, among which 2 SNPs were chosen and genotypes were analyzed with PCR-RFLP method, were found in Hsp90β gene in these 3 chicken breeds. Association analysis indicated that SNP of C.-141G>A in the 5' flanking region of the Hsp90β gene in chicken had some effect on thermo tolerance traits, which may be a potential molecular marker of thermo tolerance, and the genotype GG was the thermo tolerance genotype. Hsp90β gene mRNA expression in different tissues detected by quantitative real-time PCR assay were demonstrated to be tissue dependent, implying that different tissues have distinct sensibilities to thermo stress. Besides, it was shown time specific and varieties differences. The expression of Hsp90β mRNA in Lingshan chickens in some tissues including heart, liver, brain and spleen were significantly higher or lower than that of White Recessive Rock (WRR). In this study, we presume that these mutations could be used in marker assisted selection for anti-heat stress chickens in our breeding program, and WRR were vulnerable to tropical thermo stress whereas Lingshan chickens were well adapted.

Association of ACE, VEGF and CCL2 gene polymorphisms with Henoch-Schönlein purpura and an evaluation of the possible interaction effects of these loci in HSP patients.

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Mohammadian, Tahereh; Bonyadi, Mortaza; Nabat, Elahe; Rafeey, Mandana

2017-07-01

Henoch-Schönlein purpura (HSP) is a multisystem, small vessel, leucocytoclastic vasculitis. It is predominantly a childhood vasculitis, rarely reported in adults. Studies have shown that several different genetic factors such as genes involved in inflammatory system and renin-angiotensin system (RAS) are important in the pathogenesis of Henoch-Schönlein purpura. The purpose of this study was to evaluate the independent effect of 3 gene polymorphisms including CCL2-2518 C/T, VEGF-634G/C and ACE(I/D) with HSP disease and their possible joint interactions in developing the disease. In this case-control study 47 HSP cases and 74 unrelated healthy controls were enrolled for evaluation. All individuals were genotyped for CCL2-2518C/T, VEGF-634G/C and ACE(I/D) gene polymorphisms. The possible association of these polymorphisms with susceptibility to develop HSP disease independently and in different joint combinations was evaluated. The frequencies of TT genotype and T allele of CCL2-2518C/T gene polymorphism and CC genotype and C allele of VEGF-634G/C gene polymorphism were significantly high in HSP children (p-values = 0.005 and = 0.007 respectively). Interestingly, studying the joint interaction of these 2 genotypes (CC genotype of VEGF G-634C and TT genotype of CCL2 C-2518T) in this cohort showed a more significant effect in the development of the disease (p gene when combined with II genotype of ACE gene in HSP children was significantly higher (p gene-gene interaction effects of CCL2, VEGF and ACE genes in developing HSP disease.

Recovery from heat, salt and osmotic stress in Physcomitrella patens requires a functional small heat shock protein PpHsp16.4.

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Ruibal, Cecilia; Castro, Alexandra; Carballo, Valentina; Szabados, László; Vidal, Sabina

2013-11-05

Plant small heat shock proteins (sHsps) accumulate in response to various environmental stresses, including heat, drought, salt and oxidative stress. Numerous studies suggest a role for these proteins in stress tolerance by preventing stress-induced protein aggregation as well as by facilitating protein refolding by other chaperones. However, in vivo evidence for the involvement of sHsps in tolerance to different stress factors is still missing, mainly due to the lack of appropriate mutants in specific sHsp genes. In this study we characterized the function of a sHsp in abiotic stress tolerance in the moss Physcomitrella patens, a model for primitive land plants. Using suppression subtractive hybridization, we isolated an abscisic acid-upregulated gene from P. patens encoding a 16.4 kDa cytosolic class II sHsp. PpHsp16.4 was also induced by salicylic acid, dithiothreitol (DTT) and by exposure to various stimuli, including osmotic and salt stress, but not by oxidative stress-inducing compounds. Expression of the gene was maintained upon stress relief, suggesting a role for this protein in the recovery stage. PpHsp16.4 is encoded by two identical genes arranged in tandem in the genome. Targeted disruption of both genes resulted in the inability of plants to recover from heat, salt and osmotic stress. In vivo localization studies revealed that PpHsp16.4 localized in cytosolic granules in the vicinity of chloroplasts under non stress conditions, suggesting possible distinct roles for this protein under stress and optimal growth. We identified a member of the class II sHsp family that showed hormonal and abiotic stress gene regulation. Induction of the gene by DTT treatment suggests that damaged proteins may act as signals for the stress-induction of PpHsp16.4. The product of this gene was shown to localize in cytosolic granules near the chloroplasts, suggesting a role for the protein in association with these organelles. Our study provides the first direct genetic

Hsp70 cochaperones HspBP1 and BAG-1M differentially regulate steroid hormone receptor function.

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Regina T Knapp

Full Text Available Hsp70 binding protein 1 (HspBP1 and Bcl2-associated athanogene 1 (BAG-1, the functional orthologous nucleotide exchange factors of the heat shock protein 70 kilodalton (Hsc70/Hsp70 chaperones, catalyze the release of ADP from Hsp70 while inducing different conformational changes of the ATPase domain of Hsp70. An appropriate exchange rate of ADP/ATP is crucial for chaperone-dependent protein folding processes. Among Hsp70 client proteins are steroid receptors such as the glucocorticoid receptor (GR, the mineralocorticoid receptor (MR, and the androgen receptor (AR. BAG-1 diversely affects steroid receptor activity, while to date the influence of HspBP1 on steroid receptor function is mostly unknown. Here, we compared the influence of HspBP1 and BAG-1M on Hsp70-mediated steroid receptor folding complexes and steroid receptor activity. Coimmunoprecipitation studies indicated preferential binding of Hsp40 and the steroid receptors to BAG-1M as compared to HspBP1. Furthermore, Hsp70 binding to the ligand-binding domain of GR was reduced in the presence of HspBP1 but not in the presence of BAG-1M as shown by pull-down assays. Reporter gene experiments revealed an inhibitory effect on GR, MR, and AR at a wide range of HspBP1 protein levels and at hormone concentrations at or approaching saturation. BAG-1M exhibited a transition from stimulatory effects at low BAG-1M levels to inhibitory effects at higher BAG-1M levels. Overall, BAG-1M and HspBP1 had differential impacts on the dynamic composition of steroid receptor folding complexes and on receptor function with important implications for steroid receptor physiology.

Cloning and Expression of a Cytosolic HSP90 Gene in Chlorella vulgaris

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Zhengyi Liu

2014-01-01

Full Text Available Heat shock protein 90 (HSP90, a highly conserved molecular chaperone, plays essential roles in folding, keeping structural integrity, and regulating the subset of cytosolic proteins. We cloned the cDNA of Chlorella vulgaris HSP90 (named CvHSP90 by combining homology cloning with rapid amplification of cDNA ends (RACE. Sequence analysis indicated that CvHSP90 is a cytosolic member of the HSP90 family. Quantitative RT-PCR was applied to determine the expression level of messenger RNA (mRNA in CvHSP90 under different stress conditions. C. vulgaris was kept in different temperatures (5–45°C for 1 h. The mRNA expression level of CvHSP90 increased with temperature from 5 to 10°C, went further from 35 to 40°C, and reached the maximum at 40°C. On the other hand, for C. vulgaris kept at 35°C for different durations, the mRNA expression level of CvHSP90 increased gradually and reached the peak at 7 h and then declined progressively. In addition, the expression level of CvHSP90 at 40 or 45 in salinity (‰ was almost fourfold of that at 25 in salinity (‰ for 2 h. Therefore, CvHSP90 may be a potential biomarker to monitor environment changes.

HSF and Msn2/4p can exclusively or cooperatively activate the yeast HSP104 gene.

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Grably, Melanie R; Stanhill, Ariel; Tell, Osnat; Engelberg, David

2002-04-01

In an effort to understand how an accurate level of stress-specific expression is obtained, we studied the promoter of the yeast HSP104 gene. Through 5' deletions, we defined a 334 bp fragment upstream of the first coding AUG as sufficient and essential for maximal basal activity and a 260 bp fragment as sufficient and essential for heat shock responsiveness. These sequences contain heat shock elements (HSEs) and stress response elements (STREs) that cooperate to achieve maximal inducible expression. However, in the absence of one set of factors (e.g. in msn2Deltamsn4Delta cells) proper induction is obtained exclusively through HSEs. We also show that HSP104 is constitutively derepressed in ras2Delta cells. This derepression is achieved exclusively through activation of STREs, with no role for HSEs. Strikingly, in ras2Deltamsn2Deltamsn4Delta cells the HSP104 promoter is also derepressed, but in this strain derepression is mediated through HSEs, showing the flexibility and adaptation of the promoter. Thus, appropriate transcription of HSP104 is usually obtained through cooperation between the Msn2/4/STRE and the HSF/ HSE systems, but each factor could activate the promoter alone, backing up the other. Transcription control of HSP104 is adaptive and robust, ensuring proper expression under extreme conditions and in various mutants.

The evolutionary capacitor HSP90 buffers the regulatory effects of mammalian endogenous retroviruses.

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Hummel, Barbara; Hansen, Erik C; Yoveva, Aneliya; Aprile-Garcia, Fernando; Hussong, Rebecca; Sawarkar, Ritwick

2017-03-01

Understanding how genotypes are linked to phenotypes is important in biomedical and evolutionary studies. The chaperone heat-shock protein 90 (HSP90) buffers genetic variation by stabilizing proteins with variant sequences, thereby uncoupling phenotypes from genotypes. Here we report an unexpected role of HSP90 in buffering cis-regulatory variation affecting gene expression. By using the tripartite-motif-containing 28 (TRIM28; also known as KAP1)-mediated epigenetic pathway, HSP90 represses the regulatory influence of endogenous retroviruses (ERVs) on neighboring genes that are critical for mouse development. Our data based on natural variations in the mouse genome show that genes respond to HSP90 inhibition in a manner dependent on their genomic location with regard to strain-specific ERV-insertion sites. The evolutionary-capacitor function of HSP90 may thus have facilitated the exaptation of ERVs as key modifiers of gene expression and morphological diversification. Our findings add a new regulatory layer through which HSP90 uncouples phenotypic outcomes from individual genotypes.

HSP70 gene expression in Mytilus galloprovincialis hemocytes is triggered by moderate heat shock and Vibrio anguillarum, but not by V. splendidus or Micrococcus lysodeikticus.

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Cellura, Cinzia; Toubiana, Mylène; Parrinello, Nicolo; Roch, Philippe

2006-01-01

Complete sequence of HSP70 cDNA from the mussel, Mytilus galloprovincialis was established before quantifying its expression following moderate heat shock or injection of heat-killed bacteria. HSP70 cDNA is comprised of 2378 bp including one ORF of 654 aa, with a predicted 70 bp 5'-UTR and a 343 bp 3'-UTR (GenBank, 18 Jan 05, AY861684). Alignment identity ranged from 89% for Crassostrea ariakensis to 72% for C. virginica. Curiously, HSP70 gene and cDNA sequences from M. galloprovincialis, deposited later (03 and 27 May), show only 73% identity with the present sequence. Meanwhile, characteristic motifs of the HSP70 family were located in conserved positions. Expression of HSP70 gene was quantified on circulating hemocyte mRNA using Q-PCR after RT using random hexaprimers. Housekeeping gene was 28S rRNA. Four stresses were applied: heat shock that consisted of immersing mussels for 90 min at 30 degrees C and returning them to 20 degrees C sea water, one injection of heat-killed Gram-negative bacteria, Vibrio splendidus LGP32, one injection of heat-killed Gram-negative bacteria Vibrio anguillarum, one injection of heat-killed Gram-positive bacteria Micrococcus lysodeikticus. We found no significant modification of 28S rRNA gene expression. Significant increase of 5.2 +/- 0.4 fold the ratio HSP70/28S rRNA was observed 6 h after heat shock and was maximum at 15 h (6.1 +/- 1.1), and still significant after 24 h (1.7 +/- 0.03). Similarly, injecting V. anguillarum resulted in a significant increase of 2.7 +/- 0.1 after 12 h. Expression was maximum after 48 h (5.2 +/- 0.05) and returned to baseline after 72 h. In contrast, injecting V. splendidus or M. lysodeikticus failed to significantly modulate HSP70 gene expression at least during the first 3 days post-injection. Consequently, mussel hemocytes appeared to discriminate between pathogenic and non-pathogenic Vibrios, as well as between Gram-negative and Gram-positive bacteria.

Identification of Candida Species Using MP65 Gene and Evaluation of the Candida albicans MP65 Gene Expression in BALB/C Mice.

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Bineshian, Farahnaz; Yadegari, Mohammad Hossien; Sharifi, Zohre; Akbari Eidgahi, Mohammadreza; Nasr, Reza

2015-05-01

Systemic candidiasis is a major public health concern. In particular, in immunocompromised people, such as patients with neutropenia, patients with Acquired Immune Deficiency Syndrome (AIDS) and cancer who are undergoing antiballistic chemotherapy or bone marrow transplants, and people with diabetes. Since the clinical signs and symptoms are nonspecific, early diagnosis is often difficult. The 65-kDa mannoprotein (MP65) gene of Candida albicans is appropriate for detection and identification of systemic candidiasis. This gene encodes a putative b-glucanase mannoprotein of 65 kDa, which plays a major role in the host-fungus relationship, morphogenesis and pathogenicity. The current study aimed to identify different species of Candida (C. albicans, C. glabrata and C. parapsilosis) using the Polymerase Chain Reaction (PCR) technique and also to evaluate C. albicans MP65 gene expression in BALB/C mice. All yeast isolates were identified on cornmeal agar supplemented with tween-80, germ tube formation in serum, and assimilation of carbon sources in the API 20 C AUX yeast identification system. Polymerase Chain Reaction was performed on all samples using species-specific primers for the MP65 65 kDa gene. After RNA extraction, cDNA synthesis was performed by the Maxime RT Pre Mix kit. Candida albicans MP65 gene expression was evaluated by quantitative Real-Time (q Real-Time) and Real-Time (RT) PCR techniques. The 2-ΔΔCT method was used to analyze relative changes in gene expression of MP65. For statistical analysis, nonparametric Wilcoxon test was applied using the SPSS version 16 software. Using biochemical methods, one hundred, six and one isolates of clinical samples were determined as C. albicans, C. glabrata and C. parapsilosis, respectively. Species-specific primers for PCR experiments were applied to clinical specimens, and in all cases a single expected band for C. albicans, C. glabrata and C. parapsilosis was obtained (475, 361 and 124 base pairs, respectively

Cyclophilin 40 facilitates HSP90-mediated RISC assembly in plants.

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Iki, Taichiro; Yoshikawa, Manabu; Meshi, Tetsuo; Ishikawa, Masayuki

2012-01-18

Posttranscriptional gene silencing is mediated by RNA-induced silencing complexes (RISCs) that contain AGO proteins and single-stranded small RNAs. The assembly of plant AGO1-containing RISCs depends on the molecular chaperone HSP90. Here, we demonstrate that cyclophilin 40 (CYP40), protein phosphatase 5 (PP5), and several other proteins with the tetratricopeptide repeat (TPR) domain associates with AGO1 in an HSP90-dependent manner in extracts of evacuolated tobacco protoplasts (BYL). Intriguingly, CYP40, but not the other TPR proteins, could form a complex with small RNA duplex-bound AGO1. Moreover, CYP40 that was synthesized by in-vitro translation using BYL uniquely facilitated binding of small RNA duplexes to AGO1, and as a result, increased the amount of mature RISCs that could cleave target RNAs. CYP40 was not contained in mature RISCs, indicating that the association is transient. Addition of PP5 or cyclophilin-binding drug cyclosporine A prevented the association of endogenous CYP40 with HSP90-AGO1 complex and inhibited RISC assembly. These results suggest that a complex of AGO1, HSP90, CYP40, and a small RNA duplex is a key intermediate of RISC assembly in plants.

Hsp90 and hepatobiliary transformation during sea lamprey metamorphosis.

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Chung-Davidson, Yu-Wen; Yeh, Chu-Yin; Bussy, Ugo; Li, Ke; Davidson, Peter J; Nanlohy, Kaben G; Brown, C Titus; Whyard, Steven; Li, Weiming

2015-12-01

Biliary atresia (BA) is a human infant disease with inflammatory fibrous obstructions in the bile ducts and is the most common cause for pediatric liver transplantation. In contrast, the sea lamprey undergoes developmental BA with transient cholestasis and fibrosis during metamorphosis, but emerges as a fecund adult. Therefore, sea lamprey liver metamorphosis may serve as an etiological model for human BA and provide pivotal information for hepatobiliary transformation and possible therapeutics. We hypothesized that liver metamorphosis in sea lamprey is due to transcriptional reprogramming that dictates cellular remodeling during metamorphosis. We determined global gene expressions in liver at several metamorphic landmark stages by integrating mRNA-Seq and gene ontology analyses, and validated the results with real-time quantitative PCR, histological and immunohistochemical staining. These analyses revealed that gene expressions of protein folding chaperones, membrane transporters and extracellular matrices were altered and shifted during liver metamorphosis. HSP90, important in protein folding and invertebrate metamorphosis, was identified as a candidate key factor during liver metamorphosis in sea lamprey. Blocking HSP90 with geldanamycin facilitated liver metamorphosis and decreased the gene expressions of the rate limiting enzyme for cholesterol biosynthesis, HMGCoA reductase (hmgcr), and bile acid biosynthesis, cyp7a1. Injection of hsp90 siRNA for 4 days altered gene expressions of met, hmgcr, cyp27a1, and slc10a1. Bile acid concentrations were increased while bile duct and gall bladder degeneration was facilitated and synchronized after hsp90 siRNA injection. HSP90 appears to play crucial roles in hepatobiliary transformation during sea lamprey metamorphosis. Sea lamprey is a useful animal model to study postembryonic development and mechanisms for hsp90-induced hepatobiliary transformation.

Phylogenetic analysis of HSP70 and cyt b gene sequences for Chinese Leishmania isolates and ultrastructural characteristics of Chinese Leishmania sp.

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Yuan, Dongmei; Qin, Hanxiao; Zhang, Jianguo; Liao, Lin; Chen, Qiwei; Chen, Dali; Chen, Jianping

2017-02-01

Leishmaniasis is a worldwide epidemic disease caused by the genus Leishmania, which is still endemic in the west and northwest areas of China. Some viewpoints of the traditional taxonomy of Chinese Leishmania have been challenged by recent phylogenetic researches based on different molecular markers. However, the taxonomic positions and phylogenetic relationships of Chinese Leishmania isolates remain controversial, which need for more data and further analysis. In this study, the heat shock protein 70 (HSP70) gene and cytochrome b (cyt b) gene were used for phylogenetic analysis of Chinese Leishmania isolates from patients, dogs, gerbils, and sand flies in different geographic origins. Besides, for the interesting Leishmania sp. in China, the ultrastructure of three Chinese Leishmania sp. strains (MHOM/CN/90/SC10H2, SD, GL) were observed by transmission electron microscopy. Bayesian trees from HSP70 and cyt b congruently indicated that the 14 Chinese Leishmania isolates belong to three Leishmania species including L. donovani complex, L. gerbilli, and L. (Sauroleishmania) sp. Their identity further confirmed that the undescribed Leishmania species causing visceral Leishmaniasis (VL) in China is closely related to L. tarentolae. The phylogenetic results from HSP70 also suggested the classification of subspecies within L. donovani complex: KXG-918, KXG-927, KXG-Liu, KXG-Xu, 9044, SC6, and KXG-65 belong to L. donovani; Cy, WenChuan, and 801 were proposed to be L. infantum. Through transmission electron microscopy, unexpectedly, the Golgi apparatus were not observed in SC10H2, SD, and GL, which was similar to previous reports of reptilian Leishmania. The statistical analysis of microtubule counts separated SC10H2, SD, and GL as one group from any other reference strain (L. donovani MHOM/IN/80/DD8; L. tropica MHOM/SU/74/K27; L. gerbilli MRHO/CN/60/GERBILLI). The ultrastructural characteristics of Leishmania sp. partly lend support to the phylogenetic inference that

Identification of HSP90 gene from the Chinese oak silkworm ...

African Journals Online (AJOL)

ajl user 1

2012-06-28

Jun 28, 2012 ... College of Life Science, Anhui Agricultural University, 130 Changjiang West Road 230036, Peoples ... program consisted of 5 min at 94°C followed by 35 cycles of 94°C ... HSP90 and 73.1% identity with Bombyx mori HSP90.

Cloning and expression of the coding regions of the heat shock proteins HSP10 and HSP16 from Piscirickettsia salmonis

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VIVIAN WILHELM

2003-01-01

Full Text Available The genes encoding the heat shock proteins HSP10 and HSP16 of the salmon pathogen Piscirickettsia salmonis have been isolated and sequenced. The HSP10 coding sequence is located in an open reading frame of 291 base pairs encoding 96 aminoacids. The HSP16 coding region was isolated as a 471 base pair fragment encoding a protein of 156 aminoacids. The deduced aminoacid sequences of both proteins show a significant homology to the respective protein from other prokaryotic organisms. Both proteins were expressed in E. coli as fusion proteins with thioredoxin and purified by chromatography on Ni-column. A rabbit serum against P. salmonis total proteins reacts with the recombinant HSP10 and HSP16 proteins. Similar reactivity was determined by ELISA using serum from salmon infected with P. salmonis. The possibility of formulating a vaccine containing these two proteins is discussed

HSP90 Shapes the Consequences of Human Genetic Variation.

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Karras, Georgios I; Yi, Song; Sahni, Nidhi; Fischer, Máté; Xie, Jenny; Vidal, Marc; D'Andrea, Alan D; Whitesell, Luke; Lindquist, Susan

2017-02-23

HSP90 acts as a protein-folding buffer that shapes the manifestations of genetic variation in model organisms. Whether HSP90 influences the consequences of mutations in humans, potentially modifying the clinical course of genetic diseases, remains unknown. By mining data for >1,500 disease-causing mutants, we found a strong correlation between reduced phenotypic severity and a dominant (HSP90 ≥ HSP70) increase in mutant engagement by HSP90. Examining the cancer predisposition syndrome Fanconi anemia in depth revealed that mutant FANCA proteins engaged predominantly by HSP70 had severely compromised function. In contrast, the function of less severe mutants was preserved by a dominant increase in HSP90 binding. Reducing HSP90's buffering capacity with inhibitors or febrile temperatures destabilized HSP90-buffered mutants, exacerbating FA-related chemosensitivities. Strikingly, a compensatory FANCA somatic mutation from an "experiment of nature" in monozygotic twins both prevented anemia and reduced HSP90 binding. These findings provide one plausible mechanism for the variable expressivity and environmental sensitivity of genetic diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Sense and antisense transcripts of the developmentally regulated murine hsp70.2 gene are expressed in distinct and only partially overlapping areas in the adult brain

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Murashov, A. K.; Wolgemuth, D. J.

1996-01-01

We have examined the spatial pattern of expression of a member of the hsp70 gene family, hsp70.2, in the mouse central nervous system. Surprisingly, RNA blot analysis and in situ hybridization revealed abundant expression of an 'antisense' hsp70.2 transcript in several areas of adult mouse brain. Two different transcripts recognized by sense and antisense riboprobes for the hsp70.2 gene were expressed in distinct and only partially overlapping neuronal populations. RNA blot analysis revealed low levels of the 2.7 kb transcript of hsp70.2 in several areas of the brain, with highest signal in the hippocampus. Abundant expression of a slightly larger (approximately 2.8 kb) 'antisense' transcript was detected in several brain regions, notably in the brainstem, cerebellum, mesencephalic tectum, thalamus, cortex, and hippocampus. In situ hybridization revealed that the sense and antisense transcripts were both predominantly neuronal and localized to the same cell types in the granular layer of the cerebellum, trapezoid nucleus of the superior olivary complex, locus coeruleus and hippocampus. The hsp70.2 antisense transcripts were particularly abundant in the frontal cortex, dentate gyrus, subthalamic nucleus, zona incerta, superior and inferior colliculi, central gray, brainstem, and cerebellar Purkinje cells. Our findings have revealed a distinct cellular and spatial localization of both sense and antisense transcripts, demonstrating a new level of complexity in the function of the heat shock genes.

Production and immune response of recombinant Hsp60 and Hsp70 from the salmon pathogen Piscirickettsia salmonis

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VIVIAN WILHELM

2005-01-01

Full Text Available We have isolated and sequenced the genes encoding the heat shock proteins 60 (Hsp60 and 70 (Hsp70 of the salmon pathogen Piscirickettsia salmonis. The sequence analysis revealed the expected two open reading frames that encode proteins with calculated molecular weights of 60,060 and 70,400. The proteins exhibit a 70-80% homology with other known prokaryotic Hsp60 and Hsp70 sequences. The coding regions have been expressed in E. coli as thioredoxin fusion proteins. Both recombinant proteins were shown to elicit a humoral response when injected intraperitoneally in Atlantic salmon and also conferred protection to fish challenged with P. salmonis. The present data will facilitate further studies on the involvement of heat shock proteins in protective immunity of fish to infection by P. salmonis and their potential use in recombinants vaccines against this intracellular pathogen.

Hsf and Hsp gene families in Populus: genome-wide identification, organization and correlated expression during development and in stress responses.

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Zhang, Jin; Liu, Bobin; Li, Jianbo; Zhang, Li; Wang, Yan; Zheng, Huanquan; Lu, Mengzhu; Chen, Jun

2015-03-14

Heat shock proteins (Hsps) are molecular chaperones that are involved in many normal cellular processes and stress responses, and heat shock factors (Hsfs) are the transcriptional activators of Hsps. Hsfs and Hsps are widely coordinated in various biological processes. Although the roles of Hsfs and Hsps in stress responses have been well characterized in Arabidopsis, their roles in perennial woody species undergoing various environmental stresses remain unclear. Here, a comprehensive identification and analysis of Hsf and Hsp families in poplars is presented. In Populus trichocarpa, we identified 42 paralogous pairs, 66.7% resulting from a whole genome duplication. The gene structure and motif composition are relatively conserved in each subfamily. Microarray and quantitative real-time RT-PCR analyses showed that most of the Populus Hsf and Hsp genes are differentially expressed upon exposure to various stresses. A coexpression network between Populus Hsf and Hsp genes was generated based on their expression. Coordinated relationships were validated by transient overexpression and subsequent qPCR analyses. The comprehensive analysis indicates that different sets of PtHsps are downstream of particular PtHsfs and provides a basis for functional studies aimed at revealing the roles of these families in poplar development and stress responses.

Study of single nucleotide polymorphisms of tumour necrosis factors and HSP genes in nasopharyngeal carcinoma in North East India.

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Lakhanpal, Meena; Singh, Laishram Chandreshwor; Rahman, Tashnin; Sharma, Jagnnath; Singh, M Madhumangal; Kataki, Amal Chandra; Verma, Saurabh; Pandrangi, Santhi Latha; Singh, Y Mohan; Wajid, Saima; Kapur, Sujala; Saxena, Sunita

2016-01-01

Nasopharyngeal carcinoma (NPC) is an epithelial tumour with a distinctive racial and geographical distribution. High incidence of NPC has been reported from China, Southeast Asia, and northeast (NE) region of India. The immune mechanism plays incredibly role in pathogenesis of NPC. Tumour necrosis factors (TNFs) and heat shock protein 70 (HSP 70) constitute significant components of innate as well as adaptive host immunity. Multi-analytical approaches including logistic regression (LR), classification and regression tree (CART) and multifactor dimensionality reduction (MDR) were applied in 120 NPC cases and 100 controls to explore high order interactions among TNF-α (-308 G>A), TNF β (+252 A>G), HSP 70-1 (+190 G>C), HSP 70-hom (+2437 T>C) genes and environmental risk factors. TNF β was identified as the primary etiological factor by all three analytical approaches. Individual analysis of results showed protective effect of TNF β GG genotype (adjusted odds ratio (OR2) = 0.27, 95 % CI = 0.125-0.611, P = 0.001), HSP 70 (+2437) CC genotype (OR2 = 0.17, 95 % CI = 0.0430.69, P = 0.013), while AG genotype of TNF β was found significantly associated with risk of NPC (OR2 = 1.97, 95 % CI = 1.019-3.83, P = 0.04). Analysis of environmental factors demonstrated association of alcohol consumption, living in mud houses and use of firewood for cooking as major risk factors for NPC. Individual haplotype association analysis showed significant risk associated with GTGA haplotype (OR = 68.61, 95 % CI = 2.47-190.37, P = 0.013) while a protective effect with CCAA and GCGA haplotypes (OR = 0.19, 95 % CI = 0.05-0.75, P = 0.019; OR = 0.01 95 % CI = 0.05-0.30, P = 0.007). The multi-analytical approaches applied in this study helped in identification of distinct gene-gene and gene-environment interactions significant in risk assessment of NPC.

Reduced Hsp70 and Glutamine in Pediatric Severe Malaria Anemia

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Kempaiah, Prakasha; Dokladny, Karol; Karim, Zachary

2016-01-01

by decreased HSPA1A, a heat shock protein (Hsp) 70 coding gene. Hsp70 is a ubiquitous chaperone that regulates Nuclear Factor-kappa B (NF-κB) signaling and production of pro-inflammatory cytokines known to be important in malaria pathogenesis (e.g., IL-1β, IL-6 and TNF-α). Since the role of host Hsp70...... in malaria pathogenesis is unexplored, we investigated Hsp70 and molecular pathways in children with SMA. Validation experiments revealed that leukocytic HSP70 transcripts were reduced in SMA relative to non-severe malaria, and that intraleukocytic hemozoin (PfHz) was associated with lower HSP70. HSP70...... was correlated with reticulocyte production and Hb. Since glutamine (Gln) up-regulates Hsp70, modulates NF-κB activation, and attenuates over-expression of pro-inflammatory cytokines, circulating Gln was measured in children with malaria. Reduced Gln was associated with increased risk of developing SMA...

Hsp40s specify functions of Hsp104 and Hsp90 protein chaperone machines.

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Michael Reidy

2014-10-01

Full Text Available Hsp100 family chaperones of microorganisms and plants cooperate with the Hsp70/Hsp40/NEF system to resolubilize and reactivate stress-denatured proteins. In yeast this machinery also promotes propagation of prions by fragmenting prion polymers. We previously showed the bacterial Hsp100 machinery cooperates with the yeast Hsp40 Ydj1 to support yeast thermotolerance and with the yeast Hsp40 Sis1 to propagate [PSI+] prions. Here we find these Hsp40s similarly directed specific activities of the yeast Hsp104-based machinery. By assessing the ability of Ydj1-Sis1 hybrid proteins to complement Ydj1 and Sis1 functions we show their C-terminal substrate-binding domains determined distinctions in these and other cellular functions of Ydj1 and Sis1. We find propagation of [URE3] prions was acutely sensitive to alterations in Sis1 activity, while that of [PIN+] prions was less sensitive than [URE3], but more sensitive than [PSI+]. These findings support the ideas that overexpressing Ydj1 cures [URE3] by competing with Sis1 for interaction with the Hsp104-based disaggregation machine, and that different prions rely differently on activity of this machinery, which can explain the various ways they respond to alterations in chaperone function.

Heterologous Expression of the Carrot Hsp17.7 gene Increased Growth, Cell Viability, and Protein Solubility in Transformed Yeast (Saccharomyces cerevisiae) under Heat, Cold, Acid, and Osmotic Stress Conditions.

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Ko, Eunhye; Kim, Minhye; Park, Yunho; Ahn, Yeh-Jin

2017-08-01

In industrial fermentation of yeast (Saccharomyces cerevisiae), culture conditions are often modified from the optimal growth conditions of the cells to maintain large-scale cultures and/or to increase recombinant protein production. However, altered growth conditions can be stressful to yeast cells resulting in reduced cell growth and viability. In this study, a small heat shock protein gene from carrot (Daucus carota L.), Hsp17.7, was inserted into the yeast genome via homologous recombination to increase tolerance to stress conditions that can occur during industrial culture. A DNA construct, Translational elongation factor gene promoter-carrot Hsp17.7 gene-Phosphoribosyl-anthranilate isomerase gene (an auxotrophic marker), was generated by a series of PCRs and introduced into the chromosome IV of the yeast genome. Immunoblot analysis showed that carrot Hsp17.7 accumulated in the transformed yeast cell lines. Growth rates and cell viability of these cell lines were higher than control cell lines under heat, cold, acid, and hyperosmotic stress conditions. Soluble protein levels were higher in the transgenic cell lines than control cell lines under heat and cold conditions, suggesting the molecular chaperone function of the recombinant Hsp17.7. This study showed that a recombinant DNA construct containing a HSP gene from carrot was successfully expressed in yeast by homologous recombination and increased tolerances to abiotic stress conditions.

The genes coding for the hsp70(dnaK) molecular chaperone machine occur in the moderate thermophilic archaeon Methanosarcina thermophila TM-1

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Hofman-Bang, H Jacob Peider; Lange, Marianne; Ahring, Birgitte Kiær

1999-01-01

The hsp70 (dnaK) locus of the moderate thermophilic archaeon Methanosarcina thermophila TM-1 was cloned, sequenced, and tested in vitro to measure gene induction by heat and ammonia, i.e., stressors pertinent to the biotechnological ecosystem of this methanogen that plays a key role in anaerobic...... thermoautotrophicum Delta H, from another genus, in which trkA is not part of the locus. The proteins encoded in the TM-1 genes are very similar to the S-6 homologs, but considerably less similar to the Delta H proteins. The TM-1 Hsp70(DnaK) protein has the 23-amino acid deletion-by comparison with homologs from Gram...

Genome-wide identification of heat shock proteins (Hsps) and Hsp interactors in rice: Hsp70s as a case study.

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Wang, Yongfei; Lin, Shoukai; Song, Qi; Li, Kuan; Tao, Huan; Huang, Jian; Chen, Xinhai; Que, Shufu; He, Huaqin

2014-05-07

Heat shock proteins (Hsps) perform a fundamental role in protecting plants against abiotic stresses. Although researchers have made great efforts on the functional analysis of individual family members, Hsps have not been fully characterized in rice (Oryza sativa L.) and little is known about their interactors. In this study, we combined orthology-based approach with expression association data to screen rice Hsps for the expression patterns of which strongly correlated with that of heat responsive probe-sets. Twenty-seven Hsp candidates were identified, including 12 small Hsps, six Hsp70s, three Hsp60s, three Hsp90s, and three clpB/Hsp100s. Then, using a combination of interolog and expression profile-based methods, we inferred 430 interactors of Hsp70s in rice, and validated the interactions by co-localization and function-based methods. Subsequent analysis showed 13 interacting domains and 28 target motifs were over-represented in Hsp70s interactors. Twenty-four GO terms of biological processes and five GO terms of molecular functions were enriched in the positive interactors, whose expression levels were positively associated with Hsp70s. Hsp70s interaction network implied that Hsp70s were involved in macromolecular translocation, carbohydrate metabolism, innate immunity, photosystem II repair and regulation of kinase activities. Twenty-seven Hsps in rice were identified and 430 interactors of Hsp70s were inferred and validated, then the interacting network of Hsp70s was induced and the function of Hsp70s was analyzed. Furthermore, two databases named Rice Heat Shock Proteins (RiceHsps) and Rice Gene Expression Profile (RGEP), and one online tool named Protein-Protein Interaction Predictor (PPIP), were constructed and could be accessed at http://bioinformatics.fafu.edu.cn/.

Hsp90: Friends, clients and natural foes.

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Verma, Sharad; Goyal, Sukriti; Jamal, Salma; Singh, Aditi; Grover, Abhinav

2016-08-01

Hsp90, a homodimeric ATPase, is responsible for the correct folding of a number of newly synthesized polypeptides in addition to the correct folding of denatured/misfolded client proteins. It requires several co-chaperones and other partner proteins for chaperone activity. Due to the involvement of Hsp90-dependent client proteins in a variety of oncogenic signaling pathways, Hsp90 inhibition has emerged as one of the leading strategies for anticancer chemotherapeutics. Most of Hsp90 inhibitors blocks the N terminal ATP binding pocket and prevents the conformational changes which are essential for the loading of co-chaperones and client proteins. Several other inhibitors have also been reported which disrupt chaperone cycle in ways other than binding to N terminal ATP binding pocket. The Hsp90 inhibition is associated with heat shock response, mediated by HSF-1, to overcome the loss of Hsp90 and sustain cell survival. This review is an attempt to give an over view of all the important players of chaperone cycle. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Genome-wide analysis of rice ClpB/HSP100, ClpC and ClpD genes

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Mittal Dheeraj

2010-02-01

Full Text Available Abstract Background ClpB-cyt/HSP100 protein acts as chaperone, mediating disaggregation of denatured proteins. Previous studies have shown that ClpB-cyt/HSP100 gene belongs to the group class I Clp ATPase proteins and ClpB-cyt/HSP100 transcript is regulated by heat stress and developmental cues. Results Nine ORFs were noted to constitute rice class I Clp ATPases in the following manner: 3 ClpB proteins (ClpB-cyt, Os05g44340; ClpB-m, Os02g08490; ClpB-c, Os03g31300, 4 ClpC proteins (ClpC1, Os04g32560; ClpC2, Os12g12580; ClpC3, Os11g16590; ClpC4, Os11g16770 and 2 ClpD proteins (ClpD1, Os02g32520; ClpD2, Os04g33210. Using the respective signal sequences cloned upstream to GFP/CFP reporter proteins and transient expression studies with onion epidermal cells, evidence is provided that rice ClpB-m and Clp-c proteins are indeed localized to their respective cell locations mitochondria and chloroplasts, respectively. Associated with their diverse cell locations, domain structures of OsClpB-c, OsClpB-m and OsClpB-cyt proteins are noted to possess a high-level conservation. OsClpB-cyt transcript is shown to be enriched at milk and dough stages of seed development. While expression of OsClpB-m was significantly less as compared to its cytoplasmic and chloroplastic counterparts in different tissues, this transcript showed highest heat-induced expression amongst the 3 ClpB proteins. OsClpC1 and OsClpC2 are predicted to be chloroplast-localized as is the case with all known plant ClpC proteins. However, the fact that OsClpC3 protein appears mitochondrial/chloroplastic with equal probability and OsClpC4 a plasma membrane protein reflects functional diversity of this class. Different class I Clp ATPase transcripts were noted to be cross-induced by a host of different abiotic stress conditions. Complementation assays of Δhsp104 mutant yeast cells showed that OsClpB-cyt, OsClpB-m, OsClpC1 and OsClpD1 have significantly positive effects. Remarkably, OsClpD1 gene

Down-regulation of HSP40 gene family following OCT4B1 suppression in human tumor cell lines

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Mohammad Reza Mirzaei

2016-02-01

Full Text Available Objective(s: The OCT4B1, as one of OCT4 variants, is expressed in cancer cell lines and tissues more than other variants and plays an important role in apoptosis and stress (heat shock protein pathways. The present study was designed to determine the effects of OCT4B1 silencing on expressional profile of HSP40 gene family expression in three different human tumor cell lines. Materials and Methods: The OCT4B1 expression was suppressed by specific siRNA transfection in AGS (gastric adenocarcinoma, 5637 (bladder tumor and U-87MG (brain tumor cell lines employing Lipofectamine reagent. Real-time PCR array technique was employed for RNA qualification. The fold changes were calculated using RT2 Profiler PCR array data analysis software version 3.5. Results: Our results indicated that fifteen genes (from 36 studied genes were down-regulated and two genes (DNAJC11 and DNAJC5B were up-regulated in all three studied tumor cell lines by approximately more than two folds. The result of other studied genes (19 genes showed different expressional pattern (up or down-expression based on tumor cell lines. Conclusion: According to the findings of the present study, we may suggest that there is a direct correlation between OCT4B1 expression in tumor cell lines (and tissues and HSP40 family gene expressions to escape from apoptosis and cancer expansion.

Organization and evolution of hsp70 clusters strikingly differ in two species of Stratiomyidae (Diptera inhabiting thermally contrasting environments

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Bettencourt Brian R

2011-03-01

Full Text Available Abstract Background Previously, we described the heat shock response in dipteran species belonging to the family Stratiomyidae that develop in thermally and chemically contrasting habitats including highly aggressive ones. Although all species studied exhibit high constitutive levels of Hsp70 accompanied by exceptionally high thermotolerance, we also detected characteristic interspecies differences in heat shock protein (Hsp expression and survival after severe heat shock. Here, we analyzed genomic libraries from two Stratiomyidae species from thermally and chemically contrasting habitats and determined the structure and organization of their hsp70 clusters. Results Although the genomes of both species contain similar numbers of hsp70 genes, the spatial distribution of hsp70 copies differs characteristically. In a population of the eurytopic species Stratiomys singularior, which exists in thermally variable and chemically aggressive (hypersaline conditions, the hsp70 copies form a tight cluster with approximately equal intergenic distances. In contrast, in a population of the stenotopic Oxycera pardalina that dwells in a stable cold spring, we did not find hsp70 copies in tandem orientation. In this species, the distance between individual hsp70 copies in the genome is very large, if they are linked at all. In O. pardalina we detected the hsp68 gene located next to a hsp70 copy in tandem orientation. Although the hsp70 coding sequences of S. singularior are highly homogenized via conversion, the structure and general arrangement of the hsp70 clusters are highly polymorphic, including gross aberrations, various deletions in intergenic regions, and insertion of incomplete Mariner transposons in close vicinity to the 3'-UTRs. Conclusions The hsp70 gene families in S. singularior and O. pardalina evolved quite differently from one another. We demonstrated clear evidence of homogenizing gene conversion in the S. singularior hsp70 genes, which form

Genetics of human longevity with emphasis on the relevance of HSP70 as candidate genes

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Singh, Ripudaman; Kølvrå, Steen; Rattan, Suresh I S

2007-01-01

Human longevity is determined to a certain extent by genetic factors. Several candidate genes have been studied for their association with human longevity, but the data collected so far are inconclusive. One of the reasons is the choice of the candidate genes in addition to the choice...... of an appropriate study design and methodology. Since aging is characterized by a progressive accumulation of molecular damage and an attenuation of the cellular defense mechanisms, the focus of studies on human longevity association with genes has now shifted to the pathways of cellular maintenance and repair...... mechanisms. One such pathway includes the battery of stress response genes, especially the heat shock protein HSP70 genes. Three such genes, HSPA1A, HSPA1B and HSPA1L, are present within the MHC-III region on the short arm of chromosome 6. We and others have found alleles, genotypes and haplotypes which have...

Heat shock protein Hsp90-2 expression in the Arabidopsis thaliana seedlings under clinorotation

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Kozeko, Liudmyla

Heat shock proteins 90 kDa (Hsp90) are abundant under normal conditions and induced by stress. This family is distinguished from other chaperones in that most of its substrates are signal transduction proteins. Previously, we determined some time-dependent increase in the Hsp90 level in pea seedlings in response to simulated microgravity that indicated a stress-reaction. However, expression of the individual members of the Hsp90 family have specific pattern. The purpose of this study was to investigate possible alterations in the gene expression pattern of cytosolic Hsp90-2 in Arabidopsis thaliana seedlings under 2D-clinorotation. To obtain detailed expression pattern of the HSP90-2 genes we used seeds that provides a resource of loss-of-function mutations gene expression patterns via translational fusions with the reporter gene, GUS (a line N 166718, NASC). There were two variants of the experiment: 1) seedlings grew under clinorotation for 10, 12, 14 d; 2) seedlings grew in the stationary conditions for 10 d followed by clinorotation for 3 h -at 22o C and 16h light cycle. The seedlings grown in the stationary conditions were used as a control. GUS staining showed that HSP90-2 expression was regulated during seedling development and affected by clinorotation in the heterozygous mutant plants. In the homozygous for the mutation plants, HSP90-2 expression was stable during seedling development and not affected by clinorotation. GUS staining was observed in cotyledons, leaves and hypocotyls of the seedlings (especially intense in vascular bundles), indicating intensive cellular processes with participation of this chaperone. Possible pathways of influence of clinorotation on HSP90-2 expression are discussed.

Reduced heat shock response in human mononuclear cells during aging and its association with polymorphisms in HSP70 genes

DEFF Research Database (Denmark)

Singh, Ripudaman; Kølvraa, Steen; Bross, Peter

2006-01-01

Age-dependent changes in heat shock response (HSR) were studied in mononuclear cells (monocytes and lymphocytes) collected from young (mean age = 22.6 +/- 1.7 years) and middle-aged (mean age = 56.3 +/- 4.7 years) subjects after 1 hour of heat shock at 42 degrees C. Genotype-specific HSR...... was measured by genotyping the subjects for 3 single nucleotide polymorphisms, HSPA1A(A-110C), HSPA1B(A1267G), and HSPA1L(T2437C), 1 each in the 3 HSP70 genes. A significant age-related decrease in the induction of Hsp70 occurred after heat shock in both monocytes and lymphocytes. The noninducible...

Phenotypic Plasticity of HSP70s Gene Expression during Diapause: Signs of Evolutionary Responses to Cold Stress among Soybean Pod Borer Populations (Leguminivora glycinivorella) in Northeast of China

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Han, Lanlan; Fan, Dong; Zhao, Kuijun

2014-01-01

The soybean pod borer (Leguminivora glycinivorella Matsumura) successfully survives the winter because of its high expression of 70-kDa heat shock proteins (HSP70s) during its overwintering diapause. The amount of HSP70s is different under different environmental stresses. In this study, inducible heat shock protein 70 and its constitutive heat shock cognate 70 were cloned by RT-PCR and RACE. These genes were named Lg-hsp70 and Lg-hsc70, respectively. Gene transcription and protein expression after cold stress treatment (5°C to −5°C) were analyzed by western blotting and by qRT-PCR for four populations that were sampled in the northeast region of China, including Shenyang, Gongzhuling, Harbin and Heihe, when the soybean pod borer was in diapause. As the cold shock temperature decreased, the levels of Lg-HSP70s were significantly up-regulated. The amount of cold-induced Lg-HSP70s was highest in the southernmost population (Shenyang, 41°50′N) and lowest in the northernmost population (Heihe, 50°22′N). These results support the hypothesis that the soybean pod borer in the northeast region of China displays phenotypic plasticity, and the accumulation of Lg-HSP70s is a strategy for overcoming environmental stress. These results also suggest that the induction of HSP70 synthesis, which is a complex physiological adaptation, can evolve quickly and inherit stability. PMID:25330365

Characterization of CaHsp70-1, a Pepper Heat-Shock Protein Gene in Response to Heat Stress and Some Regulation Exogenous Substances in Capsicum annuum L.

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Guo, Meng; Zhai, Yu-Fei; Lu, Jin-Ping; Chai, Lin; Chai, Wei-Guo; Gong, Zhen-Hui; Lu, Ming-Hui

2014-01-01

Pepper (Capsicum annuum L.) is sensitive to heat stress (HS). Heat shock proteins 70 (Hsp70s) play a crucial role in protecting plant cells against HS and control varies characters in different plants. However, CaHsp70-1 gene was not well characterized in pepper. In this study, CaHsp70-1 was cloned from the pepper thermotolerant line R9, which encoded a protein of 652 amino acids, with a molecular weight of 71.54 kDa and an isoelectric point of 5.20. CaHsp70-1 belongs to the cytosolic Hsp70 subgroup, and best matched with tomato SlHsp70. CaHsp70-1 was highly induced in root, stem, leaf and flower in R9 with HS treatment (40 °C for 2 h). In both thermosensitive line B6 and thermotolerant line R9, CaHsp70-1 significantly increased after 0.5 h of HS (40 °C), and maintained in a higher level after 4 h HS. The expression of CaHsp70-1 induced by CaCl2, H2O2 and putrescine (Put) under HS were difference between B6 and R9 lines. The different expression patterns may be related to the differences in promoters of CaHsp70-1 from the two lines. These results suggest that CaHsp70-1 as a member of cytosolic Hsp70 subgroup, may be involved in HS defense response via a signal transduction pathway contained Ca2+, H2O2 and Put. PMID:25356507

Molecular karyotype and chromosomal localization of genes encoding ß-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli

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CB Toaldo

2001-01-01

Full Text Available The molecular karyotype of nine Trypanosoma rangeli strains was analyzed by contour-clamped homogeneous electric field electrophoresis, followed by the chromosomal localization of ß-tubulin, cysteine proteinase, 70 kDa heat shock protein (hsp 70 and actin genes. The T. rangeli strains were isolated from either insects or mammals from El Salvador, Honduras, Venezuela, Colombia, Panama and southern Brazil. Also, T. cruzi CL-Brener clone was included for comparison. Despite the great similarity observed among strains from Brazil, the molecular karyotype of all T. rangeli strains analyzed revealed extensive chromosome polymorphism. In addition, it was possible to distinguish T. rangeli from T. cruzi by the chromosomal DNA electrophoresis pattern. The localization of ß-tubulin genes revealed differences among T. rangeli strains and confirmed the similarity between the isolates from Brazil. Hybridization assays using probes directed to the cysteine proteinase, hsp 70 and actin genes discriminated T. rangeli from T. cruzi, proving that these genes are useful molecular markers for the differential diagnosis between these two species. Numerical analysis based on the molecular karyotype data revealed a high degree of polymorphism among T. rangeli strains isolated from southern Brazil and strains isolated from Central and the northern South America. The T. cruzi reference strain was not clustered with any T. rangeli strain.

Hsp90 Is Essential under Heat Stress in the Bacterium Shewanella oneidensis

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Flora Ambre Honoré

2017-04-01

Full Text Available The Hsp90 chaperone is essential in eukaryotes and activates a large array of client proteins. In contrast, its role is still elusive in bacteria, and only a few Hsp90 bacterial clients are known. Here, we found that Hsp90 is essential in the model bacterium Shewanella oneidensis under heat stress. A genetic screen for Hsp90 client proteins identified TilS, an essential protein involved in tRNA maturation. Overexpression of TilS rescued the growth defect of the hsp90 deletion strain under heat stress. In vivo, the activity and the amount of TilS were significantly reduced in the absence of Hsp90 at high temperature. Furthermore, we showed that Hsp90 interacts with TilS, and Hsp90 prevents TilS aggregation in vitro at high temperature. Together, our results indicate that TilS is a client of Hsp90 in S. oneidensis. Therefore, our study links the essentiality of bacterial Hsp90 at high temperature with the identification of a client.

Crosstalk between p38, Hsp25 and Akt in spinal motor neurons after sciatic nerve injury

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Murashov, A. K.; Ul Haq, I.; Hill, C.; Park, E.; Smith, M.; Wang, X.; Wang, X.; Goldberg, D. J.; Wolgemuth, D. J.

2001-01-01

The p38 stress-activated protein kinase pathway is involved in regulation of phosphorylation of Hsp25, which in turn regulates actin filament dynamic in non-neuronal cells. We report that p38, Hsp25 and Akt signaling pathways were specifically activated in spinal motor neurons after sciatic nerve axotomy. The activation of the p38 kinase was required for induction of Hsp25 expression. Furthermore, Hsp25 formed a complex with Akt, a member of PI-3 kinase pathway that prevents neuronal cell death. Together, our observations implicate Hsp25 as a central player in a complex system of signaling that may both promote regeneration of nerve fibers and prevent neuronal cell death in the injured spinal cord.

Signaling pathways involved in HSP32 induction by hyperbaric oxygen in rat spinal neurons

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Guoyang Huang

2016-12-01

Full Text Available Spinal cord injury (SCI is a debilitating disease, effective prevention measures are in desperate need. Our previous work found that hyperbaric oxygen (HBO preconditioning significantly protected rats from SCI after stimulated diving, and in vitro study further testified that HBO protected primary cultured rat spinal neurons from oxidative insult and oxygen glucose deprivation injury via heat shock protein (HSP 32 induction. In this study, underlying molecular mechanisms were further investigated. The results showed that a single exposure to HBO significantly increased intracellular levels of reactive oxygen species (ROS and nitric oxide (NO and activated MEK1/2, ERK1/2, p38 MAPK, CREB, Bach1 and Nrf2. The induction of HSP32 by HBO was significantly reversed by pretreatment neurons with ROS scavenger N-Acetyl-L-cysteine, p38 MAPK inhibitor or Nrf2 gene knockdown, enhanced by MEK1/2 inhibitors or gene knockdown but not by ERK1/2 inhibitor. CREB knockdown did not change the expression of HSP32 induced by HBO. N-Acetyl-L-cysteine significantly inhibited the activation of MEK1/2, ERK1/2, p38 MAPK, and Nrf2. Activation of Nrf2 was significantly inhibited by p38 MAPK inhibitor and the nuclear export of Bach1 was significantly enhanced by MEK1/2 inhibitor. The results demonstrated that HBO induces HSP32 expression through a ROS/p38 MAPK/Nrf2 pathway and the MEK1/2/Bach1 pathway contributes to negative regulation in the process. More importantly, as we know, this is the first study to delineate that ERK1/2 is not the only physiological substrates of MEK1/2.

RNAi knockdown of Hop (Hsp70/Hsp90 organising protein) decreases invasion via MMP-2 down regulation.

LENUS (Irish Health Repository)

Walsh, Naomi

2011-07-28

We previously identified Hop as over expressed in invasive pancreatic cancer cell lines and malignant tissues of pancreatic cancer patients, suggesting an important role for Hop in the biology of invasive pancreatic cancer. Hop is a co-chaperone protein that binds to both Hsp70\\/Hsp90. We hypothesised that by targeting Hop, signalling pathways modulating invasion and client protein stabilisation involving Hsp90-dependent complexes may be altered. In this study, we show that Hop knockdown by small interfering (si)RNA reduces the invasion of pancreatic cancer cells, resulting in decreased expression of the downstream target gene, matrix metalloproteinases-2 (MMP-2). Hop in conditioned media co-immunoprecipitates with MMP-2, implicating a possible extracellular function for Hop. Knockdown of Hop expression also reduced expression levels of Hsp90 client proteins, HER2, Bcr-Abl, c-MET and v-Src. Furthermore, Hop is strongly expressed in high grade PanINs compared to lower PanIN grades, displaying differential localisation in invasive ductal pancreatic cancer, indicating that the localisation of Hop is an important factor in pancreatic tumours. Our data suggests that the attenuation of Hop expression inactivates key signal transduction proteins which may decrease the invasiveness of pancreatic cancer cells possibly through the modulation of Hsp90 activity. Therefore, targeting Hop in pancreatic cancer may constitute a viable strategy for targeted cancer therapy.

Association of HSP70 and its co-chaperones with Alzheimer's disease

NARCIS (Netherlands)

L. Broer (Linda); M.A. Ikram (Arfan); M. Schuur (Maaike); A.L. DeStefano (Anita); J.C. Bis (Joshua); F. Liu (Fan); F. Rivadeneira Ramirez (Fernando); A.G. Uitterlinden (André); A. Beiser (Alexa); W.T. Longstreth Jr; A. Hofman (Albert); Y.S. Aulchenko (Yurii); S. Seshadri (Sudha); A.L. Fitzpatrick (Annette); B.A. Oostra (Ben); M.M.B. Breteler (Monique); P. Tikka-Kleemola (Päivi)

2011-01-01

textabstractThe heat shock protein (HSP) 70 family has been implicated in the pathology of Alzheimer's disease (AD). In this study, we examined common genetic variations in the 80 genes encoding HSP70 and its co-chaperones. We conducted a study in a series of 462 patients and 5238 unaffected

Sequential folding of UmuC by the Hsp70 and Hsp60 chaperone complexes of Escherichia coli.

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Petit, M A; Bedale, W; Osipiuk, J; Lu, C; Rajagopalan, M; McInerney, P; Goodman, M F; Echols, H

1994-09-23

Replication-blocking lesions generate a signal in Escherichia coli that leads to the induction of the multigene SOS response. Among the SOS-induced genes are umuD and umuC, whose products are necessary for the increased mutation rate in induced bacteria. The mutations are likely to result from replication across the DNA lesion, and such a bypass event has been reconstituted in vitro (Rajagopalan, M., L, C., Woodgate, R., O'Donnel, M., Goodman, M. F., Echols, H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 10777-10781). In this work, we show that the chaperone proteins promote the proper folding of UmuC protein in vitro. We treated purified and inactive UmuC with Hsp70 and Hsp60. After Hsp70 treatment, the DNA binding activity of UmuC was recovered, but the ability to promote replication across DNA lesions was not. However, lesion bypass activity was recovered upon further treatment with Hsp60. The biological significance of such a folding pathway for UmuC protein is strengthened by in vivo evidence for a role of DnaK in UV-induced mutagenesis.

Silencing of Hsp27 and Hsp72 in glioma cells as a tool for programmed cell death induction upon temozolomide and quercetin treatment

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Jakubowicz-Gil, Joanna, E-mail: jjgil@poczta.umcs.lublin.pl [Department of Comparative Anatomy and Anthropology, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin (Poland); Langner, Ewa [Department of Medical Biology, Institute of Agricultural Medicine, Jaczewskiego 2, 20-950 Lublin (Poland); Bądziul, Dorota [Department of Comparative Anatomy and Anthropology, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin (Poland); Wertel, Iwona [1st Department of Gynaecology, University School of Medicine, Staszica 16, 20-081 Lublin (Poland); Rzeski, Wojciech [Department of Medical Biology, Institute of Agricultural Medicine, Jaczewskiego 2, 20-950 Lublin (Poland); Department of Immunology and Virology, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin (Poland)

2013-12-15

The aim of the present study was to investigate whether silencing of Hsp27 or Hsp72 expression in glioblastoma multiforme T98G and anaplastic astrocytoma MOGGCCM cells increases their sensitivity to programmed cell death induction upon temozolomide and/or quercetin treatment. Transfection with specific siRNA was performed for the Hsp gene silencing. As revealed by microscopic observation and flow cytometry, the inhibition of Hsp expression was correlated with severe apoptosis induction upon the drug treatment studied. No signs of autophagy were detected. This was correlated with a decreased mitochondrial membrane potential, increased level of cytochrome c in the cytoplasm, and activation of caspase 3 and caspase 9. All these results suggest that the apoptotic signal was mediated by an internal pathway. Additionally, in a large percentage of cells treated with temozolomide, with or without quercetin, granules within the ER system were found, which was accompanied by an increased level of caspase 12 expression. This might be correlated with ER stress. Quercetin and temozolomide also changed the shape of nuclei from circular to “croissant like” in both transfected cell lines. Our results indicate that blocking of Hsp27 and Hsp72 expression makes T98G cells and MOGGCCM cells extremely vulnerable to apoptosis induction upon temozolomide and quercetin treatment and that programmed cell death is initiated by an internal signal. - Highlights: • Hsps gene silencing induced severe apoptosis upon temozolomide–quercetin treatment • Apoptosis in transfected glioma cells was initiated by internal signal • Programmed cell death was preceded by ER stress • Temozolomide–quercetin treatment changed nuclei shape in transfected glioma cells.

A Novel Therapeutic Strategy for the Treatment of Glioma, Combining Chemical and Molecular Targeting of Hsp90α

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Mehta, Adi; Shervington, Leroy; Munje, Chinmay; Shervington, Amal

2011-01-01

Hsp90α's vital role in tumour survival and progression, together with its highly inducible expression profile in gliomas and its absence in normal tissue and cell lines validates it as a therapeutic target for glioma. Hsp90α was downregulated using the post-transcriptional RNAi strategy (sihsp90α) and a post-translational inhibitor, the benzoquinone antibiotic 17-AAG. Glioblastoma U87-MG and normal human astrocyte SVGp12 were treated with sihsp90α, 17-AAG and concurrent sihsp90α/17-AAG (combined treatment). Both Hsp90α gene silencing and the protein inhibitor approaches resulted in a dramatic reduction in cell viability. Results showed that sihsp90α, 17-AAG and a combination of sihsp90α/17-AAG, reduced cell viability by 27%, 75% and 88% (p < 0.001), respectively, after 72 h. hsp90α mRNA copy numbers were downregulated by 65%, 90% and 99% after 72 h treatment with sihsp90α, 17-AAG and sihsp90α/17-AAG, respectively. The relationship between Hsp90α protein expression and its client Akt kinase activity levels were monitored following treatment with sihsp90α, 17-AAG and sihsp90α/17-AAG. Akt kinase activity was downregulated as a direct consequence of Hsp90α inhibition. Both Hsp90α and Akt kinase levels were significantly downregulated after 72 h. Although, 17-AAG when used as a single agent reduces the Hsp90α protein and the Akt kinase levels, the efficacy demonstrated by combinatorial treatment was found to be far more effective. Combination treatment reduced the Hsp90α protein and Akt kinase levels to 4.3% and 43%, respectively, after 72 h. hsp90α mRNA expression detected in SVGp12 was negligible compared to U87-MG, also, the combination treatment did not compromise the normal cell viability. Taking into account the role of Hsp90α in tumour progression and the involvement of Akt kinase in cell signalling and the anti-apoptotic pathways in tumours, this double targets treatment infers a novel therapeutic strategy

Hsp90-downregulation influences the heat-shock response, innate immune response and onset of oocyte development in nematodes.

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Julia Eckl

Full Text Available Hsp90 is a molecular chaperone involved in the regulation and maturation of kinases and transcription factors. In Caenorhabditis elegans, it contributes to the development of fertility, maintenance of muscle structure, the regulation of heat-shock response and dauer state. To understand the consequences of Hsp90-depletion, we studied Hsp90 RNAi-treated nematodes by DNA microarrays and mass spectrometry. We find that upon development of phenotypes the levels of chaperones and Hsp90 cofactors are increased, while specific proteins related to the innate immune response are depleted. In microarrays, we further find many differentially expressed genes related to gonad and larval development. These genes form an expression cluster that is regulated independently from the immune response implying separate pathways of Hsp90-involvement. Using fluorescent reporter strains for the differentially expressed immune response genes skr-5, dod-24 and clec-60 we observe that their activity in intestinal tissues is influenced by Hsp90-depletion. Instead, effects on the development are evident in both gonad arms. After Hsp90-depletion, changes can be observed in early embryos and adults containing fluorescence-tagged versions of SEPA-1, CAV-1 or PUD-1, all of which are downregulated after Hsp90-depletion. Our observations identify molecular events for Hsp90-RNAi induced phenotypes during development and immune responses, which may help to separately investigate independent Hsp90-influenced processes that are relevant during the nematode's life and development.

Suppressed phenylalanine ammonia-lyase activity after heat shock in transgenic Nicotiana plumbaginifolia containing an Arabidopsis HSP18.2-parsley PAL2 chimera gene.

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Moriwaki, M; Yamakawa, T; Washino, T; Kodama, T; Igarashi, Y

1999-01-01

The activity of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) after heat shock (HS) in leaves and buds of transgenic Nicotiana plumbaginifolia containing an Arabidopsis HSP18.2 promoter-parsley phenylalanine ammonia-lyase 2 (HSP18.2-PAL2) chimera gene was examined. Immediately after HS treatment at 44 degrees C for 5 h, the PAL activity in both transgenic and normal (untransformed) plants was 35-38% lower than that before HS. At normal temperature (25-26 degrees C), the PAL activity recovered within 5 h of ending the HS treatment in normal plants, but not until 12-24 h in transgenic plants containing the HSP18.2-PAL2 gene. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed the presence of parsley PAL2 mRNA in transgenic plants, which remained for 8-12 h following 5-h HS at 44 degrees C; the mRNA was not observed before HS. The content of chlorogenic acid (CGA; 3-caffeoylquinic acid) decreased drastically 8-12 h after HS in transgenic plants, but only slightly in normal plants. Thus, the decrease in PAL activity accompanied by expression of the parsley PAL2 gene after HS treatment corresponded to the decrease in CGA synthesis. These results might be attributed to post-transcriptional degradation of endogenous PAL mRNA triggered by transcription of the transgene.

Whey protein hydrolysate enhances HSP90 but does not alter HSP60 and HSP25 in skeletal muscle of rats.

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Carolina Soares Moura

Full Text Available Whey protein hydrolysate (WPH intake has shown to increase HSP70 expression. The aim of the present study was to investigate whether WPH intake would also influences HSP90, HSP60 and HSP25 expression, as well as associated parameters. Forty-eight male Wistar rats were divided into sedentary (unstressed and exercised (stressed groups, and were fed with three different sources of protein: whey protein (WP, whey protein hydrolysate (WPH and casein (CAS as a control, based on the AIN93G diet for 3 weeks. WPH intake increased HSP90 expression in both sedentary and exercised animals compared to WP or CAS, however no alteration was found from exercise or diet to HSP60 or HSP25. Co-chaperone Aha1 and p-HSF1 were also increased in the exercised animals fed with WPH in comparison with WP or CAS, consistent with enhanced HSP90 expression. VEGF and p-AKT were increased in the WPH exercised group. No alteration was found in BCKDH, PI3-Kinase (p85, GFAT, OGT or PGC for diet or exercise. The antioxidant system GPx, catalase and SOD showed different responses to diet and exercise. The data indicate that WPH intake enhanced factors related to cell survival, such as HSP90 and VEGF, but does not alter HSP60 or HSP25 in rat skeletal muscle.

Subthalamic hGAD65 Gene Therapy and Striatum TH Gene Transfer in a Parkinson’s Disease Rat Model

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Zheng, Deyu; Jiang, Xiaohua; Zhao, Junpeng; Duan, Deyi; Zhao, Huanying; Xu, Qunyuan

2013-01-01

The aim of the present study is to detect a combination method to utilize gene therapy for the treatment of Parkinson’s disease (PD). Here, a PD rat model is used for the in vivo gene therapy of a recombinant adeno-associated virus (AAV2) containing a human glutamic acid decarboxylase 65 (rAAV2-hGAD65) gene delivered to the subthalamic nucleus (STN). This is combined with the ex vivo gene delivery of tyrosine hydroxylase (TH) by fibroblasts injected into the striatum. After the treatment, the rotation behavior was improved with the greatest efficacy in the combination group. The results of immunohistochemistry showed that hGAD65 gene delivery by AAV2 successfully led to phenotypic changes of neurons in STN. And the levels of glutamic acid and GABA in the internal segment of the globus pallidus (GPi) and substantia nigra pars reticulata (SNr) were obviously lower than the control groups. However, hGAD65 gene transfer did not effectively protect surviving dopaminergic neurons in the SNc and VTA. This study suggests that subthalamic hGAD65 gene therapy and combined with TH gene therapy can alleviate symptoms of the PD model rats, independent of the protection the DA neurons from death. PMID:23738148

Targeting Hsp27/eIF4E interaction with phenazine compound: a promising alternative for castration-resistant prostate cancer treatment.

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Hajer, Ziouziou; Claudia, Andrieu; Erik, Laurini; Sara, Karaki; Maurizio, Fermeglia; Ridha, Oueslati; David, Taieb; Michel, Camplo; Olivier, Siri; Sabrina, Pricl; Maria, Katsogiannou; Palma, Rocchi

2017-09-29

The actual strategy to improve current therapies in advanced prostate cancer involves targeting genes activated by androgen withdrawal, either to delay or prevent the emergence of the castration-refractory phenotype. However, these genes are often implicated in several physiological processes, and long-term inhibition of survival proteins might be accompanied with cytotoxic effects. To avoid this problem, an alternative therapeutic strategy relies on the identification and use of compounds that disrupt specific protein-protein interactions involved in androgen withdrawal. Specifically, the interaction of the chaperone protein Hsp27 with the initiation factor eIF4E leads to the protection of protein synthesis initiation process and enhances cell survival during cell stress induced by castration or chemotherapy. Thus, in this work we aimed at i) identifying the interaction site of the Hsp27/eIF4E complex and ii) interfere with the relevant protein/protein association mechanism involved in castration-resistant progression of prostate cancer. By a combination of experimental and modeling techniques, we proved that eIF4E interacts with the C-terminal part of Hsp27, preferentially when Hsp27 is phosphorylated. We also observed that the loss of this interaction increased cell chemo-and hormone-sensitivity. In order to find a potential inhibitor of Hsp27/eIF4E interaction, BRET assays in combination with molecular simulations identified the phenazine derivative 14 as the compound able to efficiently interfere with this protein/protein interaction, thereby inhibiting cell viability and increasing cell death in chemo- and castration-resistant prostate cancer models in vitro and in vivo .

Plasmodium falciparum Hsp70-z, an Hsp110 homologue, exhibits independent chaperone activity and interacts with Hsp70-1 in a nucleotide-dependent fashion.

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Zininga, Tawanda; Achilonu, Ikechukwu; Hoppe, Heinrich; Prinsloo, Earl; Dirr, Heini W; Shonhai, Addmore

2016-05-01

The role of molecular chaperones, among them heat shock proteins (Hsps), in the development of malaria parasites has been well documented. Hsp70s are molecular chaperones that facilitate protein folding. Hsp70 proteins are composed of an N-terminal nucleotide binding domain (NBD), which confers them with ATPase activity and a C-terminal substrate binding domain (SBD). In the ADP-bound state, Hsp70 possesses high affinity for substrate and releases the folded substrate when it is bound to ATP. The two domains are connected by a conserved linker segment. Hsp110 proteins possess an extended lid segment, a feature that distinguishes them from canonical Hsp70s. Plasmodium falciparum Hsp70-z (PfHsp70-z) is a member of the Hsp110 family of Hsp70-like proteins. PfHsp70-z is essential for survival of malaria parasites and is thought to play an important role as a molecular chaperone and nucleotide exchange factor of its cytosolic canonical Hsp70 counterpart, PfHsp70-1. Unlike PfHsp70-1 whose functions are fairly well established, the structure-function features of PfHsp70-z remain to be fully elucidated. In the current study, we established that PfHsp70-z possesses independent chaperone activity. In fact, PfHsp70-z appears to be marginally more effective in suppressing protein aggregation than its cytosol-localized partner, PfHsp70-1. Furthermore, based on coimmunoaffinity chromatography and surface plasmon resonance analyses, PfHsp70-z associated with PfHsp70-1 in a nucleotide-dependent fashion. Our findings suggest that besides serving as a molecular chaperone, PfHsp70-z could facilitate the nucleotide exchange function of PfHsp70-1. These dual functions explain why it is essential for parasite survival.

Stress-induced localization of HSPA6 (HSP70B') and HSPA1A (HSP70-1) proteins to centrioles in human neuronal cells.

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Khalouei, Sam; Chow, Ari M; Brown, Ian R

2014-05-01

The localization of yellow fluorescent protein (YFP)-tagged HSP70 proteins was employed to identify stress-sensitive sites in human neurons following temperature elevation. Stable lines of human SH-SY5Y neuronal cells were established that expressed YFP-tagged protein products of the human inducible HSP70 genes HSPA6 (HSP70B') and HSPA1A (HSP70-1). Following a brief period of thermal stress, YFP-tagged HSPA6 and HSPA1A rapidly appeared at centrioles in the cytoplasm of human neuronal cells, with HSPA6 demonstrating a more prolonged signal compared to HSPA1A. Each centriole is composed of a distal end and a proximal end, the latter linking the centriole doublet. The YFP-tagged HSP70 proteins targeted the proximal end of centrioles (identified by γ-tubulin marker) rather than the distal end (centrin marker). Centrioles play key roles in cellular polarity and migration during neuronal differentiation. The proximal end of the centriole, which is involved in centriole stabilization, may be stress-sensitive in post-mitotic, differentiating human neurons.

EFFECT OF STRESS ON THE PERCENT BODY WEIGHT CHANGE AND MRNA EXPRESSION OF IGF-1, SURVIVINE AND HSP-70 GENE IN THE HIERARCHIAL FOLLICLES OF JAPANESE QUAIL

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N Shit

2014-12-01

Full Text Available The present study was carried out to explore the effect of stress on body weight and the mRNA expression of IGF-1, Survivine and HSP-70 gene in the hierarchial follicles of Japanese quail. A total 24 birds (10 weeks were taken and stress was induced by immobilization daily for 2hrs (between 9.00 - 11.00 AM throughout the study (10 days. Four birds were sacrificed on 1, 2, 4, 6, 8 and 10 days of the treatment. Hierarchial follicles (F1, F2 & F3 were aseptically collected to quantify the expression of IGF-1, Survivine and HSP-70 gene using real-time PCR technique. The percent body weight reduction increased and reached highest (21.30% on 10th day. The fold expression of IGF-1 gene was significantly ((P=0.05 down regulated in advance to the time of experiment. However, the fold expression of survivine gene was significantly (P=0.05 up regulated and the intensity was highest (17 fold in F-3 follicle on 4th day of experiment. No significant change in the mRNA expression of HSP-70 gene was evident in this study. From this study it may be concluded that stress brings physio-molecular change through HPA activation, which not only causes tissue regression also modifies the cellular mechanism.

Expression analysis of HSP70 in the testis of Octopus tankahkeei under thermal stress.

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Long, Ling-Li; Han, Ying-Li; Sheng, Zhang; Du, Chen; Wang, You-Fa; Zhu, Jun-Quan

2015-09-01

The gene encoding heat shock protein 70 (HSP70) was identified in Octopus tankahkeei by homologous cloning and rapid amplification of cDNA ends (RACE). The full-length cDNA (2471 bp) consists of a 5'-untranslated region (UTR) (89 bp), a 3'-UTR (426 bp), and an open reading frame (1956 bp) that encodes 651 amino acid residues with a predicted molecular mass of 71.8 kDa and an isoelectric point of 5.34. Based on the amino acid sequence analysis and multiple sequence alignment, this cDNA is a member of cytoplasmic hsp70 subfamily of the hsp70 family and was designated as ot-hsp70. Tissue expression analysis showed that HSP70 expression is highest in the testes when all examined organs were compared. Immunohistochemistry analysis, together with hematoxylin-eosin staining, revealed that the HSP70 protein was expressed in all spermatogenic cells, but not in fibroblasts. In addition, O. tankahkeei were heat challenged by exposure to 32 °C seawater for 2 h, then returned to 13 °C for various recovery time (0-24 h). Relative expression of ot-hsp70 mRNA in the testes was measured at different time points post-challenge by quantitative real-time PCR. A clear time-dependent mRNA expression of ot-hsp70 after thermal stress indicates that the HSP70 gene is inducible. Ultrastructural changes of the heat-stressed testis were observed by transmission electron microscopy. We suggest that HSP70 plays an important role in spermatogenesis and testis protection against thermal stress in O. tankahkeei. Copyright © 2015 Elsevier Inc. All rights reserved.

HIF-1α-induced HSP70 regulates anabolic responses in articular chondrocytes under hypoxic conditions.

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Tsuchida, Shinji; Arai, Yuji; Takahashi, Kenji A; Kishida, Tsunao; Terauchi, Ryu; Honjo, Kuniaki; Nakagawa, Shuji; Inoue, Hiroaki; Ikoma, Kazuya; Ueshima, Keiichiro; Matsuki, Tomohiro; Mazda, Osam; Kubo, Toshikazu

2014-08-01

We assessed whether heat shock protein 70 (HSP70) is involved in hypoxia inducible factor 1 alpha (HIF-1α)-dependent anabolic pathways in articular chondrocytes under hypoxic conditions. Primary rabbit chondrocytes were cultured under normoxia (20% oxygen condition) or hypoxia (1% oxygen condition). Alternatively, cells cultured under normoxia were treated with CoCl2 , which induces HIF-1α, to simulate hypoxia, or transfected with siRNAs targeting HIF-1α (si-HIF-1α) and HSP70 (si-HSP70) under hypoxia. HSP70 expression was enhanced by the increased expression of HIF-1α under hypoxia or simulated hypoxia, but not in the presence of si-HIF-1α. Hypoxia-induced overexpression of ECM genes was significantly suppressed by si-HIF-1α or si-HSP70. Cell viability positively correlated with hypoxia, but transfection with si-HIF-1α or si-HSP70 abrogated the chondroprotective effects of hypoxia. Although LDH release from sodium nitroprusside-treated cells and the proportion of TUNEL positive cells were decreased under hypoxia, transfection with si-HIF-1α or si-HSP70 almost completely blocked these effects. These findings indicated that HIF-1α-induced HSP70 overexpression increased the expression levels of ECM genes and cell viability, and protected chondrocytes from apoptosis. HIF-1α may regulate the anabolic effects of chondrocytes under hypoxic conditions by regulating HSP70 expression. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Sequence analysis of the 3’-untranslated region of HSP70 (type I genes in the genus Leishmania: its usefulness as a molecular marker for species identification

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Requena Jose M

2012-04-01

Full Text Available Abstract Background The Leishmaniases are a group of clinically diverse diseases caused by parasites of the genus Leishmania. To distinguish between species is crucial for correct diagnosis and prognosis as well as for treatment decisions. Recently, sequencing of the HSP70 coding region has been applied in phylogenetic studies and for identifying of Leishmania species with excellent results. Methods In the present study, we analyzed the 3’-untranslated region (UTR of Leishmania HSP70-type I gene from 24 strains representing eleven Leishmania species in the belief that this non-coding region would have a better discriminatory capacity for species typing than coding regions. Results It was observed that there was a remarkable degree of sequence conservation in this region, even between species of the subgenus Leishmania and Viannia. In addition, the presence of many microsatellites was a common feature of the 3´-UTR of HSP70-I genes in the Leishmania genus. Finally, we constructed dendrograms based on global sequence alignments of the analyzed Leishmania species and strains, the results indicated that this particular region of HSP70 genes might be useful for species (or species complex typing, improving for particular species the discrimination capacity of phylogenetic trees based on HSP70 coding sequences. Given the large size variation of the analyzed region between the Leishmania and Viannia subgenera, direct visualization of the PCR amplification product would allow discrimination between subgenera, and a HaeIII-PCR-RFLP analysis might be used for differentiating some species within each subgenera. Conclusions Sequence and phylogenetic analyses indicated that this region, which is readily amplified using a single pair of primers from both Old and New World Leishmania species, might be useful as a molecular marker for species discrimination.

Functional analysis of the Hikeshi-like protein and its interaction with HSP70 in Arabidopsis

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Koizumi, Shinya; Ohama, Naohiko; Mizoi, Junya [Laboratory of Plant Molecular Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Shinozaki, Kazuo [RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, Kanagawa 230-0045 (Japan); Yamaguchi-Shinozaki, Kazuko, E-mail: akys@mail.ecc.u-tokyo.ac.jp [Laboratory of Plant Molecular Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan)

2014-07-18

Highlights: • HKL, a Hikeshi homologous gene is identified in Arabidopsis. • HKL interacts with two HSP70 isoforms and regulates the subcellular localization of HSC70-1. • The two HSP70 translocate into nucleus in response to heat stress. • Overexpression of HKL confers thermotolerance in transgenic plants. - Abstract: Heat shock proteins (HSPs) refold damaged proteins and are an essential component of the heat shock response. Previously, the 70 kDa heat shock protein (HSP70) has been reported to translocate into the nucleus in a heat-dependent manner in many organisms. In humans, the heat-induced translocation of HSP70 requires the nuclear carrier protein Hikeshi. In the Arabidopsis genome, only one gene encodes a protein with high homology to Hikeshi, and we named this homolog Hikeshi-like (HKL) protein. In this study, we show that two Arabidopsis HSP70 isoforms accumulate in the nucleus in response to heat shock and that HKL interacts with these HSP70s. Our histochemical analysis revealed that HKL is predominantly expressed in meristematic tissues, suggesting the potential importance of HKL during cell division in Arabidopsis. In addition, we show that HKL regulates HSP70 localization, and HKL overexpression conferred thermotolerance to transgenic Arabidopsis plants. Our results suggest that HKL plays a positive role in the thermotolerance of Arabidopsis plants and cooperatively interacts with HSP70.

Insulated hsp70B' promoter: stringent heat-inducible activity in replication-deficient, but not replication-competent adenoviruses.

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Rohmer, Stanimira; Mainka, Astrid; Knippertz, Ilka; Hesse, Andrea; Nettelbeck, Dirk M

2008-04-01

Key to the realization of gene therapy is the development of efficient and targeted gene transfer vectors. Therapeutic gene transfer by replication-deficient or more recently by conditionally replication-competent/oncolytic adenoviruses has shown much promise. For specific applications, however, it will be advantageous to provide vectors that allow for external control of gene expression. The efficient cellular heat shock system in combination with available technology for focused and controlled hyperthermia suggests heat-regulated transcription control as a promising tool for this purpose. We investigated the feasibility of a short fragment of the human hsp70B' promoter, with and without upstream insulator elements, for the regulation of transgene expression by replication-deficient or oncolytic adenoviruses. Two novel adenoviral vectors with an insulated hsp70B' promoter were developed and showed stringent heat-inducible gene expression with induction ratios up to 8000-fold. In contrast, regulation of gene expression from the hsp70B' promoter without insulation was suboptimal. In replication-competent/oncolytic adenoviruses regulation of the hsp70B' promoter was lost specifically during late replication in permissive cells and could not be restored by the insulators. We developed novel adenovirus gene transfer vectors that feature improved and stringent regulation of transgene expression from the hsp70B' promoter using promoter insulation. These vectors have potential for gene therapy applications that benefit from external modulation of therapeutic gene expression or for combination therapy with hyperthermia. Furthermore, our study reveals that vector replication can deregulate inserted cellular promoters, an observation which is of relevance for the development of replication-competent/oncolytic gene transfer vectors. (c) 2008 John Wiley & Sons, Ltd.

Expression of HSP70 in cerebral ischemia and neuroprotetive action of hypothermia and ketoprofen Expressão de HSP70 na isquemia cerebral e a ação neuroprotetora da hipotermia e do cetoprofeno

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Daniela Pretti da Cunha Tirapelli

2010-08-01

Full Text Available Heat shock proteins (HSPs are molecular chaperones that bind to other proteins to shepherd them across membranes and direct them to specific locations within a cell. Several injurious stimuli can induce Hsp70 expression, including ischemia. This study aimed to investigate the pattern of expression of protein (immunohistochemistry and gene (real-time PCR Hsp70 in experimental focal cerebral ischemia in rats by occlusion of the middle cerebral artery for 1 hour and the role of neuroprotection with hypothermia (H and ketoprofen (K. The infarct volume was measured using morphometric analysis defined by triphenyl tetrazolium chloride. It was observed increases in the protein (p=0.0001 and gene (p=0.0001 Hsp70 receptor in the ischemic areas that were reduced by H (protein and gene: pProteínas de choque térmico (HSPs são chaperones moleculares que se ligam a outras proteínas para atravessar as membranas e encaminhá-las para locais específicos dentro de uma célula. Vários estímulos nocivos podem induzir a expressão de Hsp70, incluindo isquemia. Este estudo teve como objetivo investigar o padrão de expressão protéica (imunohistoquímica e gênica (PCR em tempo real de Hsp70 na isquemia cerebral focal experimental em ratos pela oclusão da artéria cerebral média durante 1 hora e o papel da neuroproteção com hipotermia (H e cetoprofeno (C. O volume de infarto foi calculado através da análise morfométrica definido por cloreto de trifenil tetrazólio. Foi observado aumento na expressão proteína (p=0,0001 e gênica (p=0,0001 de Hsp70 nas áreas isquêmicas que foram reduzidas pela H (proteína e gene: p<0,05, C (proteína: p<0,001 e H+K (proteína: p<0,01 e gene: p<0,05. O aumento de Hsp70 na área isquêmica sugere que a neuroexcitotoxicidade mediada pela Hsp70 desempenha um papel importante na morte celular e que o efeito neuroprotetor tanto da H quanto do C está diretamente envolvido com a Hsp70.

Design and Construction of a Cloning Vector Containing the hspX Gene of Mycobacterium tuberculosis.

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Yaghoubi, Atieh; Aryan, Ehsan; Zare, Hosna; Alami, Shadi; Teimourpour, Roghayeh; Meshkat, Zahra

2016-10-01

Tuberculosis (TB) is a major cause of death worldwide. Finding an effective vaccine against TB is the best way to control it. Several vaccines against this disease have been developed but none are completely protective. The aim of this study was to design and construct a cloning vector containing the Mycobacterium tuberculosis (M. tuberculosis) heat shock protein X (hspX) . First, an hspX fragment was amplified by PCR and cloned into plasmid pcDNA3.1(+) and recombinant vector was confirmed. A 435 bp hspX fragment was isolated. The fragment was 100% homologous with hspX of M. tuberculosis strain H37Rv in GenBank. In this study, the cloning vector pcDNA3.1(+), containing a 435-bp hspX fragment of M. tuberculosis , was constructed. This could be used as a DNA vaccine to induce immune responses in animal models in future studies.

Increased HSF activation in muscles with a high constitutive Hsp70 expression

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Locke, Marius; Tanguay, Robert M.

1996-01-01

Stress-induced transcriptional regulation of the Hsps is mediated by trimerization and binding of a pre-existing heat shock transcription factor (HSF1) to a specific DNA sequence located in the 5′ region of hsp genes, known as the heat shock element. Hsp70 has been implicated in regulating the activation of the HSF and, according to cell culture models, high steady-state levels of Hsp70 are inversely correlated with HSF activation. To determine if this applies in an intact animal, muscles of ...

Activation of Hsp90 Enzymatic Activity and Conformational Dynamics through Rationally Designed Allosteric Ligands.

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Sattin, Sara; Tao, Jiahui; Vettoretti, Gerolamo; Moroni, Elisabetta; Pennati, Marzia; Lopergolo, Alessia; Morelli, Laura; Bugatti, Antonella; Zuehlke, Abbey; Moses, Mike; Prince, Thomas; Kijima, Toshiki; Beebe, Kristin; Rusnati, Marco; Neckers, Len; Zaffaroni, Nadia; Agard, David A; Bernardi, Anna; Colombo, Giorgio

2015-09-21

Hsp90 is a molecular chaperone of pivotal importance for multiple cell pathways. ATP-regulated internal dynamics are critical for its function and current pharmacological approaches block the chaperone with ATP-competitive inhibitors. Herein, a general approach to perturb Hsp90 through design of new allosteric ligands aimed at modulating its functional dynamics is proposed. Based on the characterization of a first set of 2-phenylbenzofurans showing stimulatory effects on Hsp90 ATPase and conformational dynamics, new ligands were developed that activate Hsp90 by targeting an allosteric site, located 65 Å from the active site. Specifically, analysis of protein responses to first-generation activators was exploited to guide the design of novel derivatives with improved ability to stimulate ATP hydrolysis. The molecules' effects on Hsp90 enzymatic, conformational, co-chaperone and client-binding properties were characterized through biochemical, biophysical and cellular approaches. These designed probes act as allosteric activators of the chaperone and affect the viability of cancer cell lines for which proper functioning of Hsp90 is necessary. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A novel Hsp70 inhibitor prevents cell intoxication with the actin ADP-ribosylating Clostridium perfringens iota toxin

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Ernst, Katharina; Liebscher, Markus; Mathea, Sebastian; Granzhan, Anton; Schmid, Johannes; Popoff, Michel R.; Ihmels, Heiko; Barth, Holger; Schiene-Fischer, Cordelia

2016-01-01

Hsp70 family proteins are folding helper proteins involved in a wide variety of cellular pathways. Members of this family interact with key factors in signal transduction, transcription, cell-cycle control, and stress response. Here, we developed the first Hsp70 low molecular weight inhibitor specifically targeting the peptide binding site of human Hsp70. After demonstrating that the inhibitor modulates the Hsp70 function in the cell, we used the inhibitor to show for the first time that the stress-inducible chaperone Hsp70 functions as molecular component for entry of a bacterial protein toxin into mammalian cells. Pharmacological inhibition of Hsp70 protected cells from intoxication with the binary actin ADP-ribosylating iota toxin from Clostridium perfringens, the prototype of a family of enterotoxins from pathogenic Clostridia and inhibited translocation of its enzyme component across cell membranes into the cytosol. This finding offers a starting point for novel therapeutic strategies against certain bacterial toxins. PMID:26839186

KADAR MDA DAN HSP 70 PADA PLASENTA PENDERITA PREEKLAMPSIA

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Arleni

2008-12-01

Full Text Available The MDA and HSP70 Concentration in Preeclamptic Patient Placenta’s. Objective: Preeclampsia is a disease in pregnancy and characterized by hypertension and proteinuria. Preeclampsia and eclampsia are the most causes of maternal and fetal mortality and morbidity in Indonesia. Placental and systemic oxidative stress caused endothelial cell dysfunction and injury. Placental oxidative stress also linked to fetal growth restriction. HSP70 is essential for cellular recovery, survival and maintenance of homeostasis. The purpose of this study was to compare the MDA, a marker for oxidative stress and HSP70 production in placental of severe preeclampsia, mild preeclampsia and normotensive pregnant women. Placenta were collected after delivery from normotensive pregnancies (N=10, severe preeclampsia (N=10 and mild preeclampsia (N=10. Placenta was cultured in RPMI and 20% FBS, and supernatant were collected in day 3. MDA was measured using spectrophotometer and absorbance read in 530nm. HSP70 was measured using enzyme-linked immunosorbent assay. The mean MDA concentration did not differ significantly between patients with severe preeclampsia (7.13+5.36 nmol/ml and mild preeclampsia (4.82+2.47 nmol/ml when compared with normotensive pregnancies (4.57+2.4 nmol/ml. The mean HSP70 concentration in mild preeclampsia is highest (10.15+12.39 nmo/ml when compared with severe preeclampsia (3.78 +3.07 nmol/ml and normotensive pregnant women (3.76+4.65nmol/ml, but the difference was not significant. Although the difference was not significant, is indicates homeostasis response in mild preclampsia women is relative good. This response was abated in severe preeclampstic women. Although MDA and HSP70 concentration did not differ significantly between groups, however the high HSP70 concentration is indicates homeostasis response relatively good in mild preeclamptic women.

Essential oil from Cymbopogon flexuosus as the potential inhibitor for HSP90.

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Gaonkar, Roopa; Shiralgi, Yallappa; Lakkappa, Dhananjaya B; Hegde, Gurumurthy

2018-01-01

The essential oil of Cymbopogon flexuosus or lemongrass oil (LO) is reported to have antibacterial, antifungal and anticancerous effects. HSP90 is one of the major chaperones responsible for the proper folding of cancer proteins. In this paper we show that the essential oil of C. flexuosus significantly suppresses the HSP90 gene expression. The cytotoxicity of the compounds was tested by MTT assay and the gene expression studies were carried out using HEK-293 and MCF-7 cells. Also we tested the efficacy of the major component of this essential oil viz. citral and geraniol in inhibiting the HSP90 expression. The oil was found to be more cytotoxic to MCF-7 cells with different IC 50 values for the oil (69.33 μg/mL), citral (140.7 μg/mL) and geraniol (117 μg/mL). The fold change of expression was calculated by RT-qPCR using ΔΔCt (2 ^-ΔΔCt ) method and it was 0.1 and 0.03 in MCF-7 cells at 80 μg/mL and 160 μg/mL of LO. Western blot results showed suppression of HSP90 protein expression and HSP90 - ATPase activity inhibition was also observed using LO. This study shows the anticancer mechanism exhibited by the essential oil of C. flexuosus is by the inhibition of the important chaperone protein HSP90.

Design and Construction of a Cloning Vector Containing the hspX Gene of Mycobacterium tuberculosis

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Atieh Yaghoubi

2016-10-01

Full Text Available Background: Tuberculosis (TB is a major cause of death worldwide. Finding an effective vaccine against TB is the best way to control it. Several vaccines against this disease have been developed but none are completely protective. The aim of this study was to design and construct a cloning vector containing the Mycobacterium tuberculosis (M. tuberculosis heat shock protein X (hspX. Methods: First, an hspX fragment was amplified by PCR and cloned into plasmid pcDNA3.1(+ and recombinant vector was confirmed. Results: A 435 bp hspX fragment was isolated. The fragment was 100% homologous with hspX of M. tuberculosis strain H37Rv in GenBank. Conclusions: In this study, the cloning vector pcDNA3.1(+, containing a 435-bp hspX fragment of M. tuberculosis, was constructed. This could be used as a DNA vaccine to induce immune responses in animal models in future studies.

One out of four: HspL but no other small heat shock protein of Agrobacterium tumefaciens acts as efficient virulence-promoting VirB8 chaperone.

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Yun-Long Tsai

Full Text Available Alpha-crystallin-type small heat shock proteins (sHsps are ubiquitously distributed in most eukaryotes and prokaryotes. Four sHsp genes named hspL, hspC, hspAT1, and hspAT2 were identified in Agrobacterium tumefaciens, a plant pathogenic bacterium capable of unique interkingdom DNA transfer via type IV secretion system (T4SS. HspL is highly expressed in virulence-induced growth condition and functions as a VirB8 chaperone to promote T4SS-mediated DNA transfer. Here, we used genetic and biochemical approaches to investigate the involvement of the other three sHsps in T4SS and discovered the molecular basis underlying the dominant function of HspL in promoting T4SS function. While single deletion of hspL but no other sHsp gene reduced T4SS-mediated DNA transfer and tumorigenesis efficiency, additional deletion of other sHsp genes in the hspL deletion background caused synergistic effects in the virulence phenotypes. This is correlated with the high induction of hspL and only modest increase of hspC, hspAT1, and hspAT2 at their mRNA and protein abundance in virulence-induced growth condition. Interestingly, overexpression of any single sHsp gene alone in the quadruple mutant caused increased T4SS-mediated DNA transfer and tumorigenesis. Thermal aggregation protecting assays in vitro indicated that all four sHsps exhibit chaperone activity for the model substrate citrate synthase but only HspL functions as efficient chaperone for VirB8. The higher VirB8 chaperone activity of HspL was also demonstrated in vivo, in which lower amounts of HspL than other sHsps were sufficient in maintaining VirB8 homeostasis in A. tumefaciens. Domain swapping between HspL and HspAT2 indicated that N-terminal, central alpha-crystallin, and C-terminal domains of HspL all contribute to HspL function as an efficient VirB8 chaperone. Taken together, we suggest that the dominant role of HspL in promoting T4SS function is based on its higher expression in virulence

First cellular approach of the effects of global warming on groundwater organisms: a study of the HSP70 gene expression.

Science.gov (United States)

Colson-Proch, Céline; Morales, Anne; Hervant, Frédéric; Konecny, Lara; Moulin, Colette; Douady, Christophe J

2010-05-01

Whereas the consequences of global warming at population or community levels are well documented, studies at the cellular level are still scarce. The study of the physiological or metabolic effects of such small increases in temperature (between +2 degrees C and +6 degrees C) is difficult because they are below the amplitude of the daily or seasonal thermal variations occurring in most environments. In contrast, subterranean biotopes are highly thermally buffered (+/-1 degrees C within a year), and underground water organisms could thus be particularly well suited to characterise cellular responses of global warming. To this purpose, we studied genes encoding chaperone proteins of the HSP70 family in amphipod crustaceans belonging to the ubiquitous subterranean genus Niphargus. An HSP70 sequence was identified in eight populations of two complexes of species of the Niphargus genus (Niphargus rhenorhodanensis and Niphargus virei complexes). Expression profiles were determined for one of these by reverse transcription and quantitative polymerase chain reaction, confirming the inducible nature of this gene. An increase in temperature of 2 degrees C seemed to be without effect on N. rhenorhodanensis physiology, whereas a heat shock of +6 degrees C represented an important thermal stress for these individuals. Thus, this study shows that although Niphargus individuals do not undergo any daily or seasonal thermal variations in underground water, they display an inducible HSP70 heat shock response. This controlled laboratory-based physiological experiment constitutes a first step towards field investigations of the cellular consequences of global warming on subterranean organisms.

Effects of Thermal Stress on the mRNA Expression of SOD, HSP90, and HSP70 in the Spotted Sea Bass ( Lateolabrax maculatus)

Science.gov (United States)

Shin, Moon-Kyeong; Park, Ho-Ra; Yeo, Won-Jun; Han, Kyung-Nam

2018-03-01

The aim of this study was to elucidate the molecular mechanisms underlying the thermal stress response in the spotted sea bass ( Lateolabrax maculatus). Spotted sea basses were exposed to 4 different water temperatures (20, 22, 24, and 28°C) in increasing increments of 2°C/h from 18°C (control) for different time periods (0, 6, 12, 24, 48, 72, and 96 h). Subsequently, 3 tissues (liver, muscle, and gill) were isolated, and the levels of SOD, HSP90, and HSP70 mRNA were assessed. SOD mRNA expression was maintained at baseline levels of control fish at all water temperatures in the liver, while muscle and gill tissue showed an increase followed by a decrease over each certain time with higher water temperature. HSP90 mRNA expression increased in the liver at ≤ 24°C over time, but maintained baseline expression at 28°C. In muscle, HSP90 mRNA expression gradually increased at all water temperatures, but increased and then decreased at ≥ 24°C in gill tissue. HSP70 mRNA expression exhibited an increase and then a decrease in liver tissue at 28°C, but mainly showed similar expression patterns to HSP90 in all tissues. These results suggest the activity of a defense mechanism using SOD, HSP90, and HSP70 in the spotted sea bass upon rapid increases in water temperature, where the expression of these genes indicated differences between tissues in the extent of the defense mechanisms. Also, these results indicate that high water temperature and long-term thermal stress exposure can inhibit physiological defense mechanisms.

Adenoviral transfer of HSP-70 into pulmonary epithelium ameliorates experimental acute respiratory distress syndrome.

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Weiss, Yoram G; Maloyan, Alina; Tazelaar, John; Raj, Nichelle; Deutschman, Clifford S

2002-09-01

The acute respiratory distress syndrome (ARDS) provokes three pathologic processes: unchecked inflammation, interstitial/alveolar protein accumulation, and destruction of pulmonary epithelial cells. The highly conserved heat shock protein HSP-70 can limit all three responses but is not appropriately expressed in the lungs after cecal ligation and double puncture (2CLP), a clinically relevant model of ARDS. We hypothesize that restoring expression of HSP-70 using adenovirus-mediated gene therapy will limit pulmonary pathology following 2CLP. We administered a vector containing the porcine HSP-70 cDNA driven by a CMV promoter (AdHSP) into the lungs of rats subjected to 2CLP or sham operation. Administration of AdHSP after either sham operation or 2CLP increased HSP-70 protein expression in lung tissue, as determined by immunohistochemistry and Western blot hybridization. Administration of AdHSP significantly attenuated interstitial and alveolar edema and protein exudation and dramatically decreased neutrophil accumulation, relative to a control adenovirus. CLP-associated mortality at 48 hours was reduced by half. Modulation of HSP-70 production reduces pathologic changes and may improve outcome in experimental ARDS.

Hsp90: A New Player in DNA Repair?

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Rosa Pennisi

2015-10-01

Full Text Available Heat shock protein 90 (Hsp90 is an evolutionary conserved molecular chaperone that, together with Hsp70 and co-chaperones makes up the Hsp90 chaperone machinery, stabilizing and activating more than 200 proteins, involved in protein homeostasis (i.e., proteostasis, transcriptional regulation, chromatin remodeling, and DNA repair. Cells respond to DNA damage by activating complex DNA damage response (DDR pathways that include: (i cell cycle arrest; (ii transcriptional and post-translational activation of a subset of genes, including those associated with DNA repair; and (iii triggering of programmed cell death. The efficacy of the DDR pathways is influenced by the nuclear levels of DNA repair proteins, which are regulated by balancing between protein synthesis and degradation as well as by nuclear import and export. The inability to respond properly to either DNA damage or to DNA repair leads to genetic instability, which in turn may enhance the rate of cancer development. Multiple components of the DNA double strand breaks repair machinery, including BRCA1, BRCA2, CHK1, DNA-PKcs, FANCA, and the MRE11/RAD50/NBN complex, have been described to be client proteins of Hsp90, which acts as a regulator of the diverse DDR pathways. Inhibition of Hsp90 actions leads to the altered localization and stabilization of DDR proteins after DNA damage and may represent a cell-specific and tumor-selective radiosensibilizer. Here, the role of Hsp90-dependent molecular mechanisms involved in cancer onset and in the maintenance of the genome integrity is discussed and highlighted.

The role of HSP70 in mediating age-dependent mortality in sepsis

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McConnell, Kevin W.; Fox, Amy C.; Clark, Andrew T.; Chang, Nai-Yuan Nicholas; Dominguez, Jessica A.; Farris, Alton B.; Buchman, Timothy G.; Hunt, Clayton R.; Coopersmith, Craig M.

2011-01-01

Sepsis is primarily a disease of the aged, with increased incidence and mortality occurring in aged hosts. Heat shock protein (HSP) 70 plays an important role in both healthy aging and the stress response to injury. The purpose of this study was to determine the role of HSP70 in mediating mortality and the host inflammatory response in aged septic hosts. Sepsis was induced in both young (6–12week old) and aged (16–17 month old) HSP70−/− and wild type (WT) mice to determine if HSP70 modulated outcome in an age-dependent fashion. Young HSP70−/− and WT mice subjected to cecal ligation and puncture (CLP), Pseudomonas aeruginosa pneumonia or Streptococcus pneumoniae pneumonia had no differences in mortality, suggesting HSP70 does not mediate survival in young septic hosts. In contrast, mortality was higher in aged HSP70−/− mice than aged WT mice subjected to CLP (p=0.01), suggesting HSP70 mediates mortality in sepsis in an age-dependent fashion. Compared to WT mice, aged septic HSP70−/− mice had increased gut epithelial apoptosis and pulmonary inflammation. In addition, HSP70−/−mice had increased systemic levels of TNF-α, IL-6, IL-10 and IL-1β compared to WT mice. These data demonstrate that HSP70 is a key determinant of mortality in aged but not young hosts in sepsis. HSP70 may play a protective role in an age-dependent response to sepsis by preventing excessive gut apoptosis and both pulmonary and systemic inflammation. PMID:21296977

ATM Is Required for the Prolactin-Induced HSP90-Mediated Increase in Cellular Viability and Clonogenic Growth After DNA Damage.

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Karayazi Atici, Ödül; Urbanska, Anna; Gopinathan, Sesha Gopal; Boutillon, Florence; Goffin, Vincent; Shemanko, Carrie S

2018-02-01

Prolactin (PRL) acts as a survival factor for breast cancer cells, but the PRL signaling pathway and the mechanism are unknown. Previously, we identified the master chaperone, heat shock protein 90 (HSP90) α, as a prolactin-Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) target gene involved in survival, and here we investigated the role of HSP90 in the mechanism of PRL-induced viability in response to DNA damage. The ataxia-telangiectasia mutated kinase (ATM) protein plays a critical role in the cellular response to double-strand DNA damage. We observed that PRL increased viability of breast cancer cells treated with doxorubicin or etoposide. The increase in cellular resistance is specific to the PRL receptor, because the PRL receptor antagonist, Δ1-9-G129R-hPRL, prevented the increase in viability. Two different HSP90 inhibitors, 17-allylamino-17-demethoxygeldanamycin and BIIB021, reduced the PRL-mediated increase in cell viability of doxorubicin-treated cells and led to a decrease in JAK2, ATM, and phosphorylated ATM protein levels. Inhibitors of JAK2 (G6) and ATM (KU55933) abolished the PRL-mediated increase in cell viability of DNA-damaged cells, supporting the involvement of each, as well as the crosstalk of ATM with the PRL pathway in the context of DNA damage. Drug synergism was detected between the ATM inhibitor (KU55933) and doxorubicin and between the HSP90 inhibitor (BIIB021) and doxorubicin. Short interfering RNA directed against ATM prevented the PRL-mediated increase in cell survival in two-dimensional cell culture, three-dimensional collagen gel cultures, and clonogenic cell survival, after doxorubicin treatment. Our results indicate that ATM contributes to the PRL-JAK2-STAT5-HSP90 pathway in mediating cellular resistance to DNA-damaging agents. Copyright © 2018 Endocrine Society.

Combined effects of thermal stress and Cd on lysosomal biomarkers and transcription of genes encoding lysosomal enzymes and HSP70 in mussels, Mytilus galloprovincialis

Energy Technology Data Exchange (ETDEWEB)

Izagirre, Urtzi; Errasti, Aitzpea; Bilbao, Eider; Múgica, María; Marigómez, Ionan, E-mail: ionan.marigomez@ehu.es

2014-04-01

Highlights: • Thermal stress and Cd caused lysosomal enlargement and membrane destabilisation. • hex, gusb and ctsl but not hsp70 were up-regulated at elevated temperature but down-regulated by Cd. • Thermal stress influenced lysosomal responses to Cd exposure. • The presence of Cd jeopardised responsiveness against thermal stress. - Abstract: In estuaries and coastal areas, intertidal organisms may be subject to thermal stress resulting from global warming, together with pollution. In the present study, the combined effects of thermal stress and exposure to Cd were investigated in the endo-lysosomal system of digestive cells in mussels, Mytilus galloprovincialis. Mussels were maintained for 24 h at 18 °C and 26 °C seawater temperature in absence and presence of 50 μg Cd/L seawater. Cadmium accumulation in digestive gland tissue, lysosomal structural changes and membrane stability were determined. Semi-quantitative PCR was applied to reveal the changes elicited by the different experimental conditions in hexosaminidase (hex), β-glucuronidase (gusb), cathepsin L (ctsl) and heat shock protein 70 (hsp70) gene transcription levels. Thermal stress provoked lysosomal enlargement whilst Cd-exposure led to fusion of lysosomes. Both thermal stress and Cd-exposure caused lysosomal membrane destabilisation. hex, gusb and ctsl genes but not hsp70 gene were transcriptionally up-regulated as a result of thermal stress. In contrast, all the studied genes were transcriptionally down-regulated in response to Cd-exposure. Cd bioaccumulation was comparable at 18 °C and 26 °C seawater temperatures but interactions between thermal stress and Cd-exposure were remarkable both in lysosomal biomarkers and in gene transcription. hex, gusb and ctsl genes, reacted to elevated temperature in absence of Cd but not in Cd-exposed mussels. Therefore, thermal stress resulting from global warming might influence the use and interpretation of lysosomal biomarkers in marine pollution

The hsp 16 Gene of the Probiotic Lactobacillus acidophilus Is Differently Regulated by Salt, High Temperature and Acidic Stresses, as Revealed by Reverse Transcription Quantitative PCR (qRT-PCR Analysis

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Daniela Fiocco

2011-08-01

Full Text Available Small heat shock proteins (sHsps are ubiquitous conserved chaperone-like proteins involved in cellular proteins protection under stressful conditions. In this study, a reverse transcription quantitative PCR (RT-qPCR procedure was developed and used to quantify the transcript level of a small heat shock gene (shs in the probiotic bacterium Lactobacillus acidophilus NCFM, under stress conditions such as heat (45 °C and 53 °C, bile (0.3% w/v, hyperosmosis (1 M and 2.5 M NaCl, and low pH value (pH 4. The shs gene of L. acidophilus NCFM was induced by salt, high temperature and acidic stress, while repression was observed upon bile stress. Analysis of the 5' noncoding region of the hsp16 gene reveals the presence of an inverted repeat (IR sequence (TTAGCACTC-N9-GAGTGCTAA homologue to the controlling IR of chaperone expression (CIRCE elements found in the upstream regulatory region of Gram-positive heat shock operons, suggesting that the hsp16 gene of L. acidophilus might be transcriptionally controlled by HrcA. In addition, the alignment of several small heat shock proteins identified so far in lactic acid bacteria, reveals that the Hsp16 of L. acidophilus exhibits a strong evolutionary relationship with members of the Lactobacillus acidophilus group.

Role of membrane Hsp70 in radiation sensitivity of tumor cells

International Nuclear Information System (INIS)

Murakami, Naoya; Kühnel, Annett; Schmid, Thomas E.; Ilicic, Katarina; Stangl, Stefan; Braun, Isabella S.; Gehrmann, Mathias; Molls, Michael; Itami, Jun; Multhoff, Gabriele

2015-01-01

The major stress-inducible heat shock protein 70 (Hsp70) is frequently overexpressed in the cytosol and integrated in the plasma membrane of tumor cells via lipid anchorage. Following stress such as non-lethal irradiation Hsp70 synthesis is up-regulated. Intracellular located Hsp70 is known to exert cytoprotective properties, however, less is known about membrane (m)Hsp70. Herein, we investigate the role of mHsp70 in the sensitivity towards irradiation in tumor sublines that differ in their cytosolic and/or mHsp70 levels. The isogenic human colon carcinoma sublines CX + with stable high and CX − with stable low expression of mHsp70 were generated by fluorescence activated cell sorting, the mouse mammary carcinoma sublines 4 T1 (4 T1 ctrl) and Hsp70 knock-down (4 T1 Hsp70 KD) were produced using the CRISPR/Cas9 system, and the Hsp70 down-regulation in human lung carcinoma sublines H1339 ctrl/H1339 HSF-1 KD and EPLC-272H ctrl/EPLC-272H HSF-1 KD was achieved by small interfering (si)RNA against Heat shock factor 1 (HSF-1). Cytosolic and mHsp70 was quantified by Western blot analysis/ELISA and flow cytometry; double strand breaks (DSBs) and apoptosis were measured by flow cytometry using antibodies against γH2AX and real-time PCR (RT-PCR) using primers and antibodies directed against apoptosis related genes; and radiation sensitivity was determined using clonogenic cell surviving assays. CX + /CX − tumor cells exhibited similar cytosolic but differed significantly in their mHsp70 levels, 4 T1 ctrl/4 T1 Hsp70 KD cells showed significant differences in their cytosolic and mHsp70 levels and H1339 ctrl/H1339 HSF-1 KD and EPLC-272H ctrl/EPLC-272H HSF-1 KD lung carcinoma cell sublines had similar mHsp70 but significantly different cytosolic Hsp70 levels. γH2AX was significantly up-regulated in irradiated CX − and 4 T1 Hsp70 KD with low basal mHsp70 levels, but not in their mHsp70 high expressing counterparts, irrespectively of their cytosolic Hsp70 content. After

Perbedaan Kadar HSP90 pada Preeklamsi Berat dengan Kehamilan Normal

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Soetrisno

2015-06-01

Full Text Available Severe pre-eclampsia is the second highest cause of maternal mortality. Free radicals that stimulate heat shock protein 90 (HSP 90 are believed to determine severe pre-eclampsia. HSP90 is an important protein that helps the establishment and maintenance of other proteins. It also increases the life time of cells after various pathological conditions (chaperone function. The chaperone function is the adaptation key factor to endogenous stress in tissues. By recognizing HSP90 level in early detection of severe pre-eclampsia, prevention and management can be started early. This study aimed to prove that the HSP90 level in pregnancy with severe pre-eclampsia is higher than normal pregnancy. This was a quantitative study using cross sectional approach by testing the HSP90 level. The study was conducted during the period of September to November 2013, at the Obstetrics and Gynecological Unit, Moewardi Hospital Surakarta and Prodia Laboratory Jakarta. The number of subjects was 30 patients, consisting of 15 normal pregnant mothers and 15 pregnant mothers with pre-eclampsia . The calculation of serum HSP90 level was conducted using enzyme-linked immunosorbent assay (ELISA. Data were analyzed using t-test using SPSS for Windows version 17 for Windows. The mean of HSP90 in the severe pre-eclampsia group was 131.91±26.66 while the mean in the normal pregnancy group was 80.28±13.39 with p=0.00 (p<0.05. Level of HSP90 serum in severe pre-eclampsia is higher than in normal pregnancy, due to the occurrence of oxidative stress in severe pre-eclampsia

HSP10 selective preference for myeloid and megakaryocytic precursors in normal human bone marrow

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F Cappello

2009-06-01

Full Text Available Heat shock proteins (HSPs constitute a heterogeneous family of proteins involved in cell homeostasis. During cell life they are involved in harmful insults, as well as in immune and inflammatory reactions. It is known that they regulate gene expression, and cell proliferation, differentiation and death. HSP60 is a mitochondrial chaperonin, highly preserved during evolution, responsible of protein folding. Its function is strictly dependent on HSP10 in both prokaryotic and eukaryotic elements. We investigated the presence and the expression of HSP60 and HSP10 in a series of 20 normal human bone marrow specimens (NHBM by the means of immunohistochemistry. NHBM showed no expression of HSP60, probably due to its being below the detectable threshold, as already demonstrated in other normal human tissues. By contrast, HSP10 showed a selective positivity for myeloid and megakaryocytic lineages. The positivity was restricted to precursor cells, while mature elements were constantly negative.We postulate that HSP10 plays a role in bone marrow cell differentiation other than being a mitochondrial co-chaperonin. The present data emphasize the role of HSP10 during cellular homeostasis and encourage further investigations in this field.

Hsp90 depletion goes wild

OpenAIRE

Siegal, Mark L; Masel, Joanna

2012-01-01

Abstract Hsp90 reveals phenotypic variation in the laboratory, but is Hsp90 depletion important in the wild? Recent work from Chen and Wagner in BMC Evolutionary Biology has discovered a naturally occurring Drosophila allele that downregulates Hsp90, creating sensitivity to cryptic genetic variation. Laboratory studies suggest that the exact magnitude of Hsp90 downregulation is important. Extreme Hsp90 depletion might reactivate transposable elements and/or induce aneuploidy, in addition to r...

Role of Subunit Exchange and Electrostatic Interactions on the Chaperone Activity of Mycobacterium leprae HSP18

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Nandi, Sandip Kumar; Panda, Alok Kumar; Chakraborty, Ayon; Ray, Sougata Sinha; Biswas, Ashis

2015-01-01

Mycobacterium leprae HSP18, a major immunodominant antigen of M. leprae pathogen, is a small heat shock protein. Previously, we reported that HSP18 is a molecular chaperone that prevents aggregation of different chemically and thermally stressed client proteins and assists refolding of denatured enzyme at normal temperature. We also demonstrated that it can efficiently prevent the thermal killing of E. coli at higher temperature. However, molecular mechanism behind the chaperone function of HSP18 is still unclear. Therefore, we studied the structure and chaperone function of HSP18 at normal temperature (25°C) as well as at higher temperatures (31–43°C). Our study revealed that the chaperone function of HSP18 is enhanced significantly with increasing temperature. Far- and near-UV CD experiments suggested that its secondary and tertiary structure remain intact in this temperature range (25–43°C). Besides, temperature has no effect on the static oligomeric size of this protein. Subunit exchange study demonstrated that subunits of HSP18 exchange at 25°C with a rate constant of 0.018 min-1. Both rate of subunit exchange and chaperone activity of HSP18 is found to increase with rise in temperature. However, the surface hydrophobicity of HSP18 decreases markedly upon heating and has no correlation with its chaperone function in this temperature range. Furthermore, we observed that HSP18 exhibits diminished chaperone function in the presence of NaCl at 25°C. At elevated temperatures, weakening of interactions between HSP18 and stressed client proteins in the presence of NaCl results in greater reduction of its chaperone function. The oligomeric size, rate of subunit exchange and structural stability of HSP18 were also found to decrease when electrostatic interactions were weakened. These results clearly indicated that subunit exchange and electrostatic interactions play a major role in the chaperone function of HSP18. PMID:26098662

Role of Subunit Exchange and Electrostatic Interactions on the Chaperone Activity of Mycobacterium leprae HSP18.

Science.gov (United States)

Nandi, Sandip Kumar; Panda, Alok Kumar; Chakraborty, Ayon; Sinha Ray, Sougata; Biswas, Ashis

2015-01-01

Mycobacterium leprae HSP18, a major immunodominant antigen of M. leprae pathogen, is a small heat shock protein. Previously, we reported that HSP18 is a molecular chaperone that prevents aggregation of different chemically and thermally stressed client proteins and assists refolding of denatured enzyme at normal temperature. We also demonstrated that it can efficiently prevent the thermal killing of E. coli at higher temperature. However, molecular mechanism behind the chaperone function of HSP18 is still unclear. Therefore, we studied the structure and chaperone function of HSP18 at normal temperature (25°C) as well as at higher temperatures (31-43°C). Our study revealed that the chaperone function of HSP18 is enhanced significantly with increasing temperature. Far- and near-UV CD experiments suggested that its secondary and tertiary structure remain intact in this temperature range (25-43°C). Besides, temperature has no effect on the static oligomeric size of this protein. Subunit exchange study demonstrated that subunits of HSP18 exchange at 25°C with a rate constant of 0.018 min(-1). Both rate of subunit exchange and chaperone activity of HSP18 is found to increase with rise in temperature. However, the surface hydrophobicity of HSP18 decreases markedly upon heating and has no correlation with its chaperone function in this temperature range. Furthermore, we observed that HSP18 exhibits diminished chaperone function in the presence of NaCl at 25°C. At elevated temperatures, weakening of interactions between HSP18 and stressed client proteins in the presence of NaCl results in greater reduction of its chaperone function. The oligomeric size, rate of subunit exchange and structural stability of HSP18 were also found to decrease when electrostatic interactions were weakened. These results clearly indicated that subunit exchange and electrostatic interactions play a major role in the chaperone function of HSP18.

Hsp10, Hsp70, and Hsp90 immunohistochemical levels change in ulcerative colitis after therapy

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G. Tomasello

2011-10-01

Full Text Available Ulcerative colitis (UC is a form of inflammatory bowel disease (IBD characterized by damage of large bowel mucosa and frequent extra-intestinal autoimmune comorbidities. The role played in IBD pathogenesis by molecular chaperones known to interact with components of the immune system involved in inflammation is unclear. We previously demonstrated that mucosal Hsp60 decreases in UC patients treated with conventional therapies (mesalazine, probiotics, suggesting that this chaperonin could be a reliable biomarker useful for monitoring response to treatment, and that it might play a role in pathogenesis. In the present work we investigated three other heat shock protein/molecular chaperones: Hsp10, Hsp70, and Hsp90. We found that the levels of these proteins are increased in UC patients at the time of diagnosis and decrease after therapy, supporting the notion that these proteins deserve attention in the study of the mechanisms that promote the development and maintenance of IBD, and as biomarkers of this disease (e.g., to monitor response to treatment at the histological level.

thermal stress and Hsp70 as selective agents

Indian Academy of Sciences (India)

Madhu Sudhan

starred in an exhaustive list of studies linking its expression to environmental ... genetic stress linked to Hsp70: polyglutamine expansion .... Twenty-five D. melanogaster genes conserved in all twelve Drosophila species (fourth column) possessing conserved ...... like growth factor-1, and vertebral bone mass in men; J. Clin.

Recovery From Radiation-induced Bone Marrow Damage by HSP25 Through Tie2 Signaling

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Lee, Hae-June [Division of Radiation Effects, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Kwon, Hee-Chung [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Chung, Hee-Yong [College of Medicine, Hanyang University, Seoul (Korea, Republic of); Lee, Yoon-Jin [Division of Radiation Effects, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Lee, Yun-Sil, E-mail: yslee0425@ewha.ac.kr [College of Pharmacy and Division of Life and Pharmaceutical Sciences, Ewha Woman' s University, Seoul (Korea, Republic of)

2012-09-01

Purpose: Whole-body radiation therapy can cause severe injury to the hematopoietic system, and therefore it is necessary to identify a novel strategy for overcoming this injury. Methods and Materials: Mice were irradiated with 4.5 Gy after heat shock protein 25 (HSP25) gene transfer using an adenoviral vector. Then, peripheral blood cell counts, histopathological analysis, and Western blotting on bone marrow (BM) cells were performed. The interaction of HSP25 with Tie2 was investigated with mouse OP9 and human BM-derived mesenchymal stem cells to determine the mechanism of HSP25 in the hematopoietic system. Results: HSP25 transfer increased BM regeneration and reduced apoptosis following whole-body exposure to ionizing radiation (IR). The decrease in Tie2 protein expression that followed irradiation of the BM was blocked by HSP25 transfer, and Tie2-positive cells were more abundant among the BM cells of HSP25-transferred mice, even after IR exposure. Following systemic RNA interference of Tie2 before IR, HSP25-mediated radioprotective effects were partially blocked in both mice and cell line systems. Stability of Tie2 was increased by HSP25, a response mediated by the interaction of HSP25 with Tie2. IR-induced tyrosine phosphorylation of Tie2 was augmented by HSP25 overexpression; downstream events in the Tie2 signaling pathway, including phosphorylation of AKT and EKR1/2, were also activated. Conclusions: HSP25 protects against radiation-induced BM damage by interacting with and stabilizing Tie2. This may be a novel strategy for HSP25-mediated radioprotection in BM.

The expression of HSP60 and HSP10 in large bowel carcinomas with lymph node metastase

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Cappello, Francesco; David, Sabrina; Rappa, Francesca; Bucchieri, Fabio; Marasà, Lorenzo; Bartolotta, Tommaso E; Farina, Felicia; Zummo, Giovanni

2005-01-01

The involvement of Heat Shock Proteins (HSP) in cancer development and progression is a widely debated topic. The objective of the present study was to evaluate the presence and expression of HSP60 and HSP10 in a series of large bowel carcinomas and locoregional lymph nodes with and without metastases. 82 Astler and Coller's stage C2 colorectal cancers, of which 48 well-differentiated and 34 poorly-differentiated, were selected along with 661 lymph nodes, including 372 with metastases and 289 with reactive hyperplasia only, from the same tumours. Primitive tumours and both metastatic and reactive lymph nodes were studied; specifically, three different compartments of the lymph nodes, secondary follicle, paracortex and medullary sinus, were also analysed. An immunohistochemical research for HSP60 and HSP10 was performed and the semiquantitative results were analysed by statistical analysis to determine the correlation between HSPs expression and 1) tumour grading; 2) degree of inflammation; 3) number of lymph nodes involved; 4) lymph node compartment hyperplasia. Moreover, western blotting was performed on a smaller group of samples to confirm the immunohistochemical results. Our data show that the expression of HSP60, in both primary tumour and lymph node metastasis, is correlated with the tumoral grade, while the HSP10 expression is not. Nevertheless, the levels of HSP10 are commonly higher than the levels of HSP60. In addition, statistical analyses do not show any correlation between the degree of inflammation and the immunopositivity for both HSP60 and HSP10. Moreover, we find a significant correlation between the presence of lymph node metastases and the positivity for both HSP60 and HSP10. In particular, metastatic lymph nodes show a higher percentage of cells positive for both HSP60 and HSP10 in the secondary follicles, and for HSP10 in the medullary sinuses, when compared with hyperplastic lymph nodes. HSP60 and HSP10 may have diagnostic and prognostic

Cloning and Expression of HSP of Taenia Solium Oncosphere%猪带绦虫六钩蚴 HSP 的克隆和表达

Institute of Scientific and Technical Information of China (English)

王哲; 赵权

2013-01-01

　　The HSP gene was separately amplified from total RNA of activated Taenia solium oncosphere by RT -PCR.The PCR products were cloned into pGH vector,recombinant positive clones was sequenced after restriction enzyme digestion.The HSP gene was subcloned into pET28a expression vector,the recombinant pET28a-HSP infected into E.coliBL21.IPTG was added to induce fusion expression and the expression products was identified by SDS-PAGE and Western-blot.One fusion protein band about 35 kDa was identified by SDS -PAGE after inducible expression after inducible expression,The result would lay foundations for the mechanism of invasion of between oncosphere and host,and the design of new vaccine anti-porcine cysticercosis and taeniasis.%　　提取猪带绦虫激活和未激活六钩蚴总 RNA，RT-PCR 扩增 HSP 目的基因，将目的基因与 pGH 克隆载体连接，经酶切鉴定后，将阳性重组质粒进行测序，结果扩增出激活的六钩蚴的目的片段。将目的基因亚克隆到原核表达载体 pET-28a-HSP中，并将获得的 pET28a-HSP 阳性重组子转化至宿主菌 E.coliBL21，IPTG 进行诱导表达，并对重组抗原 pET-28a-HSP 进行SDS-PAGE 和 Western-blot 检测。结果表明重组经 SDS-PAGE 分析可见一条约35 kDa 大小的融合蛋白条带的抗原，Western-blot 结果显示其能被囊虫病人阳性血清识别。这将为进一步阐明六钩蚴入侵中间宿主的机理、设计新型抗猪囊虫病和绦虫病疫苗打下基础。

Mobile phone base station-emitted radiation does not induce phosphorylation of Hsp27.

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Hirose, H; Sakuma, N; Kaji, N; Nakayama, K; Inoue, K; Sekijima, M; Nojima, T; Miyakoshi, J

2007-02-01

An in vitro study focusing on the effects of low-level radiofrequency (RF) fields from mobile radio base stations employing the International Mobile Telecommunication 2000 (IMT-2000) cellular system was conducted to test the hypothesis that modulated RF fields act to induce phosphorylation and overexpression of heat shock protein hsp27. First, we evaluated the responses of human cells to microwave exposure at a specific absorption rate (SAR) of 80 mW/kg, which corresponds to the limit of the average whole-body SAR for general public exposure defined as a basic restriction in the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines. Second, we investigated whether continuous wave (CW) and Wideband Code Division Multiple Access (W-CDMA) modulated signal RF fields at 2.1425 GHz induced activation or gene expression of hsp27 and other heat shock proteins (hsps). Human glioblastoma A172 cells were exposed to W-CDMA radiation at SARs of 80 and 800 mW/kg for 2-48 h, and CW radiation at 80 mW/kg for 24 h. Human IMR-90 fibroblasts from fetal lungs were exposed to W-CDMA at 80 and 800 mW/kg for 2 or 28 h, and CW at 80 mW/kg for 28 h. Under the RF field exposure conditions described above, no significant differences in the expression levels of phosphorylated hsp27 at serine 82 (hsp27[pS82]) were observed between the test groups exposed to W-CDMA or CW signal and the sham-exposed negative controls, as evaluated immediately after the exposure periods by bead-based multiplex assays. Moreover, no noticeable differences in the gene expression of hsps were observed between the test groups and the negative controls by DNA Chip analysis. Our results confirm that exposure to low-level RF field up to 800 mW/kg does not induce phosphorylation of hsp27 or expression of hsp gene family.

High mobility group protein DSP1 negatively regulates HSP70 transcription in Crassostrea hongkongensis

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Miao, Zongyu; Xu, Delin; Cui, Miao; Zhang, Qizhong, E-mail: zhangqzdr@126.com

2016-06-10

HSP70 acts mostly as a molecular chaperone and plays important roles in facilitating the folding of nascent peptides as well as the refolding or degradation of the denatured proteins. Under stressed conditions, the expression level of HSP70 is upregulated significantly and rapidly, as is known to be achieved by various regulatory factors controlling the transcriptional level. In this study, a high mobility group protein DSP1 was identified by DNA-affinity purification from the nuclear extracts of Crassostrea hongkongensis using the ChHSP70 promoter as a bait. The specific interaction between the prokaryotically expressed ChDSP1 and the FITC-labeled ChHSP70 promoter was confirmed by EMSA analysis. ChDSP1 was shown to negatively regulate ChHSP70 promoter expression by Luciferase Reporter Assay in the heterologous HEK293T cells. Both ChHSP70 and ChDSP1 transcriptions were induced by either thermal or CdCl{sub 2} stress, while the accumulated expression peaks of ChDSP1 were always slightly delayed when compared with that of ChHSP70. This indicates that ChDSP1 is involved, very likely to exert its suppressive role, in the recovery of the ChHSP70 expression from the induced level to its original state. This study is the first to report negative regulator of HSP70 gene transcription, and provides novel insights into the mechanisms controlling heat shock protein expression. -- Highlights: •HMG protein ChDSP1 shows affinity to ChHSP70 promoter in Crassostrea hongkongensis. •ChDSP1 negatively regulates ChHSP70 transcription. •ChHSP70 and ChDSP1 transcriptions were coordinately induced by thermal/Cd stress. •ChDSP1 may contribute to the recovery of the induced ChHSP70 to its original state. •This is the first report regarding negative regulator of HSP70 transcription.

Hsp90 depletion goes wild

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Siegal Mark L

2012-02-01

Full Text Available Abstract Hsp90 reveals phenotypic variation in the laboratory, but is Hsp90 depletion important in the wild? Recent work from Chen and Wagner in BMC Evolutionary Biology has discovered a naturally occurring Drosophila allele that downregulates Hsp90, creating sensitivity to cryptic genetic variation. Laboratory studies suggest that the exact magnitude of Hsp90 downregulation is important. Extreme Hsp90 depletion might reactivate transposable elements and/or induce aneuploidy, in addition to revealing cryptic genetic variation. See research article http://wwww.biomedcentral.com/1471-2148/12/25

Molecular analysis of Hsp70 mechanisms in plants and their function in response to stress.

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Usman, Magaji G; Rafii, Mohd Y; Martini, Mohammad Y; Yusuff, Oladosu A; Ismail, Mohd R; Miah, Gous

2017-04-01

Studying the strategies of improving abiotic stress tolerance is quite imperative and research under this field will increase our understanding of response mechanisms to abiotic stress such as heat. The Hsp70 is an essential regulator of protein having the tendency to maintain internal cell stability like proper folding protein and breakdown of unfolded proteins. Hsp70 holds together protein substrates to help in movement, regulation, and prevent aggregation under physical and or chemical pressure. However, this review reports the molecular mechanism of heat shock protein 70 kDa (Hsp70) action and its structural and functional analysis, research progress on the interaction of Hsp70 with other proteins and their interaction mechanisms as well as the involvement of Hsp70 in abiotic stress responses as an adaptive defense mechanism.

Lactobacillus strain diversity based on partial hsp60 gene sequences and design of PCR-restriction fragment length polymorphism assays for species identification and differentiation.

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Blaiotta, Giuseppe; Fusco, Vincenzina; Ercolini, Danilo; Aponte, Maria; Pepe, Olimpia; Villani, Francesco

2008-01-01

A phylogenetic tree showing diversities among 116 partial (499-bp) Lactobacillus hsp60 (groEL, encoding a 60-kDa heat shock protein) nucleotide sequences was obtained and compared to those previously described for 16S rRNA and tuf gene sequences. The topology of the tree produced in this study showed a Lactobacillus species distribution similar, but not identical, to those previously reported. However, according to the most recent systematic studies, a clear differentiation of 43 single-species clusters was detected/identified among the sequences analyzed. The slightly higher variability of the hsp60 nucleotide sequences than of the 16S rRNA sequences offers better opportunities to design or develop molecular assays allowing identification and differentiation of either distant or very closely related Lactobacillus species. Therefore, our results suggest that hsp60 can be considered an excellent molecular marker for inferring the taxonomy and phylogeny of members of the genus Lactobacillus and that the chosen primers can be used in a simple PCR procedure allowing the direct sequencing of the hsp60 fragments. Moreover, in this study we performed a computer-aided restriction endonuclease analysis of all 499-bp hsp60 partial sequences and we showed that the PCR-restriction fragment length polymorphism (RFLP) patterns obtainable by using both endonucleases AluI and TacI (in separate reactions) can allow identification and differentiation of all 43 Lactobacillus species considered, with the exception of the pair L. plantarum/L. pentosus. However, the latter species can be differentiated by further analysis with Sau3AI or MseI. The hsp60 PCR-RFLP approach was efficiently applied to identify and to differentiate a total of 110 wild Lactobacillus strains (including closely related species, such as L. casei and L. rhamnosus or L. plantarum and L. pentosus) isolated from cheese and dry-fermented sausages.

Altered Cross-linking of HSP27 by Zerumbone as a Novel Strategy for Overcoming HSP27- mediated Resistance

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Choi, Seo Hyun; Lee, Yoon Jin; Lee, Hae June; Lee, Yun Sil; Kim, Joon; Seo, Woo Duck; Nam, Joo Won; Lee, Yoo Jin; Seo, Eun Kyung

2010-01-01

HSPs have diverse roles in the regulation of signal transduction and in numerous aspects of cell growth and death. Indeed, HSP90, HSP70, and HSP27 have each been implicated in promoting cancer. Most HSP27 exists as large oligomeric complexes ranging from 100- 800 kDa, which are probably stabilized by complex interactions between dimeric building blocks. The functional properties of HSP27 are dependent on the quaternary structure of the protein. For example, HSP27 acts as a chaperone and binds to cytochrome c or Daxx as a dimer. Therefore, the oligomerization pattern of HPS27 is believed to have HSP27-mediated protective functions. In this study, zerumbone (ZER), the cytotoxic component isolated from Zingiber zerumbet Smith, induced cross-linking of HSP27 protein by its insertion between the disulfide bond of HSP27, and ZERmediated altered cross-linking of HSP27 modified normal HSP27 dimerization, which resulted in a sensitizing effect to tumors after treatment with radiation. Therefore, altered cross-linking by ZER may be a novel strategy for inhibition of HSP27-mediated resistance

An RNA aptamer specific to Hsp70-ATP conformation inhibits its ATPase activity independent of Hsp40.

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Thirunavukarasu, Deepak; Shi, Hua

2015-04-01

The highly conserved and ubiquitous molecular chaperone heat shock protein 70 (Hsp70) plays a critical role in protein homeostasis (proteostasis). Controlled by its ATPase activity, Hsp70 cycles between two conformations, Hsp70-ATP and Hsp70-ADP, to bind and release its substrate. Chemical tools with distinct modes of action, especially those capable of modulating the ATPase activity of Hsp70, are being actively sought after in the mechanistic dissection of this system. Here, we report a conformation-specific RNA aptamer that binds only to Hsp70-ATP but not to Hsp70-ADP. We have refined this aptamer and demonstrated its inhibitory effect on Hsp70's ATPase activity. We have also shown that this inhibitory effect on Hsp70 is independent of its interaction with the Hsp40 co-chaperone. As Hsp70 is increasingly being recognized as a drug target in a number of age related diseases such as neurodegenerative, protein misfolding diseases and cancer, this aptamer is potentially useful in therapeutic applications. Moreover, this work also demonstrates the feasibility of using aptamers to target ATPase activity as a general therapeutic strategy.

[Gene Expression Profile of Apoptosis in Leukemia Cells Induced by Hsp90 Selective inhibitor 17-AAG].

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Wang, Na-Na; Li, Zhi-Heng; Tao, Yan-Fang; Xu, Li-Xiao; Pan, Jian; Hu, Shao-Yan

2016-06-01

To investigate the apoptotic effects of Hsp90 selective inhibitor 17-AAG on human leukemia HL-60 and NB4 cells and analyse its possible mechanism. CCK-8 assay was used to quantify the growth inhibition of cells after exposure to 17-AAG for 24 hours. Flow cytometrve with annexin V/propidium iodide staining was used to detect apoptosis of leukemia cells. Then Western blot was used to detect the activation of apoptosis related protein caspase-3 and PARP level. Gene expression profile of NB4 cells treated with 17-AAG was analyzed with real-time PCR arrays. The inhibition of leukemia cell proliferation displayed a dose-dependent manner. Annexin V assay, cell cycle analysis and activation of PARP demonstrate that 17-AAG induced apoptosis leukemia cells. Real-time PCR array analysis showed that expression of 56 genes significantly up-regulated and expression of 23 genes were significantly down-regulated after 17-AAG treatment. The 17-AAG can inhibit the proliferation and induce the apoptosis of leukemia cells. After leukemia cells are treated with 17-AAG, the significant changes of apoptosis-related genes occured, and the cell apoptosis occurs via activating apoptosis related signaling pathway.

Conformational Activation of Argonaute by Distinct yet Coordinated Actions of the Hsp70 and Hsp90 Chaperone Systems.

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Tsuboyama, Kotaro; Tadakuma, Hisashi; Tomari, Yukihide

2018-05-17

Loading of small RNAs into Argonaute, the core protein in RNA silencing, requires the Hsp70/Hsp90 chaperone machinery. This machinery also activates many other clients, including steroid hormone receptors and kinases, but how their structures change during chaperone-dependent activation remains unclear. Here, we utilized single-molecule Förster resonance energy transfer (smFRET) to probe the conformational changes of Drosophila Ago2 mediated by the chaperone machinery. We found that empty Ago2 exists in various closed conformations. The Hsp70 system (Hsp40 and Hsp70) and the Hsp90 system (Hop, Hsp90, and p23) together render Ago2 into an open, active form. The Hsp70 system, but not the Hsp90 system alone, is sufficient for Ago2 to partially populate the open form. Instead, the Hsp90 system is required to extend the dwell time of Ago2 in the open state, which must be transiently primed by the Hsp70 system. Our data uncover distinct and coordinated actions of the chaperone machinery, where the Hsp70 system expands the structural ensembles of Ago2 and the Hsp90 system captures and stabilizes the active form. Copyright © 2018 Elsevier Inc. All rights reserved.

Transformation of heat shock protein gene (HspB-C) of helicobacter pylori into sweet potato varieties

International Nuclear Information System (INIS)

Wu Jie; Yan Wenzhao; Zhou Yu; Zhang Xuemei

2010-01-01

Sweet potato which is one of the most important crops in the world has many advantages as a new bioreactor. Helicobacter pylori, as a kind of cancer-causing factor by the World Health Organization, has a strong immunogenicity, and its monoclonal antibody has bactericidal activity, which has the possibility as the vaccine components. In this research, we have constructed the plant expression vector with heat shock protein gene (HspB-C) of Helicobacter pylori. This vector was transformed by agrobactrium tumefaciens EHA105 into four sweet potato varieties. After callus-induction and re-differentiation, we got the transgenic plants from sweet potato variety of Nancy holl. (authors)

Mesothelioma Cells Escape Heat Stress by Upregulating Hsp40/Hsp70 Expression via Mitogen-Activated Protein Kinases

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Michael Roth

2009-01-01

Full Text Available Therapy with hyperthermal chemotherapy in pleural diffuse malignant mesothelioma had limited benefits for patients. Here we investigated the effect of heat stress on heat shock proteins (HSP, which rescue tumour cells from apoptosis. In human mesothelioma and mesothelial cells heat stress (39–42°C induced the phosphorylation of two mitogen activated kinases (MAPK Erk1/2 and p38, and increased Hsp40, and Hsp70 expression. Mesothelioma cells expressed more Hsp40 and were less sensitive to heat stress compared to mesothelial cells. Inhibition of Erk1/2 MAPK by PD98059 or by Erk1 siRNA down-regulated heat stress-induced Hsp40 and Hsp70 expression and reduced mesothelioma cell survival. Inhibition of p38MAPK by SB203580 or siRNA reduced Hsp40, but not Hsp70, expression and also increased mesothelioma cell death. Thus hyperthermia combined with suppression of p38 MAPK or Hsp40 may represent a novel approach to improve mesothelioma therapy.

Targeting Hsp90-Cdc37: A Promising Therapeutic Strategy by Inhibiting Hsp90 Chaperone Function.

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Wang, Lei; Li, Li; Gu, Kai; Xu, Xiao-Li; Sun, Yuan; You, Qi-Dong

2017-01-01

The Hsp90 chaperone protein regulates the folding, maturation and stability of a wide variety of oncoproteins. In recent years, many Hsp90 inhibitors have entered into the clinical trials while all of them target ATPase showing similar binding capacity and kinds of side-effects so that none have reached to the market. During the regulation progress, numerous protein- protein interactions (PPI) such as Hsp90 and client proteins or cochaperones are involved. With the Hsp90-cochaperones PPI networks being more and more clear, many cancerous proteins have been reported to be tightly correlated to Hsp90-cochaperones PPI. Among them, Hsp90-Cdc37 PPI has been widely reported to associate with numerous protein kinases, making it a novel target for the treatment of cancers. In this paper, we briefly review the strategies and modulators targeting Hsp90-Cdc37 complex including direct and indirect regulation mechanism. Through these discussions we expect to present inspirations for new insights into an alternative way to inhibit Hsp90 chaperone function. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Intracellular dynamics of the Hsp90 co-chaperone p23 is dictated by Hsp90

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Picard, Didier

2006-01-01

p23 is a component of the Hsp90 molecular chaperone machine. It binds and stabilizes the ATP-bound dimeric form of Hsp90. Since Hsp90 binds protein substrates in the ATP conformation, p23 has been proposed to stabilize Hsp90-substrate complexes. In addition, p23 can also function as a molecular chaperone by itself and even possesses an unrelated enzymatic activity. Whether it fulfills the latter functions in cells while bound to Hsp90 remains unknown and is difficult to extrapolate from cell-free biochemical experiments. Using the 'fluorescence recovery after photobleaching' (FRAP) technology, I have examined the dynamics of human p23, expressed as a fusion protein with the green fluorescent protein (GFP), in living human HeLa cells. GFP-p23 is distributed throughout the cell, and its mobility is identical in the cytoplasm and in the nucleus. When the Hsp90 interaction is disrupted either with the Hsp90 inhibitor geldanamycin or by introduction of point mutations into p23, the mobility of p23 is greatly accelerated. Under these conditions, its intracellular movement may be diffusion-controlled. In contrast, when wild-type p23 is able to bind Hsp90, a more complex FRAP behavior is observed, suggesting that it is quantitatively bound in Hsp90 complexes undergoing a multitude of other interactions

The molecular chaperone Hsp90 is required for cell cycle exit in Drosophila melanogaster.

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Jennifer L Bandura

Full Text Available The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. However, the full complement of mechanisms necessary to restrain E2F/DP and Cyclin/Cdk activities in differentiating cells are not known. Here, we have performed a genetic screen in Drosophila melanogaster, designed to identify genes required for cell cycle exit. This screen utilized a PCNA-miniwhite(+ reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C, suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, we propose that Cdh1/Fzr is a client protein of Hsp83. Our results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis.

The molecular chaperone Hsp90 is required for cell cycle exit in Drosophila melanogaster.

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Bandura, Jennifer L; Jiang, Huaqi; Nickerson, Derek W; Edgar, Bruce A

2013-01-01

The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. However, the full complement of mechanisms necessary to restrain E2F/DP and Cyclin/Cdk activities in differentiating cells are not known. Here, we have performed a genetic screen in Drosophila melanogaster, designed to identify genes required for cell cycle exit. This screen utilized a PCNA-miniwhite(+) reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C), suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, we propose that Cdh1/Fzr is a client protein of Hsp83. Our results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis.

Synergistic role of HSP90α and HSP90β to promote myofibroblast persistence in lung fibrosis.

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Bellaye, Pierre-Simon; Shimbori, Chiko; Yanagihara, Toyoshi; Carlson, David A; Hughes, Philip; Upagupta, Chandak; Sato, Seidai; Wheildon, Nolan; Haystead, Timothy; Ask, Kjetil; Kolb, Martin

2018-02-01

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of the lung parenchyma, causing significant morbidity through worsening dyspnoea and overall functional decline. IPF is characterised by apoptosis-resistant myofibroblasts, which are a major source for the excessive production of extracellular matrix (ECM) overtaking normal lung tissue. We sought to study the role of heat shock protein (HSP) isoforms HSP90α and HSP90β, whose distinct roles in lung fibrogenesis remain elusive.We determined the level of circulating HSP90α in IPF patients (n=31) and age-matched healthy controls (n=9) by ELISA. The release of HSP90α and HSP90β was evaluated in vitro in primary IPF and control lung fibroblasts and ex vivo after mechanical stretch on fibrotic lung slices from rats receiving adenovector-mediated transforming growth factor-β1.We demonstrate that circulating HSP90α is upregulated in IPF patients in correlation with disease severity. The release of HSP90α is enhanced by the increase in mechanical stress of the fibrotic ECM. This increase in extracellular HSP90α signals through low-density lipoprotein receptor-related protein 1 (LRP1) to promote myofibroblast differentiation and persistence. In parallel, we demonstrate that the intracellular form of HSP90β stabilises LRP1, thus amplifying HSP90α extracellular action.We believe that the specific inhibition of extracellular HSP90α is a promising therapeutic strategy to reduce pro-fibrotic signalling in IPF. Copyright ©ERS 2018.

A 65‑gene signature for prognostic prediction in colon adenocarcinoma.

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Jiang, Hui; Du, Jun; Gu, Jiming; Jin, Liugen; Pu, Yong; Fei, Bojian

2018-04-01

The aim of the present study was to examine the molecular factors associated with the prognosis of colon cancer. Gene expression datasets were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus databases to screen differentially expressed genes (DEGs) between colon cancer samples and normal samples. Survival‑related genes were selected from the DEGs using the Cox regression method. A co‑expression network of survival‑related genes was then constructed, and functional clusters were extracted from this network. The significantly enriched functions and pathways of the genes in the network were identified. Using Bayesian discriminant analysis, a prognostic prediction system was established to distinguish the positive from negative prognostic samples. The discrimination efficacy of the system was validated in the GSE17538 dataset using Kaplan‑Meier survival analysis. A total of 636 and 1,892 DEGs between the colon cancer samples and normal samples were screened from the TCGA and GSE44861 dataset, respectively. There were 155 survival‑related genes selected. The co‑expression network of survival‑related genes included 138 genes, 534 lines (connections) and five functional clusters, including the signaling pathway, cellular response to cAMP, and immune system process functional clusters. The molecular function, cellular components and biological processes were the significantly enriched functions. The peroxisome proliferator‑activated receptor signaling pathway, Wnt signaling pathway, B cell receptor signaling pathway, and cytokine‑cytokine receptor interactions were the significant pathways. A prognostic prediction system based on a 65‑gene signature was established using this co‑expression network. Its discriminatory effect was validated in the TCGA dataset (P=3.56e‑12) and the GSE17538 dataset (P=1.67e‑6). The 65‑gene signature included kallikrein‑related peptidase 6 (KLK6), collagen type XI α1 (COL11A1), cartilage

The Malarial Exported PFA0660w Is an Hsp40 Co-Chaperone of PfHsp70-x.

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Michael O Daniyan

Full Text Available Plasmodium falciparum, the human pathogen responsible for the most dangerous malaria infection, survives and develops in mature erythrocytes through the export of proteins needed for remodelling of the host cell. Molecular chaperones of the heat shock protein (Hsp family are prominent members of the exportome, including a number of Hsp40s and a Hsp70. PFA0660w, a type II Hsp40, has been shown to be exported and possibly form a complex with PfHsp70-x in the infected erythrocyte cytosol. However, the chaperone properties of PFA0660w and its interaction with human and parasite Hsp70s are yet to be investigated. Recombinant PFA0660w was found to exist as a monomer in solution, and was able to significantly stimulate the ATPase activity of PfHsp70-x but not that of a second plasmodial Hsp70 (PfHsp70-1 or a human Hsp70 (HSPA1A, indicating a potential specific functional partnership with PfHsp70-x. Protein binding studies in the presence and absence of ATP suggested that the interaction of PFA0660w with PfHsp70-x most likely represented a co-chaperone/chaperone interaction. Also, PFA0660w alone produced a concentration-dependent suppression of rhodanese aggregation, demonstrating its chaperone properties. Overall, we have provided the first biochemical evidence for the possible role of PFA0660w as a chaperone and as co-chaperone of PfHsp70-x. We propose that these chaperones boost the chaperone power of the infected erythrocyte, enabling successful protein trafficking and folding, and thereby making a fundamental contribution to the pathology of malaria.

Geranylgeranylacetone ameliorates lung ischemia/reperfusion injury by HSP70 and thioredoxin redox system: NF-kB pathway involved.

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Cao, Weijun; Li, Manhui; Li, Jianxiong; Li, Chengwei; Xu, Xin; Gu, Weiqing

2015-06-01

Geranylgeranylacetone (GGA) has been clinically used as an anti-ulcer drug. In the present study, we explored the protective effects of GGA on lung ischemia/reperfusion injury (IRI) and the underlying mechanism. The results demonstrated that GGA ameliorated the lung biochemical and histological alterations induced by IRI, which was reversed by HSP70 inhibition. To further explore the mechanism of GGA action, we focused on NF-kB and thioredoxin (Trx) redox system. It was shown that GGA induced the HSP70 and Trx-1 expression, NF-kB nuclear translocation and activated thioredoxin reductase (TrxR). The Trx-1 expression and TrxR activity was suppressed by HSP70 and NF-kB inhibition, while the nuclear NF-kB p65 expression was suppressed by HSP70 inhibitor. These results indicated that GGA may protect rat lung against IRI by HSP70 and Trx redox system, in which NF-kB pathway may be involved. Copyright © 2015 Elsevier Ltd. All rights reserved.

Differences in the curing of [PSI+] prion by various methods of Hsp104 inactivation.

Directory of Open Access Journals (Sweden)

Yang-Nim Park

Full Text Available [PSI(+] yeast, containing the misfolded amyloid conformation of Sup35 prion, is cured by inactivation of Hsp104. There has been controversy as to whether inactivation of Hsp104 by guanidine treatment or by overexpression of the dominant negative Hsp104 mutant, Hsp104-2KT, cures [PSI(+] by the same mechanism- inhibition of the severing of the prion seeds. Using live cell imaging of Sup35-GFP, overexpression of Hsp104-2KT caused the foci to increase in size, then decrease in number, and finally disappear when the cells were cured, similar to that observed in cells cured by depletion of Hsp104. In contrast, guanidine initially caused an increase in foci size but then the foci disappeared before the cells were cured. By starving the yeast to make the foci visible in cells grown with guanidine, the number of cells with foci was found to correlate exactly with the number of [PSI(+] cells, regardless of the curing method. Therefore, the fluorescent foci are the prion seeds required for maintenance of [PSI(+] and inactivation of Hsp104 cures [PSI(+] by preventing severing of the prion seeds. During curing with guanidine, the reduction in seed size is an Hsp104-dependent effect that cannot be explained by limited severing of the seeds. Instead, in the presence of guanidine, Hsp104 retains an activity that trims or reduces the size of the prion seeds by releasing Sup35 molecules that are unable to form new prion seeds. This Hsp104 activity may also occur in propagating yeast.

Expression profiling of Plasmodium berghei HSP70 genes for generation of bright red fluorescent parasites.

Directory of Open Access Journals (Sweden)

Marion Hliscs

Full Text Available Live cell imaging of recombinant malarial parasites encoding fluorescent probes provides critical insights into parasite-host interactions and life cycle progression. In this study, we generated a red fluorescent line of the murine malarial parasite Plasmodium berghei. To allow constitutive and abundant expression of the mCherry protein we profiled expression of all members of the P. berghei heat shock protein 70 (HSP70 family. We identified PbHSP70/1, an invariant ortholog of Plasmodium falciparum HSP70-1, as the protein with the highest expression levels during Plasmodium blood, mosquito, and liver infection. Stable allelic insertion of a mCherry expression cassette into the PbHsp70/1 locus created constitutive red fluorescent P. berghei lines, termed Pbred. We show that these parasites can be used for live imaging of infected host cells and organs, including hepatocytes, erythrocytes, and whole Anopheles mosquitoes. Quantification of the fluorescence intensity of several Pbred parasite stages revealed significantly enhanced signal intensities in comparison to GFP expressed under the control of the constitutive EF1alpha promoter. We propose that systematic transcript profiling permits generation of reporter parasites, such as the Pbred lines described herein.

Progranulin Recruits HSP70 to ?-Glucocerebrosidase and Is Therapeutic Against Gaucher Disease

OpenAIRE

Jian, Jinlong; Tian, Qing-Yun; Hettinghouse, Aubryanna; Zhao, Shuai; Liu, Helen; Wei, Jianlu; Grunig, Gabriele; Zhang, Wujuan; Setchell, Kenneth D.R.; Sun, Ying; Overkleeft, Herman S.; Chan, Gerald L.; Liu, Chuan-ju

2016-01-01

Highlights ? PGRN is required for lysosomal appearance of GCase and PGRN deficiency causes GCase/LIMP2 aggregation upon stress ? PGRN directly binds to GCase through a two-site mechanism ? PGRN recruits HSP70 to GCase and prevents GCase aggregation in response to stress ? PGRN derivative Pcgin binds to GCase and HSP70 and is therapeutic against Gaucher disease In this study, we demonstrate that PGRN directly binds to GCase and is required for the lysosomal appearance of GCase. In addition, HS...

A comparative examination of cortisol effects on muscle myostatin and HSP90 gene expression in salmonids.

Science.gov (United States)

Galt, Nicholas J; McCormick, Stephen D; Froehlich, Jacob Michael; Biga, Peggy R

2016-10-01

Cortisol, the primary corticosteroid in teleost fishes, is released in response to stressors to elicit local functions, however little is understood regarding muscle-specific responses to cortisol in these fishes. In mammals, glucocorticoids strongly regulate the muscle growth inhibitor, myostatin, via glucocorticoid response elements (GREs) leading to muscle atrophy. Bioinformatics methods suggest that this regulatory mechanism is conserved among vertebrates, however recent evidence suggests some fishes exhibit divergent regulation. Therefore, the aim of this study was to evaluate the conserved actions of cortisol on myostatin and hsp90 expression to determine if variations in cortisol interactions have emerged in salmonid species. Representative salmonids; Chinook salmon (Oncorhynchus tshawytscha), cutthroat trout (Oncorhynchus clarki), brook trout (Salvelinus fontinalis), and Atlantic salmon (Salmo salar); were injected intraperitoneally with a cortisol implant (50μg/g body weight) and muscle gene expression was quantified after 48h. Plasma glucose and cortisol levels were significantly elevated by cortisol in all species, demonstrating physiological effectiveness of the treatment. HSP90 mRNA levels were elevated by cortisol in brook trout, Chinook salmon, and Atlantic salmon, but were decreased in cutthroat trout. Myostatin mRNA levels were affected in a species, tissue (muscle type), and paralog specific manner. Cortisol treatment increased myostatin expression in brook trout (Salvelinus) and Atlantic salmon (Salmo), but not in Chinook salmon (Oncorhynchus) or cutthroat trout (Oncorhynchus). Interestingly, the VC alone increased myostatin mRNA expression in Chinook and Atlantic salmon, while the addition of cortisol blocked the response. Taken together, these results suggest that cortisol affects muscle-specific gene expression in species-specific manners, with unique Oncorhynchus-specific divergence observed, that are not predictive solely based upon

Altered Cross-Linking of HSP27 by Zerumbone as a Novel Strategy for Overcoming HSP27-Mediated Radioresistance

International Nuclear Information System (INIS)

Choi, Seo-Hyun; Lee, Yoon-Jin; Seo, Woo Duck; Lee, Hae-June; Nam, Joo-Won; Lee, Yoo Jin; Kim, Joon; Seo, Eun-Kyoung; Lee, Yun-Sil

2011-01-01

Purpose: HSP27 or HSP25 negatively regulates apoptosis pathways after radiation or chemotherapeutic agents. Abrogation of HSP27 function may be a candidate target for overcoming radio- and chemoresistance. Methods and Materials: Zerumbone (ZER), a cytotoxic component isolated from Zingiber zerumbet smith. Clonogenic survival assay and flow cytometry after Annexin V staining were performed to determine in vitro sensitization effects of ZER with ionizing radiation. A nude mouse xenografting system was also applied to detect in vivo radiosensitizing effects of ZER. Results: ZER produced cross-linking of HSP27, which was dependent on inhibition of the monomeric form of HSP27. ZER was directly inserted between the disulfide bond in the HSP27 dimer and modified normal HSP27 dimerization. Pretreatment with ZER before radiation inhibited the binding affinity between HSP27 and apoptotic molecules, such as cytochrome c and PKCδ, and induced sensitization in vitro and in an in vivo xenografted nude mouse system. Structural analogs lacking only the carbonyl group in ZER, such as α-humulene (HUM) and 8-hydroxy-humulen (8-OH-HUM), did not affect normal cross-linking of HSP27 and did not induce radiosensitization. Conclusions: We suggest that altered cross-linking of HSP27 by ZER is a good strategy for abolishing HSP27-mediated resistance.

Imaging of Hsp70-positive tumors with cmHsp70.1 antibody-conjugated gold nanoparticles

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Gehrmann MK

2015-09-01

Full Text Available Mathias K Gehrmann,1 Melanie A Kimm,2 Stefan Stangl,1 Thomas E Schmid,1 Peter B Noël,2 Ernst J Rummeny,2 Gabriele Multhoff11Department of Radiation Oncology, 2Department of Diagnostic and Interventional Radiology, Klinikum rechts der Isar, Technische Universität München, Munich, GermanyAbstract: Real-time imaging of small tumors is still one of the challenges in cancer diagnosis, prognosis, and monitoring of clinical outcome. Targeting novel biomarkers that are selectively expressed on a large variety of different tumors but not normal cells has the potential to improve the imaging capacity of existing methods such as computed tomography. Herein, we present a novel technique using cmHsp70.1 monoclonal antibody-conjugated spherical gold nanoparticles for quantification of the targeted uptake of gold nanoparticles into membrane Hsp70-positive tumor cells. Upon binding, cmHsp70.1-conjugated gold nanoparticles but not nanoparticles coupled to an isotype-matched IgG1 antibody or empty nanoparticles are rapidly taken up by highly malignant Hsp70 membrane-positive mouse tumor cells. After 24 hours, the cmHsp70.1-conjugated gold nanoparticles are found to be enriched in the perinuclear region. Specificity for membrane Hsp70 was shown by using an Hsp70 knockout tumor cell system. Toxic side effects of the cmHsp70.1-conjugated nanoparticles are not observed at a concentration of 1–10 µg/mL. Experiments are ongoing to evaluate whether cmHsp70.1 antibody-conjugated gold nanoparticles are suitable for the detection of membrane-Hsp70-positive tumors in vivo.Keywords: heat shock protein 70, tumor biomarker, theranostics, multimodal CT, multispectral CT, k-edge

Detection of irradiation-induced, membrane heat shock protein 70 (Hsp70) in mouse tumors using Hsp70 Fab fragment

International Nuclear Information System (INIS)

Stangl, Stefan; Themelis, George; Friedrich, Lars; Ntziachristos, Vasilis; Sarantopoulos, Athanasios; Molls, Michael; Skerra, Arne; Multhoff, Gabriele

2011-01-01

Background and purpose: The major stress-inducible heat shock protein 70 (Hsp70) is frequently overexpressed in highly aggressive tumors, and elevated intracellular Hsp70 levels mediate protection against apoptosis. Following therapeutic intervention, such as ionizing irradiation, translocation of cytosolic Hsp70 to the plasma membrane is selectively increased in tumor cells and therefore, membrane Hsp70 might serve as a therapy-inducible, tumor-specific target structure. Materials and methods: Based on the IgG1 mouse monoclonal antibody (mAb) cmHsp70.1, we produced the Hsp70-specific recombinant Fab fragment (Hsp70 Fab), as an imaging tool for the detection of membrane Hsp70 positive tumor cells in vitro and in vivo. Results: The binding characteristics of Hsp70 Fab towards mouse colon (CT26) and pancreatic (1048) carcinoma cells at 4 deg. C were comparable to that of cmHsp70.1 mAb, as determined by flow cytometry. Following a temperature shift to 37 deg. C, Hsp70 Fab rapidly translocates into subcellular vesicles of mouse tumor cells. Furthermore, in tumor-bearing mice Cy5.5-conjugated Hsp70 Fab, but not unrelated IN-1 control Fab fragment (IN-1 ctrl Fab), gradually accumulates in CT26 tumors between 12 and 55 h after i.v. injection. Conclusions: In summary, the Hsp70 Fab provides an innovative, low immunogenic tool for imaging of membrane Hsp70 positive tumors, in vivo.

Hsp105 family proteins suppress staurosporine-induced apoptosis by inhibiting the translocation of Bax to mitochondria in HeLa cells

International Nuclear Information System (INIS)

Yamagishi, Nobuyuki; Ishihara, Keiichi; Saito, Youhei; Hatayama, Takumi

2006-01-01

Hsp105 (Hsp105α and Hsp105β), major heat shock proteins in mammalian cells, belong to a subgroup of the HSP70 family, HSP105/110. Previously, we have shown that Hsp105α has completely different effects on stress-induced apoptosis depending on cell type. However, the molecular mechanisms by which Hsp105α regulates stress-induced apoptosis are not fully understood. Here, we established HeLa cells that overexpress either Hsp105α or Hsp105β by removing doxycycline and examined how Hsp105 modifies staurosporine (STS)-induced apoptosis in HeLa cells. Apoptotic features such as the externalization of phosphatidylserine on the plasma membrane and nuclear morphological changes were induced by the treatment with STS, and the STS-induced apoptosis was suppressed by overexpression of Hsp105α or Hsp105β. In addition, we found that overexpression of Hsp105α or Hsp105β suppressed the activation of caspase-3 and caspase-9 by preventing the release of cytochrome c from mitochondria. Furthermore, the translocation of Bax to mitochondria, which results in the release of cytochrome c from the mitochondria, was also suppressed by the overexpression of Hsp105α or Hsp105β. Thus, it is suggested that Hsp105 suppresses the stress-induced apoptosis at its initial step, the translocation of Bax to mitochondria in HeLa cells

In vivo effects of UV radiation on multiple endpoints and expression profiles of DNA repair and heat shock protein (Hsp) genes in the cycloid copepod Paracyclopina nana

International Nuclear Information System (INIS)

Won, Eun-Ji; Han, Jeonghoon; Lee, Yeonjung; Kumar, K. Suresh; Shin, Kyung-Hoon; Lee, Su-Jae; Park, Heum Gi; Lee, Jae-Seong

2015-01-01

Highlights: • UV-B radiation induced a significant reduction of the re-brooding rate of ovigerous females. • A dose-dependent decrease in food ingestion and the rate of assimilation to the body upon UV radiation. • Expression of base excision repair-associated and hsp chaperoning genes was significantly increased upon UV radiation in P. nana. - Abstract: To evaluate the effects of ultraviolet (UV) radiation on energy acquisition and consumption, the copepod Paracyclopina nana was irradiated with several doses (0–3 kJ/m 2 ) of UV. After UV radiation, we measured the re-brooding success, growth pattern of newly hatched nauplii, ingestion rate, and assimilation of diet. In addition, we checked the modulated patterns of DNA repair and heat shock protein (hsp) chaperoning genes of P. nana. UV-B radiation induced a significant reduction (7–87%) of the re-brooding rate of ovigerous females, indicating that UV-induced egg sac damage is closely correlated with a reduction in the hatching rate of UV-irradiated ovigerous female offspring. Using chlorophyll a and stable carbon isotope incubation experiments, we found a dose-dependent decrease (P < 0.05) in food ingestion and the rate of assimilation to the body in response to UV radiation, implying that P. nana has an underlying ability to shift its balanced-energy status from growth and reproduction to DNA repair and adaptation. Also, expression of P. nana base excision repair (BER)-associated genes and hsp chaperoning genes was significantly increased in response to UV radiation in P. nana. These findings indicate that even 1 kJ/m 2 of UV radiation induces a reduction in reproduction and growth patterns, alters the physiological balance and inhibits the ability to cope with UV-induced damage in P. nana

In vivo effects of UV radiation on multiple endpoints and expression profiles of DNA repair and heat shock protein (Hsp) genes in the cycloid copepod Paracyclopina nana

Energy Technology Data Exchange (ETDEWEB)

Won, Eun-Ji; Han, Jeonghoon [Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Lee, Yeonjung; Kumar, K. Suresh; Shin, Kyung-Hoon [Department of Marine Sciences and Convergent Technology, College of Science and Technology, Hanyang University, Ansan 426-791 (Korea, Republic of); Lee, Su-Jae [Department of Life Sciences, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Park, Heum Gi, E-mail: hgpark@gwnu.ac.kr [Department of Marine Resource Development, College of Life Sciences, Gangneung-Wonju National University, Gangneung 210-702 (Korea, Republic of); Lee, Jae-Seong, E-mail: jslee2@skku.edu [Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of)

2015-08-15

Highlights: • UV-B radiation induced a significant reduction of the re-brooding rate of ovigerous females. • A dose-dependent decrease in food ingestion and the rate of assimilation to the body upon UV radiation. • Expression of base excision repair-associated and hsp chaperoning genes was significantly increased upon UV radiation in P. nana. - Abstract: To evaluate the effects of ultraviolet (UV) radiation on energy acquisition and consumption, the copepod Paracyclopina nana was irradiated with several doses (0–3 kJ/m{sup 2}) of UV. After UV radiation, we measured the re-brooding success, growth pattern of newly hatched nauplii, ingestion rate, and assimilation of diet. In addition, we checked the modulated patterns of DNA repair and heat shock protein (hsp) chaperoning genes of P. nana. UV-B radiation induced a significant reduction (7–87%) of the re-brooding rate of ovigerous females, indicating that UV-induced egg sac damage is closely correlated with a reduction in the hatching rate of UV-irradiated ovigerous female offspring. Using chlorophyll a and stable carbon isotope incubation experiments, we found a dose-dependent decrease (P < 0.05) in food ingestion and the rate of assimilation to the body in response to UV radiation, implying that P. nana has an underlying ability to shift its balanced-energy status from growth and reproduction to DNA repair and adaptation. Also, expression of P. nana base excision repair (BER)-associated genes and hsp chaperoning genes was significantly increased in response to UV radiation in P. nana. These findings indicate that even 1 kJ/m{sup 2} of UV radiation induces a reduction in reproduction and growth patterns, alters the physiological balance and inhibits the ability to cope with UV-induced damage in P. nana.

Hsp90 inhibitor 17-AAG inhibits progression of LuCaP35 xenograft prostate tumors to castration resistance.

Science.gov (United States)

O'Malley, Katherine J; Langmann, Gabrielle; Ai, Junkui; Ramos-Garcia, Raquel; Vessella, Robert L; Wang, Zhou

2012-07-01

Advanced prostate cancer is currently treated with androgen deprivation therapy (ADT). ADT initially results in tumor regression; however, all patients eventually relapse with castration-resistant prostate cancer. New approaches to delay the progression of prostate cancer to castration resistance are in desperate need. This study addresses whether targeting Heat shock protein 90 (HSP90) regulation of androgen receptor (AR) can inhibit prostate cancer progression to castration resistance. The HSP90 inhibitor 17-AAG was injected intraperitoneally into nude mice bearing LuCaP35 xenograft tumors to determine the effect of HSP90 inhibition on prostate cancer progression to castration resistance and host survival. Administration of 17-AAG maintained androgen-sensitivity, delayed the progression of LuCaP35 xenograft tumors to castration resistance, and prolonged the survival of host. In addition, 17-AAG prevented nuclear localization of endogenous AR in LuCaP35 xenograft tumors in castrated nude mice. Targeting Hsp90 or the mechanism by which HSP90 regulates androgen-independent AR nuclear localization and activation may lead to new approaches to prevent and/or treat castration-resistant prostate cancer. Copyright © 2011 Wiley Periodicals, Inc.

Identification of the gene encoding the 65-kilodalton DNA-binding protein of herpes simplex virus type 1

International Nuclear Information System (INIS)

Parris, D.S.; Cross, A.; Orr, A.; Frame, M.C.; Murphy, M.; McGeoch, D.J.; Marsden, H.S.; Haarr, L.

1988-01-01

Hybrid arrest of in vitro translation was used to localize the region of the herpes simplex virus type 1 genome encoding the 65-kilodalton DNA-binding protein (65K DBP ) to between genome coordinates 0.592 and 0.649. Knowledge of the DNA sequence of this region allowed us to identify three open reading frames as likely candidates for the gene encoding 65K DBP . Two independent approaches were used to determine which of these three open reading frames encoded the protein. For the first approach a monoclonal antibody, MAb 6898, which reacted specifically with 65K DBP , was isolated. This antibody was used, with the techniques of hybrid arrest of in vitro translation and in vitro translation of selected mRNA, to identify the gene encoding 65K DBP . The second approach involved preparation of antisera directed against oligopeptides corresponding to regions of the predicted amino acid sequence of this gene. These antisera reacted specifically with 65K DBP , thus confirming the gene assignment

AsHSP17, a creeping bentgrass small heat shock protein modulates plant photosynthesis and ABA-dependent and independent signalling to attenuate plant response to abiotic stress.

Science.gov (United States)

Sun, Xinbo; Sun, Chunyu; Li, Zhigang; Hu, Qian; Han, Liebao; Luo, Hong

2016-06-01

Heat shock proteins (HSPs) are molecular chaperones that accumulate in response to heat and other abiotic stressors. Small HSPs (sHSPs) belong to the most ubiquitous HSP subgroup with molecular weights ranging from 12 to 42 kDa. We have cloned a new sHSP gene, AsHSP17 from creeping bentgrass (Agrostis stolonifera) and studied its role in plant response to environmental stress. AsHSP17 encodes a protein of 17 kDa. Its expression was strongly induced by heat in both leaf and root tissues, and by salt and abscisic acid (ABA) in roots. Transgenic Arabidopsis plants constitutively expressing AsHSP17 exhibited enhanced sensitivity to heat and salt stress accompanied by reduced leaf chlorophyll content and decreased photosynthesis under both normal and stressed conditions compared to wild type. Overexpression of AsHSP17 also led to hypersensitivity to exogenous ABA and salinity during germination and post-germinative growth. Gene expression analysis indicated that AsHSP17 modulates expression of photosynthesis-related genes and regulates ABA biosynthesis, metabolism and ABA signalling as well as ABA-independent stress signalling. Our results suggest that AsHSP17 may function as a protein chaperone to negatively regulate plant responses to adverse environmental stresses through modulating photosynthesis and ABA-dependent and independent signalling pathways. © 2015 John Wiley & Sons Ltd.

Hsp27 gene in Drosophila ananassae subgroup was split by a ...

Indian Academy of Sciences (India)

To investigate the molecular evolution of Hsp27 in the .... genetic evolution, as base composition bias is known to dis- tort determination of phylogenetic relationships in simulated datasets ... examined using unbiased tests with CONSEL (Shimodaira .... ACD is still unclear, however, mutations of a conserved argi- nine in the ...

Suppression of cadmium-induced JNK/p38 activation and HSP70 family gene expression by LL-Z1640-2 in NIH3T3 cells

International Nuclear Information System (INIS)

Sugisawa, Nobusuke; Matsuoka, Masato; Okuno, Takeo; Igisu, Hideki

2004-01-01

When NIH3T3 cells were exposed to CdCl 2 , the three major mitogen-activated protein kinases (MAPKs), extracellular signal-regulated protein kinase (ERK), c-Jun NH 2 -terminal kinase (JNK), and p38, were phosphorylated in a time (1-9 h)- and dose (1-20 μM)-dependent manner. Treatment with a macrocyclic nonaketide compound, LL-Z1640-2 (10-100 ng/ml), suppressed the phosphorylation of MAPKs without affecting the total protein level in cells exposed to 10 μM CdCl 2 for 6 h. CdCl 2 -induced phosphorylation of c-Jun on Ser63 and that on Ser73, and resultant accumulation of total c-Jun protein were also suppressed by LL-Z1640-2 treatment. The in vitro kinase assays also showed significant inhibitory effects of LL-Z1640-2 (at 10 or 25 ng/ml) on JNK and p38 but less markedly. In contrast to JNK and p38, ERK activity was inhibited moderately only at 50 or 100 ng/ml LL-Z1640-2. On the other hand, other JNK inhibitors, SP600125 and L-JNKI1, failed to suppress CdCl 2 -induced activation of the JNK pathway. Among the mouse stress response genes upregulated in response to CdCl 2 exposure, the expressions of hsp68 (encoding for heat shock 70 kDa protein 1; Hsp70-1) and grp78 (encoding for 78 kDa glucose-regulated protein; Grp78) genes were suppressed by treatment with 25 ng/ml LL-Z1640-2. Thus, LL-Z1640-2 could suppress CdCl 2 -induced activation of JNK/p38 pathways and expression of HSP70 family genes in NIH3T3 cells. LL-Z1640-2 seems to be useful to analyze functions of toxic metal-induced JNK/p38 activation

C-terminal sequences of hsp70 and hsp90 as non-specific anchors for tetratricopeptide repeat (TPR) proteins.

Science.gov (United States)

Ramsey, Andrew J; Russell, Lance C; Chinkers, Michael

2009-10-12

Steroid-hormone-receptor maturation is a multi-step process that involves several TPR (tetratricopeptide repeat) proteins that bind to the maturation complex via the C-termini of hsp70 (heat-shock protein 70) and hsp90 (heat-shock protein 90). We produced a random T7 peptide library to investigate the roles played by the C-termini of the two heat-shock proteins in the TPR-hsp interactions. Surprisingly, phages with the MEEVD sequence, found at the C-terminus of hsp90, were not recovered from our biopanning experiments. However, two groups of phages were isolated that bound relatively tightly to HsPP5 (Homo sapiens protein phosphatase 5) TPR. Multiple copies of phages with a C-terminal sequence of LFG were isolated. These phages bound specifically to the TPR domain of HsPP5, although mutation studies produced no evidence that they bound to the domain's hsp90-binding groove. However, the most abundant family obtained in the initial screen had an aspartate residue at the C-terminus. Two members of this family with a C-terminal sequence of VD appeared to bind with approximately the same affinity as the hsp90 C-12 control. A second generation pseudo-random phage library produced a large number of phages with an LD C-terminus. These sequences acted as hsp70 analogues and had relatively low affinities for hsp90-specific TPR domains. Unfortunately, we failed to identify residues near hsp90's C-terminus that impart binding specificity to individual hsp90-TPR interactions. The results suggest that the C-terminal sequences of hsp70 and hsp90 act primarily as non-specific anchors for TPR proteins.

Heat shock proteins (Hsp 70) response is not systematic to cell stress

International Nuclear Information System (INIS)

Hassen, Wafa; Ayed-Boussema, Imen; Bouslimi, Amel; Bacha, Hassen

2007-01-01

Ochratoxin A (OTA) is a mycotoxin routinely detected in improperly stored animal and human food supplies as well as in human sera worldwide. OTA has multiple toxic effects; however, the most prominent is nephrotoxicity. Thus, OTA is involved in the pathogenesis of human nephropathy in Balkan areas. In this study, we address the question of the appropriate functioning of the basal cellular defense mechanisms, after exposure to OTA, which, up to now, has not been investigated satisfactorily. In this context, we have monitored the effect of OTA on (i) the inhibition of cell viability, (ii) the oxidative damage using the GSH depletion, (iii) the inhibition of protein synthesis through the incorporation of [ 3 H] Leucine and (iv) the induction of Hsp 70 gene expression as a parameter of cytotoxicity, oxidative damage and particularly as a protective and adaptative response. This study was conducted using the Human Hep G2 hepatocytes and monkey kidney Vero cells under exposure conditions ranging from non-cytotoxic to sub-lethal. Our results clearly showed that OTA inhibits cell proliferation, strongly reduces protein synthesis and induces the decrease of GSH in concentration-dependent manner in both Hep G2 and Vero cells. However, although cytotoxicity and oxidative damage (main inducers of Hsp expression) occur, no change was observed in Hsp 70 level under the multiple tested conditions. Inhibition of protein synthesis could not explain the absence of Hsp 70 response since concentrations, which did not influence protein synthesis, also failed to display the expected Hsp 70 response. Our data are consistent with recently published reports where considerable differences were noticed in the ability of relevant toxicants to induce Hsp. These results raised doubt about the universal character of Hsp induction which seems to be more complex than originally envisioned. It could be concluded that Hsp 70 induction is not systematic to cell stress

Hsp72 preserves muscle function and slows progression of severe muscular dystrophy.

Science.gov (United States)

Gehrig, Stefan M; van der Poel, Chris; Sayer, Timothy A; Schertzer, Jonathan D; Henstridge, Darren C; Church, Jarrod E; Lamon, Severine; Russell, Aaron P; Davies, Kay E; Febbraio, Mark A; Lynch, Gordon S

2012-04-04

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder caused by mutations in the dystrophin gene that result in the absence of the membrane-stabilizing protein dystrophin. Dystrophin-deficient muscle fibres are fragile and susceptible to an influx of Ca(2+), which activates inflammatory and muscle degenerative pathways. At present there is no cure for DMD, and existing therapies are ineffective. Here we show that increasing the expression of intramuscular heat shock protein 72 (Hsp72) preserves muscle strength and ameliorates the dystrophic pathology in two mouse models of muscular dystrophy. Treatment with BGP-15 (a pharmacological inducer of Hsp72 currently in clinical trials for diabetes) improved muscle architecture, strength and contractile function in severely affected diaphragm muscles in mdx dystrophic mice. In dko mice, a phenocopy of DMD that results in severe spinal curvature (kyphosis), muscle weakness and premature death, BGP-15 decreased kyphosis, improved the dystrophic pathophysiology in limb and diaphragm muscles and extended lifespan. We found that the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA, the main protein responsible for the removal of intracellular Ca(2+)) is dysfunctional in severely affected muscles of mdx and dko mice, and that Hsp72 interacts with SERCA to preserve its function under conditions of stress, ultimately contributing to the decreased muscle degeneration seen with Hsp72 upregulation. Treatment with BGP-15 similarly increased SERCA activity in dystrophic skeletal muscles. Our results provide evidence that increasing the expression of Hsp72 in muscle (through the administration of BGP-15) has significant therapeutic potential for DMD and related conditions, either as a self-contained therapy or as an adjuvant with other potential treatments, including gene, cell and pharmacological therapies.

An ELISA using recombinant TmHSP70 for the diagnosis of Taenia multiceps infections in goats.

Science.gov (United States)

Wang, Yu; Nie, Huaming; Gu, Xiaobin; Wang, Tao; Huang, Xing; Chen, Lin; Lai, Weimin; Peng, Xuerong; Yang, Guangyou

2015-09-15

Infections with the tapeworm Taenia multiceps are problematic for ruminant farming worldwide. Here we develop a novel and rapid method for serodiagnosis of T. multiceps infections via an indirect ELISA (iELISA) that uses a heat shock protein, namely, TmHSP70. We extracted the total RNA of T. multiceps from the protoscoleces of cysts dissected from the brains of infected goats. Subsequently, we successfully amplified, cloned and expressed the TmHSP70 gene in Escherichia coli BL21 (DE3). Western blot analysis showed that the recombinant protein (∼34 kDa molecular weight) was recognized by the coenurosis positive serum. Given these initial, robust immunogenic properties for recombinant TmHSP protein, we assessed the ELISA-based serodiagnostic potential of this gene. The indirect ELISA was then optimized to 2.70 μg/well dilution for antigen and 1:80 dilution for serum,while the cut-off value is 0.446. We report that our novel TmHSP ELISA detected T. multiceps sera with a sensitivity of 1:10240 and a specificity of 83.3% (5/6). In a preliminary application, this assay correctly confirmed T. multiceps infection in 30 infected goats, consistent with the clinical examination. This study has revealed that our novel iELISA, which uses the rTmHSP protein, provides a rapid test for diagnosing coenurosis. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparative baseline levels of mercury, Hsp 70 and Hsp 60 in subsistence fish from the Yukon-Kuskokwim delta region of Alaska.

Science.gov (United States)

Duffy, L K; Scofield, E; Rodgers, T; Patton, M; Bowyer, R T

1999-10-01

In subsistence fish; northern pike (Esox lucius), burbot (Lota lota), whitefish (Coregonus nelsoni), grayling (Thymallus arcticus) and sheefish (Stenodus lencichthys), we determined the Hsp 60 and Hsp 70 levels in 31 samples from adult fish gills. A dot-blot analysis using antibodies to either Hsp 70 or Hsp 60 showed the average Hsp 70 concentration was 9.1 microg/mg protein, while the average Hsp 60 concentration was 147.4 microg/mg protein. Mercury levels in muscle tissue in these fish averaged 0.382 ppm. Using a subset of samples (n = 24), we determined that the major component in the muscle of Alaskan subsistence fish was methyl mercury. No correlation was observed between Hsp 60 or Hsp 70 expression in gill tissue and mercury concentrations in muscle tissue. Hsp 60 and Hsp 70 protein levels in the gills were correlated.

Energy sources and levels influenced on performance parameters, thyroid hormones, and HSP70 gene expression of broiler chickens under heat stress.

Science.gov (United States)

Raghebian, Majid; Sadeghi, Ali Asghar; Aminafshar, Mehdi

2016-12-01

The present study was conducted to evaluate the effects of energy sources and levels on body and organs weights, thyroid hormones, and heat shock protein (HSP70) gene expression in broilers under heat stress. In a completely randomized design, 600 1-day-old Cobb chickens were assigned to five dietary treatments and four replicates. The chickens were fed diet based on corn as main energy source and energy level based on Cobb standard considered as control (C), corn-based diet with 3 % lesser energy than the control (T1), corn-based diet with 6 % lesser energy than the control (T2), corn and soybean oil-based diet according to Cobb standard (T3), and corn and soybean oil-based diet with 3 % upper energy than the control (T4). Temperature was increased to 34 °C for 8 h daily from days 12 to 41 of age to induce heat stress. The chickens in T1 and T2 had lower thyroid hormones and corticosterone levels than those in C, T3, and T4. The highest liver weight was for C and the lowest one was for T4. The highest gene expression was found in chickens fed T4 diet, and the lowest gene expression was for those in T2 group. The highest feed intake and worse feed conversion ratio was related to chickens in T2. The chickens in T3 and T4 had higher feed intake and weight gain than those in C. The results showed that the higher energy level supplied from soybean oil could enhance gene expression of HSP70 and decline the level of corticosterone and thyroid hormones and consequently improved performance.

Genetic variation and recombination of RdRp and HSP 70h genes of Citrus tristeza virus isolates from orange trees showing symptoms of citrus sudden death disease

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Pappas Georgios J

2008-01-01

Full Text Available Abstract Background Citrus sudden death (CSD, a disease that rapidly kills orange trees, is an emerging threat to the Brazilian citrus industry. Although the causal agent of CSD has not been definitively determined, based on the disease's distribution and symptomatology it is suspected that the agent may be a new strain of Citrus tristeza virus (CTV. CTV genetic variation was therefore assessed in two Brazilian orange trees displaying CSD symptoms and a third with more conventional CTV symptoms. Results A total of 286 RNA-dependent-RNA polymerase (RdRp and 284 heat shock protein 70 homolog (HSP70h gene fragments were determined for CTV variants infecting the three trees. It was discovered that, despite differences in symptomatology, the trees were all apparently coinfected with similar populations of divergent CTV variants. While mixed CTV infections are common, the genetic distance between the most divergent population members observed (24.1% for RdRp and 11.0% for HSP70h was far greater than that in previously described mixed infections. Recombinants of five distinct RdRp lineages and three distinct HSP70h lineages were easily detectable but respectively accounted for only 5.9 and 11.9% of the RdRp and HSP70h gene fragments analysed and there was no evidence of an association between particular recombinant mosaics and CSD. Also, comparisons of CTV population structures indicated that the two most similar CTV populations were those of one of the trees with CSD and the tree without CSD. Conclusion We suggest that if CTV is the causal agent of CSD, it is most likely a subtle feature of population structures within mixed infections and not merely the presence (or absence of a single CTV variant within these populations that triggers the disease.

Requirement of Hsp105 in CoCl{sub 2}-induced HIF-1α accumulation and transcriptional activation

Energy Technology Data Exchange (ETDEWEB)

Mikami, Hiroki; Saito, Youhei, E-mail: ysaito@mb.kyoto-phu.ac.jp; Okamoto, Namiko; Kakihana, Ayana; Kuga, Takahisa; Nakayama, Yuji, E-mail: nakayama@mb.kyoto-phu.ac.jp

2017-03-15

The mammalian stress protein Hsp105α protects cells from stress conditions. Several studies have indicated that Hsp105α is overexpressed in many types of solid tumors, which contain hypoxic microenvironments. However, the role of Hsp105α in hypoxic tumors remains largely unknown. We herein demonstrated the involvement of Hsp105α in HIF-1 functions induced by the hypoxia-mimetic agent CoCl{sub 2}. While Hsp105α is mainly localized in the cytoplasm under normal conditions, a treatment with CoCl{sub 2} induces the nuclear localization of Hsp105α, which correlated with HIF-1α expression levels. The overexpression of degradation-resistant HIF-1α enhances the nuclear localization of Hsp105α without the CoCl{sub 2} treatment. The CoCl{sub 2}-dependent transcriptional activation of HIF-1, which is measured using a reporter gene containing a HIF-responsive element, is reduced by the knockdown of Hsp105α. Furthermore, the CoCl{sub 2}-induced accumulation of HIF-1α is enhanced by heat shock, which results in the nuclear localization of Hsp105, and is suppressed by the knockdown of Hsp105. Hsp105 associates with HIF-1α in CoCl{sub 2}-treated cells. These results suggest that Hsp105α plays an important role in the functions of HIF-1 under hypoxic conditions, in which Hsp105α enhances the accumulation and transcriptional activity of HIF-1 through the HIF-1α-mediated nuclear localization of Hsp105α. - Highlights: • Hsp105α is required for the CoCl{sub 2}-induced transcriptional activation and accumulation of HIF-1. • Hsp105α localizes to the nucleus and interacts with HIF-1α in CoCl{sub 2}-treated cells. • Hsp105 enhances the CoCl{sub 2}-induced accumulation of HIF-1α under heat shock conditions.

Heat shock protein (Hsp) 40 mutants inhibit Hsp70 in mammalian cells

NARCIS (Netherlands)

Michels, AA; Kanon, B; Bensaude, O; Kampinga, HH

1999-01-01

Heat shock protein (Hsp) 70 and Hsp40 expressed in mammalian cells had been previously shown to cooperate in accelerating the reactivation of heat-denatured firefly luciferase (Michels, A. A., Kanon, B., Konings, A. W. T., Ohtsuka, K,, Bensaude, O., and Kampinga, H. H. (1997) J. Biol. Chem. 272,

Deletion in HSP110 T17: correlation with wild-type HSP110 expression and prognostic significance in microsatellite-unstable advanced gastric cancers.

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Kim, Kyung-Ju; Lee, Tae Hun; Kim, Jung Ho; Cho, Nam-Yun; Kim, Woo Ho; Kang, Gyeong Hoon

2017-09-01

Deletion of the HSP110 T 17 mononucleotide repeat has recently been identified as a prognostic marker that is correlated with wild-type HSP110 (HSP110wt) expression in microsatellite instability-high (MSI-H) colorectal cancers. The aim of this study was to assess the correlation between deletion of the HSP110 T 17 repeat and expression of HSP110wt using DNA testing and immunohistochemistry and to determine the prognostic implications of HSP110 T 17 deletion in MSI-H advanced gastric cancers (GCs). The status of HSP110wt expression was evaluated by immunohistochemistry using an HSP110wt-specific antibody in 142 MSI-H advanced GCs. The size of the HSP110 T 17 repeat deletion was analyzed in 96 MSI-H advanced GCs; deletions were divided into small (0-2base pairs) and large deletions (3-5base pairs). Low and high expressions of HSP110wt were detected in 38 (26.8%) and 104 (73.2%) of the 142 cases, respectively. The HSP110 T 17 deletion was observed in 45 (46.9%) of the 96 MSI-H GC samples. Tumors with high expression of HSP110wt showed a tendency to have small or no deletion of HSP110 T 17 . In Kaplan-Meier survival analysis, tumors with a large HSP110 T 17 deletion were associated with favorable overall survival and disease-free survival compared with those with small/no deletion of HSP110 T 17 . However, HSP110 T 17 deletion size was not an independent prognostic factor in multivariate analysis. In summary, deletion of the HSP110 T 17 repeat was frequently observed in MSI-H GCs, and HSP110 T 17 deletion size was inversely correlated with HSP110wt expression status. Large HSP110 T 17 was not a prognostic indicator in MSI-H GCs. Copyright © 2017 Elsevier Inc. All rights reserved.

Jasmonate signalling in Arabidopsis involves SGT1b-HSP70-HSP90 chaperone complexes.

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Zhang, Xue-Cheng; Millet, Yves A; Cheng, Zhenyu; Bush, Jenifer; Ausubel, Frederick M

Plant hormones play pivotal roles in growth, development and stress responses. Although it is essential to our understanding of hormone signalling, how plants maintain a steady state level of hormone receptors is poorly understood. We show that mutation of the Arabidopsis thaliana co-chaperone SGT1b impairs responses to the plant hormones jasmonate, auxin and gibberellic acid, but not brassinolide and abscisic acid, and that SGT1b and its homologue SGT1a are involved in maintaining the steady state levels of the F-box proteins COI1 and TIR1, receptors for jasmonate and auxin, respectively. The association of SGT1b with COI1 is direct and is independent of the Arabidopsis SKP1 protein, ASK1. We further show that COI1 is a client protein of SGT1b-HSP70-HSP90 chaperone complexes and that the complexes function in hormone signalling by stabilizing the COI1 protein. This study extends the SGT1b-HSP90 client protein list and broadens the functional scope of SGT1b-HSP70-HSP90 chaperone complexes.

Molecular identification of clinical Nocardia isolates from India.

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Rudramurthy, Shivaprakash M; Honnavar, Prasanna; Kaur, Harsimran; Samanta, Palash; Ray, Pallab; Ghosh, Anup; Chakrabarti, Arunaloke

2015-10-01

The epidemiology of nocardiosis is evolving with increasing number of Nocardia spp. causing human infection. In recent years, molecular techniques have been used to identify Nocardia spp. There are limited data available on the spectrum of Nocardia spp. isolated from clinical samples in India. Here, a molecular study was carried on 30 clinical isolates maintained in our National Culture Collection to evaluate the techniques used for identifying the agents. The isolates were identified by sequencing two promising genes: the 16S rRNA gene and hsp65. Both hsp65 and the 16S rRNA gene could reliably identify 90 % of Nocardia isolates, i.e. N. farcinica, N. cyriacigeorgica, N. brasiliensis, N. otitidiscaviarum, N. amamiensis and N. pneumoniae. The mean percentage dissimilarity of sequence identification was higher using the hsp65 gene (4 %, range 0-7.9 %) compared with the 16S rRNA gene (2.3 %, range 0-8.9 %). Two isolates that showed ambiguous results in both the short segment of the 16S rRNA gene and hsp65 sequences could be resolved by sequencing a larger fragment (∼1000 bp) of the 16S rRNA gene. Both of these isolates were identified as N. beijingensis with similarities of 99.8 and 100 % compared with the standard strain. Genotyping of N. cyriacigeorgica strains was performed using hsp65 gene sequences and compared with previously described genotypes. Our N. cyriacigeorgica isolates belonged to genotype 1 (n = 4) and genotype 2 (n = 2). The present study highlights a wide spectrum of Nocardia spp. in India and emphasizes the need for molecular techniques for identification to the species level.

Sequence features and phylogenetic analysis of the stress protein Hsp90α in chinook salmon Oncorhynchus tshawytscha, a poikilothermic vertebrate

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Palmisano, Aldo N.; Winton, James R.; Dickhoff, Walton W.

1999-01-01

We cloned and sequenced a chinook salmon Hsp90 cDNA; sequence analysis shows it to be Hsp90??. Phylogenetic analysis supports the hypothesis that ?? and ?? paralogs of Hsp90 arose as a result of a gene duplication event and that they diverged early in the evolution of vertebrates, before tetrapods separated from the teleost lineage. Among several differences distinguishing poikilothermic Hsp90?? sequences from their bird and mammal orthologs, the teleost versions specifically lack a characteristic QTQDQP phosphorylation site near the N-terminus. We used the cDNA to develop an RNA (Northern) blot to quantify cellular Hsp90 mRNA levels. Chinook salmon embryonic (CHSE-214) cells responded to heat shock with a rapid rise in Hsp90 mRNA through 4 h, followed by a gradual decline over the next 20 h. Hsp90 mRNA level may be useful as a stress indicator, especially in a laboratory setting or in response to acute heat stress.

The Hsp90 co-chaperones Sti1, Aha1, and P23 regulate adaptive responses to antifungal azoles

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Xiaokui Gu

2016-10-01

Full Text Available Heat Shock Protein 90 (Hsp90 is essential for tumor progression in humans and drug resistance in fungi. However, the roles of its many co-chaperones in antifungal resistance are unknown. In this study, by susceptibility test of Neurospora crassa mutants lacking each of 18 Hsp90/Calcineurin system member genes (including 8 Hsp90 co-chaperone genes to antifungal drugs and other stresses, we demonstrate that the Hsp90 co-chaperones Sti1 (Hop1 in yeast, Aha1, and P23 (Sba1 in yeast were required for the basal resistance to antifungal azoles and heat stress. Deletion of any of them resulted in hypersensitivity to azoles and heat. Liquid chromatography–mass spectrometry (LC-MS analysis showed that the toxic sterols eburicol and 14α-methyl-3,6-diol were significantly accumulated in the sti1 and p23 deletion mutants after ketoconazole treatment, which has been shown before to led to cell membrane stress. At the transcriptional level, Aha1, Sti1, and P23 positively regulate responses to ketoconazole stress by erg11 and erg6, key genes in the ergosterol biosynthetic pathway. Aha1, Sti1, and P23 are highly conserved in fungi, and sti1 and p23 deletion also increased the susceptibility to azoles in Fusarium verticillioides. These results indicate that Hsp90-cochaperones Aha1, Sti1, and P23 are critical for the basal azole resistance and could be potential targets for developing new antifungal agents.

Active participation of Hsp90 in the biogenesis of the trimeric reovirus cell attachment protein sigma1.

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Gilmore, R; Coffey, M C; Lee, P W

1998-06-12

The reovirus cell attachment protein, sigma1, is a lollipop-shaped homotrimer with an N-terminal fibrous tail and a C-terminal globular head. Biogenesis of this protein involves two trimerization events: N-terminal trimerization, which occurs cotranslationally and is Hsp70/ATP-independent, and C-terminal trimerization, which occurs posttranslationally and is Hsp70/ATP-dependent. To determine if Hsp90 also plays a role in sigma1 biogenesis, we analyzed sigma1 synthesized in rabbit reticulocyte lysate. Coprecipitation experiments using anti-Hsp90 antibodies revealed that Hsp90 was associated with immature sigma1 trimers (hydra-like intermediates with assembled N termini and unassembled C termini) but not with mature trimers. The use of truncated sigma1 further demonstrated that only the C-terminal half of sigma1 associated with Hsp90. In the presence of the Hsp90 binding drug geldanamycin, N-terminal trimerization proceeded normally, but C-terminal trimerization was blocked. Geldanamycin did not inhibit the association of Hsp90 with sigma 1 but prevented the subsequent release of Hsp90 from the immature sigma1 complex. We also examined the status of p23, an Hsp90-associated cochaperone. Like Hsp90, p23 only associated with immature sigma1 trimers, and this association was mapped to the C-terminal half of sigma1. However, unlike Hsp90, p23 was released from the sigma1 complex upon the addition of geldanamycin. These results highlight an all-or-none concept of chaperone involvement in different oligomerization domains within a single protein and suggest a possible common usage of chaperones in the regulation of general protein folding and of steroid receptor activation.

HSP60, a protein downregulated by IGFBP7 in colorectal carcinoma

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Lin Jie

2010-04-01

Full Text Available Abstract Background In our previous study, it was well defined that IGFBP7 was an important tumor suppressor gene in colorectal cancer (CRC. We aimed to uncover the downstream molecules responsible for IGFBP7's behaviour in this study. Methods Differentially expressed protein profiles between PcDNA3.1(IGFBP7-transfected RKO cells and the empty vector transfected controls were generated by two-dimensional gel electrophoresis (2-DE and mass spectrometry (MS identification. The selected differentially expressed protein induced by IGFBP7 was confirmed by western blot and ELISA. The biological behaviour of the protein was explored by cell growth assay and colony formation assay. Results Six unique proteins were found differentially expressed in PcDNA3.1(IGFBP7-transfected RKO cells, including albumin (ALB, 60 kDa heat shock protein(HSP60, Actin cytoplasmic 1 or 2, pyruvate kinase muscle 2(PKM2, beta subunit of phenylalanyl-tRNA synthetase(FARSB and hypothetical protein. The downregulation of HSP60 by IGFBP7 was confirmed by western blot and ELISA. Recombinant human HSP60 protein could increase the proliferation rate and the colony formation ability of PcDNA3.1(IGFBP7-RKO cells. Conclusion HSP60 was an important downstream molecule of IGFBP7. The downregulation of HSP60 induced by IGFBP7 may be, at least in part, responsible for IGFBP7's tumor suppressive biological behaviour in CRC.

Regulated expression of the human cytomegalovirus pp65 gene: Octamer sequence in the promoter is required for activation by viral gene products

International Nuclear Information System (INIS)

Depto, A.S.; Stenberg, R.M.

1989-01-01

To better understand the regulation of late gene expression in human cytomegalovirus (CMV)-infected cells, the authors examined expression of the gene that codes for the 65-kilodalton lower-matrix phosphoprotein (pp65). Analysis of RNA isolated at 72 h from cells infected with CMV Towne or ts66, a DNA-negative temperature-sensitive mutant, supported the fact that pp65 is expressed at low levels prior to viral DNA replication but maximally expressed after the initiation of viral DNA replication. To investigate promoter activation in a transient expression assay, the pp65 promoter was cloned into the indicator plasmid containing the gene for chloramphenicol acetyltransferase (CAT). Transfection of the promoter-CAT construct and subsequent superinfection with CMV resulted in activation of the promoter at early times after infection. Cotransfection with plasmids capable of expressing immediate-early (IE) proteins demonstrated that the promoter was activated by IE proteins and that both IE regions 1 and 2 were necessary. These studies suggest that interactions between IE proteins and this octamer sequence may be important for the regulation and expression of this CMV gene

Can RNAi-mediated hsp90α knockdown in combination with 17-AAG be a therapy for glioma?

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Mehta, Adi; Shervington, Amal; Howl, John; Jones, Sarah; Shervington, Leroy

2013-01-01

Heat shock protein 90 promotes tumor progression and survival and has emerged as a vital therapeutic target. Previously we reported that the combinatorial treatment of 17AAG/sihsp90α significantly downregulated Hsp90α mRNA and protein levels in Glioblastoma Multiforme (GBM). Here we investigated the ability of cell penetrating peptide (Tat48-60 CPP)-mediated siRNA-induced hsp90α knockdown as a single agent and in combination with 17-allylamino-17-demethoxygeldanamycin (17-AAG) to induce tumor growth inhibition in GBM and whether it possessed therapeutic implications. GBM and non-tumorigenic cells exposed to siRNA and/or 17-AAG were subsequently assessed by qRT-PCR, immunofluorescence, FACS analysis, quantitative Akt, LDH leakage and cell viability assays. PAGE was performed for serum stability assessment. A combination of siRNA/17-AAG treatment significantly induced Hsp90α gene and protein knockdown by 95% and 98%, respectively, concomitant to 84% Akt kinase activity attenuation, induced cell cycle arrest and tumor-specific cytotoxicity by 88%. Efficient complex formation between CPP and siRNA exhibited improved serum stability of the siRNA with minimal intrinsic toxicity in vitro. The preliminary in vivo results showed that combination therapy induced hsp90α knockdown and attenuated Akt kinase activity in intracranial glioblastoma mouse models. The results imply that RNAi-mediated hsp90α knockdown increases 17-AAG treatment efficacy in GBM. In addition, the cytotoxic response observed was the consequence of downregulation of hsp90α gene expression, reduced Akt kinase activity and S-G2/M cell cycle arrest. These results are novel and highlight the ability of Tat to efficiently deliver siRNA in GBM and suggest that the dual inhibition of Hsp90 has therapeutic potentials.

Andrographolide induces degradation of mutant p53 via activation of Hsp70.

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Sato, Hirofumi; Hiraki, Masatsugu; Namba, Takushi; Egawa, Noriyuki; Baba, Koichi; Tanaka, Tomokazu; Noshiro, Hirokazu

2018-05-22

The tumor suppressor gene p53 encodes a transcription factor that regulates various cellular functions, including DNA repair, apoptosis and cell cycle progression. Approximately half of all human cancers carry mutations in p53 that lead to loss of tumor suppressor function or gain of functions that promote the cancer phenotype. Thus, targeting mutant p53 as an anticancer therapy has attracted considerable attention. In the current study, a small-molecule screen identified andrographlide (ANDRO) as a mutant p53 suppressor. The effects of ANDRO, a small molecule isolated from the Chinese herb Andrographis paniculata, on tumor cells carrying wild-type or mutant p53 were examined. ANDRO suppressed expression of mutant p53, induced expression of the cyclin-dependent kinase inhibitor p21 and pro-apoptotic proteins genes, and inhibited the growth of cancer cells harboring mutant p53. ANDRO also induced expression of the heat-shock protein (Hsp70) and increased binding between Hsp70 and mutant p53 protein, thus promoting proteasomal degradation of p53. These results provide novel insights into the mechanisms regulating the function of mutant p53 and suggest that activation of Hsp70 may be a new strategy for the treatment of cancers harboring mutant p53.

RPE65 gene: multiplex PCR and mutation screening in patients from ...

Indian Academy of Sciences (India)

Unknown

The RPE65 protein is believed to play an important role in the metabolism of vitamin A in the ... PCR and mutation screening in patients from India with retinal degenerative diseases. ..... Bennett J. 2001 Gene therapy restores vision in a canine.

Unique spectrum of SPAST variants in Estonian HSP patients: presence of benign missense changes but lack of exonic rearrangements

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Gross-Paju Katrin

2010-03-01

Full Text Available Abstract Background Hereditary spastic paraplegia (HSP is a clinically and genetically heterogeneous disorder that can be an autosomal-dominant, autosomal-recessive, or X-linked disease. The most common autosomal-dominant form of the disease derives from mutations in the SPAST gene. Methods The aim of this study was to analyze 49 patients diagnosed with HSP from the Estonian population for sequence variants of the SPAST gene and to describe the associated phenotypes. Healthy control individuals (n = 100 with no family history of HSP were also analyzed. All patient samples were screened using denaturing high performance liquid chromatography (DHPLC and multiplex ligation-dependent probe amplification (MLPA assay. Samples with abnormal DHPLC and MLPA profiles were sequenced, with the same regions sequenced in control samples. Results Sequence variants of SPAST were identified in 19/49 HSP patients (38.8%, twelve among them had pathogenic mutations. Within the latter group there was one sporadic case. Eight patients had pure, and four - complex HSP. The twelve variants were identified: seven pathogenic (c.1174-1G>C, c.1185delA, c.1276C>T, c.1352_1356delGAGAA, c.1378C>A, c.1518_1519insTC, c.1841_1842insA and five non-pathogenic (c.131C>T, c.484G>A, c.685A>G, c.1245+202delG, c.1245+215G>C. Only 2 of these mutations had previously been described (c.131C>T, c.1245+202delG. Three mutations, c.1174-1G>C, c.1276 C>T, c.1378C>A, showed intrafamilial segregation. Conclusion This study identified new variants of the SPAST gene which included benign missense variants and short insertions/deletions. No large rearrangements were found. Based on these data, 7 new pathogenic variants of HSP are associated with clinical phenotypes.

A rat retinal damage model predicts for potential clinical visual disturbances induced by Hsp90 inhibitors

International Nuclear Information System (INIS)

Zhou, Dan; Liu, Yuan; Ye, Josephine; Ying, Weiwen; Ogawa, Luisa Shin; Inoue, Takayo; Tatsuta, Noriaki; Wada, Yumiko; Koya, Keizo; Huang, Qin; Bates, Richard C.; Sonderfan, Andrew J.

2013-01-01

In human trials certain heat shock protein 90 (Hsp90) inhibitors, including 17-DMAG and NVP-AUY922, have caused visual disorders indicative of retinal dysfunction; others such as 17-AAG and ganetespib have not. To understand these safety profile differences we evaluated histopathological changes and exposure profiles of four Hsp90 inhibitors, with or without clinical reports of adverse ocular effects, using a rat retinal model. Retinal morphology, Hsp70 expression (a surrogate marker of Hsp90 inhibition), apoptotic induction and pharmacokinetic drug exposure analysis were examined in rats treated with the ansamycins 17-DMAG and 17-AAG, or with the second-generation compounds NVP-AUY922 and ganetespib. Both 17-DMAG and NVP-AUY922 induced strong yet restricted retinal Hsp70 up-regulation and promoted marked photoreceptor cell death 24 h after the final dose. In contrast, neither 17-AAG nor ganetespib elicited photoreceptor injury. When the relationship between drug distribution and photoreceptor degeneration was examined, 17-DMAG and NVP-AUY922 showed substantial retinal accumulation, with high retina/plasma (R/P) ratios and slow elimination rates, such that 51% of 17-DMAG and 65% of NVP-AUY922 present at 30 min post-injection were retained in the retina 6 h post-dose. For 17-AAG and ganetespib, retinal elimination was rapid (90% and 70% of drugs eliminated from the retina at 6 h, respectively) which correlated with lower R/P ratios. These findings indicate that prolonged inhibition of Hsp90 activity in the eye results in photoreceptor cell death. Moreover, the results suggest that the retina/plasma exposure ratio and retinal elimination rate profiles of Hsp90 inhibitors, irrespective of their chemical class, may predict for ocular toxicity potential. - Highlights: • In human trials some Hsp90 inhibitors cause visual disorders, others do not. • Prolonged inhibition of Hsp90 in the rat eye results in photoreceptor cell death. • Retina/plasma ratio and retinal

Overexpression of a heat shock protein (ThHSP18.3) from Tamarix hispida confers stress tolerance to yeast.

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Gao, Caiqiu; Jiang, Bo; Wang, Yucheng; Liu, Guifeng; Yang, Chuanping

2012-04-01

It is well known that plant heat shock proteins (HSPs) play important roles both in response to adverse environmental conditions and in various developmental processes. However, among plant HSPs, the functions of tree plant HSPs are poorly characterized. To improve our understanding of tree HSPs, we cloned and characterized an HSP gene (ThHSP18.3) from Tamarix hispida. Sequence alignment reveals that ThHSP18.3 belongs to the class I small heat shock protein family. A transient expression assay showed that ThHSP18.3 protein was targeted to the cell nucleus. Treatment of Tamarix hispida with cold and heat shock highly induced ThHSP18.3 expression in all studied leaves, roots and stems, whereas, treatment of T. hispida with NaCl, NaHCO(3), and PEG induced ThHSP18.3 expression in leaves and decreased its expression in roots and stems. Further, to study the role of ThHSP18.3 in stress tolerance under different stress conditions, we cloned ThHSP18.3 into the pYES2 vector, transformed and expressed the vector in yeast Saccharomyces cerevisiae. Yeast cells transformed with an empty pYES2 vector were employed as a control. Compared to the control, yeast cells expressing ThHSP18.3 showed greater tolerance to salt, drought, heavy metals, and both low and high temperatures, indicating that ThHSP18.3 confers tolerance to these stress conditions. These results suggested that ThHSP18.3 is involved in tolerance to a variety of stress conditions in T. hispida.

BCG and BCG/DNAhsp65 Vaccinations Promote Protective Effects without Deleterious Consequences for Experimental Autoimmune Encephalomyelitis

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Sofia Fernanda Gonçalves Zorzella-Pezavento

2013-01-01

Full Text Available A prime-boost strategy conserving BCG is considered the most promising vaccine to control tuberculosis. A boost with a DNA vaccine containing the mycobacterial gene of a heat shock protein (pVAXhsp65 after BCG priming protected mice against experimental tuberculosis. However, anti-hsp65 immunity could worsen an autoimmune disease due to molecular mimicry. In this investigation, we evaluated the effect of a previous BCG or BCG/pVAXhsp65 immunization on experimental autoimmune encephalomyelitis (EAE development. Female Lewis rats were immunized with BCG or BCG followed by pVAXhsp65 boosters. The animals underwent EAE induction and were daily evaluated for weight loss and clinical score. They were euthanized during recovery phase to assess immune response and inflammatory infiltration at the central nervous system. Previous immunization did not aggravate or accelerate clinical score or weight loss. In addition, this procedure clearly decreased inflammation in the brain. BCG immunization modulated the host immune response by triggering a significant reduction in IL-10 and IFN-γ levels induced by myelin basic protein. These data indicated that vaccination protocols with BCG or BCG followed by boosters with pVAXhsp65 did not trigger a deleterious effect on EAE evolution.

Targeting GRP75 improves HSP90 inhibitor efficacy by enhancing p53-mediated apoptosis in hepatocellular carcinoma.

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Weiwei Guo

Full Text Available Heat shock protein 90 (HSP90 inhibitors are potential drugs for cancer therapy. The inhibition of HSP90 on cancer cell growth largely through degrading client proteins, like Akt and p53, therefore, triggering cancer cell apoptosis. Here, we show that the HSP90 inhibitor 17-AAG can induce the expression of GRP75, a member of heat shock protein 70 (HSP70 family, which, in turn, attenuates the anti-growth effect of HSP90 inhibition on cancer cells. Additionally, 17-AAG enhanced binding of GRP75 and p53, resulting in the retention of p53 in the cytoplasm. Blocking GRP75 with its inhibitor MKT-077 potentiated the anti-tumor effects of 17-AAG by disrupting the formation of GRP75-p53 complexes, thereby facilitating translocation of p53 into the nuclei and leading to the induction of apoptosis-related genes. Finally, dual inhibition of HSP90 and GRP75 was found to significantly inhibit tumor growth in a liver cancer xenograft model. In conclusion, the GRP75 inhibitor MKT-077 enhances 17-AAG-induced apoptosis in HCCs and increases p53-mediated inhibition of tumor growth in vivo. Dual targeting of GRP75 and HSP90 may be a useful strategy for the treatment of HCCs.

Identification of the key structural motifs involved in HspB8/HspB6-Bag3 interaction

NARCIS (Netherlands)

Fuchs, Margit; Poirier, Dominic J.; Seguin, Samuel J.; Lambert, Herman; Carra, Serena; Charette, Steve J.; Landry, Jacques

2010-01-01

The molecular chaperone HspB8 [Hsp (heat-shock protein) B8] is member of the B-group of Hsps. These proteins bind to unfolded or misfolded proteins and protect them from aggregation. HspB8 has been reported to form a stable molecular complex with the chaperone cohort protein Bag3 (Bcl-2-associated

Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells

International Nuclear Information System (INIS)

Banzet, N.; Richaud, C.; Deveaux, Y.; Kazmaier, M.; Gagnon, J.; Triantaphylides, C.

1998-01-01

Changes in gene expression, by application of H2O2, O2.- generating agents (methyl viologen, digitonin) and gamma irradiation to tomato suspension cultures, were investigated and compared to the well-described heat shock response. Two-dimensional gel protein mapping analyses gave the first indication that at least small heat shock proteins (smHSP) accumulated in response to application of H2O2 and gamma irradiation, but not to O2.- generating agents. While some proteins seemed to be induced specifically by each treatment, only part of the heat shock response was observed. On the basis of Northern hybridization experiments performed with four heterologous cDNA, corresponding to classes I-IV of pea smHSP, it could be concluded that significant amounts of class I and II smHSP mRNA are induced by H2O2 and by irradiation. Taken together, these results demonstrate that in plants some HSP genes are inducible by oxidative stresses, as in micro-organisms and other eukaryotic cells. HSP22, the main stress protein that accumulates following H2O2 action or gamma irradiation, was also purified. Sequence homology of amino terminal and internal sequences, and immunoreactivity with Chenopodium rubrum mitochondrial smHSP antibody, indicated that the protein belongs to the recently discovered class of plant mitochondrial smHSP. Heat shock or a mild H2O2 pretreatment was also shown to lead to plant cell protection against oxidative injury. Therefore, the synthesis of these stress proteins can be considered as an adaptive mechanism in which mitochondrial protection could be essential

Chromosomal assignment of six genes (EIF4G3, HSP90, RBBP6, IL8, TERT, and TERC) in four species of the genus Equus.

Science.gov (United States)

Vidale, Pamela; Piras, Francesca M; Nergadze, Solomon G; Bertoni, Livia; Verini-Supplizi, Andrea; Adelson, David; Guérin, Gérard; Giulotto, Elena

2011-01-01

We mapped six genes (EIF4G3, HSP90, RBBP6, IL8, TERT, and TERC) on the chromosomes of Equus caballus, Equus asinus, Equus grevyi, and Equus burchelli by fluorescence in situ hybridization. Our results add six type I markers to the cytogenetic map of these species and provide new information on the comparative genomics of the genus Equus. Copyright © Taylor & Francis Group, LLC

The Heat Shock Protein 26 Gene is Required for Ethanol Tolerance in Drosophila

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Awoyemi A. Awofala

2011-01-01

Full Text Available Stress plays an important role in drug- and addiction-related behaviours. However, the mechanisms underlying these behavioural responses are still poorly understood. In the light of recent reports that show consistent regulation of many genes encoding stress proteins including heat shock proteins following ethanol exposure in Drosophila , it was hypothesised that transition to alcohol dependence may involve the dysregulation of the circuits that mediate behavioural responses to stressors. Thus, behavioural genetic methodologies were used to investigate the role of the Drosophila hsp26 gene, a small heat shock protein coding gene which is induced in response to various stresses, in the development of rapid tolerance to ethanol sedation. Rapid tolerance was quantified as the percentage difference in the mean sedation times between the second and first ethanol exposure. Two independently isolated P-element mutations near the hsp26 gene eliminated the capacity for tolerance. In addition, RNAi-mediated functional knockdown of hsp26 expression in the glial cells and the whole nervous system also caused a defect in tolerance development. The rapid tolerance phenotype of the hsp26 mutants was rescued by the expression of the wild-type hsp26 gene in the nervous system. None of these manipulations of the hsp26 gene caused changes in the rate of ethanol absorption. Hsp26 genes are evolutionary conserved, thus the role of hsp26 in ethanol tolerance may present a new direction for research into alcohol dependency.

Cloning, characterization, and heat stress-induced redistribution of a protein homologous to human hsp27 in the zebrafish Danio rerio

International Nuclear Information System (INIS)

Mao Li; Bryantsev, Anton L.; Chechenova, Maria B.; Shelden, Eric A.

2005-01-01

Hsp27 is a small heat shock protein (shsp) regulating stress tolerance and increasingly thought to play roles in tissue homeostasis and differentiation. The zebrafish Danio rerio is an important model for the study of developmental processes, but little is known regarding shsps in this animal. Here, we report the sequence, expression, regulation, and function of a zebrafish protein (zfHsp27) homologous to human Hsp27. zfHsp27 contains three conserved phosphorylatable serines and a cysteine important for regulation of apoptosis, but it lacks much of a C-terminal tail domain and shows low homology in two putative actin interacting domains that are features of mammalian Hsp27. zfHsp27 mRNA is most abundant in adult skeletal muscle and heart and is upregulated during early embryogenesis. zfHsp27 expressed in mammalian fibroblasts was phosphorylated in response to heat stress and anisomycin, and this phosphorylation was prevented by treatment with SB202190, an inhibitor of p38 MAPK. Expression of zfHsp27 and human Hsp27 in mammalian fibroblasts promoted a similar degree of tolerance to heat stress. zfHsp27 fusion proteins entered the nucleus and associated with the cytoskeleton of heat stressed cells in vitro and in zebrafish embryos. These results reveal conservation in regulation and function of mammalian and teleost Hsp27 proteins and define zebrafish as a new model for the study of Hsp27 function

Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

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Trevor M. Morey

2017-12-01

Full Text Available Choline acetyltransferase (ChAT synthesizes the neurotransmitter acetylcholine in cholinergic neurons, and mutations of this enzyme are linked to the neuromuscular disorder congenital myasthenic syndrome (CMS. One CMS-related mutation, V18M, reduces ChAT enzyme activity and cellular protein levels, and is located within a highly-conserved N-terminal proline-rich motif at residues 14PKLPVPP20. We showed previously that disruption of this proline-rich motif by either proline-to-alanine mutation (P17A/P19A or mutation of residue Val18 (V18M enhances ubiquitination and degradation of these mutant ChAT proteins expressed in cholinergic SN56 cells by an unknown mechanism. In this study, using proximity-dependent biotin identification (BioID, co-immunoprecipitation and in situ proximity-ligation assay (PLA, we identified the heat shock proteins (HSPs HSC/HSP70 and HSP90 as novel ChAT protein-interactors. These molecular chaperones are well-known for promoting the folding and stabilization of cellular proteins. Thus, we found that inhibition of HSPs by treatment of cells with either the HSC/HSP70 inhibitors 2-phenylethynesulfonamide (PES or VER-155008, or the HSP90 inhibitor 17-AAG reduced cellular ChAT activity and solubility, and enhanced the ubiquitination and proteasome-dependent loss of ChAT protein. Importantly, the effects of HSP inhibition were greater for mutant ChAT proteins (P17A/P19A-ChAT and CMS-related V18M- and A513T-ChAT compared to wild-type ChAT. HSPs can promote ubiquitination and degradation of terminally misfolded proteins through cooperative interaction with the E3 ubiquitin ligase CHIP/Stub1, and while we show that ChAT interacts with CHIP in situ, siRNA-mediated knock-down of CHIP had no effect on either wild-type or mutant ChAT protein levels. However, inhibition of the endoplasmic reticulum (ER- and HSP-associated co-chaperone p97/VCP prevented degradation of ubiquitinated ChAT. Together, these results identify novel mechanisms

Identification of the divergent calmodulin binding motif in yeast Ssb1/Hsp75 protein and in other HSP70 family members.

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Heinen, R C; Diniz-Mendes, L; Silva, J T; Paschoalin, V M F

2006-11-01

Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37% of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89% similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed.

Identification of the divergent calmodulin binding motif in yeast Ssb1/Hsp75 protein and in other HSP70 family members

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R.C. Heinen

2006-11-01

Full Text Available Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37% of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89% similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed.

Characterization of small HSPs from Anemonia viridis reveals insights into molecular evolution of alpha crystallin genes among cnidarians.

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Aldo Nicosia

Full Text Available Gene family encoding small Heat-Shock Proteins (sHSPs containing α-crystallin domain are found both in prokaryotic and eukaryotic organisms; however, there is limited knowledge of their evolution. In this study, two small HSP genes termed AvHSP28.6 and AvHSP27, both organized in one intron and two exons, were characterised in the Mediterranean snakelocks anemone Anemonia viridis. The release of the genome sequence of Hydra magnipapillata and Nematostella vectensis enabled a comprehensive study of the molecular evolution of α-crystallin gene family among cnidarians. Most of the H. magnipapillata sHSP genes share the same gene organization described for AvHSP28.6 and AvHSP27, differing from the sHSP genes of N. vectensis which mainly show an intronless architecture. The different genomic organization of sHSPs, the phylogenetic analyses based on protein sequences, and the relationships among Cnidarians, suggest that the A.viridis sHSPs represent the common ancestor from which H. magnipapillata genes directly evolved through segmental genome duplication. Additionally retroposition events may be considered responsible for the divergence of sHSP genes of N. vectensis from A. viridis. Analyses of transcriptional expression profile showed that AvHSP28.6 was constitutively expressed among different tissues from both ectodermal and endodermal layers of the adult sea anemones, under normal physiological conditions and also under different stress condition. Specifically, we profiled the transcriptional activation of AvHSP28.6 after challenges with different abiotic/biotic stresses showing induction by extreme temperatures, heavy metals exposure and immune stimulation. Conversely, no AvHSP27 transcript was detected in such dissected tissues, in adult whole body cDNA library or under stress conditions. Hence, the involvement of AvHSP28.6 gene in the sea anemone defensome is strongly suggested.

Characterization of small HSPs from Anemonia viridis reveals insights into molecular evolution of alpha crystallin genes among cnidarians.

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Nicosia, Aldo; Maggio, Teresa; Mazzola, Salvatore; Gianguzza, Fabrizio; Cuttitta, Angela; Costa, Salvatore

2014-01-01

Gene family encoding small Heat-Shock Proteins (sHSPs containing α-crystallin domain) are found both in prokaryotic and eukaryotic organisms; however, there is limited knowledge of their evolution. In this study, two small HSP genes termed AvHSP28.6 and AvHSP27, both organized in one intron and two exons, were characterised in the Mediterranean snakelocks anemone Anemonia viridis. The release of the genome sequence of Hydra magnipapillata and Nematostella vectensis enabled a comprehensive study of the molecular evolution of α-crystallin gene family among cnidarians. Most of the H. magnipapillata sHSP genes share the same gene organization described for AvHSP28.6 and AvHSP27, differing from the sHSP genes of N. vectensis which mainly show an intronless architecture. The different genomic organization of sHSPs, the phylogenetic analyses based on protein sequences, and the relationships among Cnidarians, suggest that the A.viridis sHSPs represent the common ancestor from which H. magnipapillata genes directly evolved through segmental genome duplication. Additionally retroposition events may be considered responsible for the divergence of sHSP genes of N. vectensis from A. viridis. Analyses of transcriptional expression profile showed that AvHSP28.6 was constitutively expressed among different tissues from both ectodermal and endodermal layers of the adult sea anemones, under normal physiological conditions and also under different stress condition. Specifically, we profiled the transcriptional activation of AvHSP28.6 after challenges with different abiotic/biotic stresses showing induction by extreme temperatures, heavy metals exposure and immune stimulation. Conversely, no AvHSP27 transcript was detected in such dissected tissues, in adult whole body cDNA library or under stress conditions. Hence, the involvement of AvHSP28.6 gene in the sea anemone defensome is strongly suggested.

Molecular characterization and expression analysis of a heat shock protein 90 gene from disk abalone (Haliotis discus).

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Wang, Ning; Whang, Ilson; Lee, Jae-Seong; Lee, Jehee

2011-06-01

Heat shock protein 90s (hsp90s) are chaperones that contribute to the proper folding of cellular proteins and help animals cope with the cellular protein damages in stress conditions. In this study, an hsp90 gene was isolated from disc abalone (Haliotis discus). The complete nucleotide sequence of the hsp90 gene contains an open reading frame of 2,184 base pairs, encoding an 84 kDa protein. Disk abalone hsp90 shares high sequence similarity with other hsp90 family proteins. Although the phylogenetic analysis did not classify it into the hsp90α group, the inductivity of this gene was confirmed by heat shock and lipopolysaccharide (LPS) challenge test. Disk abalone hsp90 gene displayed a rapid and reversible induction response to both an exposure of typical heat shock and the LPS challenge. Once given the sublethal heat shock treatment, the transcription of disk abalone hsp90 gene was significantly up-regulated. With a recovery of 12 h, the transcription of disk abalone hsp90 gene gradually attenuated to the control level. These observations reflected the feedback regulation of abalone heat shock responses faithfully. In response to LPS challenge, the transcription of disk abalone hsp90 gene was significantly increased within 2 h and it approached maximum induction at 4 h later and recovered finally the reference level in 24 h. Take all together, the cloning and expression analysis of disk abalone hsp90 gene provided useful molecular information of abalone responses in stress conditions and potential ways to monitor the chronic stressors in abalone culture environments and diagnose the animal health status.

Identification of non-tuberculous mycobacteria from the Central Public Health Laboratory from Mato Grosso do Sul and analysis of clinical relevance Identificação de micobactérias não-tuberculosas do Laboratório Central de Saúde Pública de Mato Grosso de Sul e análise de dados clínicos dos pacientes

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Paulo Ricardo de Souza Moraes

2008-06-01

Full Text Available Non-tuberculous mycobacteria isolated at the Central Public Health Laboratory from Mato Grosso do Sul in 2003 and 2004 were identified by conventional phenotypic methods (TI and by PCR-Restriction Enzyme Analysis (PRA using the hsp65 gene as target (PRA-hsp65. With 15 of the 32 analysed isolates, results of both methods were concordant, being 8 Mycobacterium avium, 3 M. fortutium, 1 M. kansasii, 1 M. flavescens, 1 M. peregrinum and 1 Nocardia brasiliensis. TI of 12 isolates was inconclusive. Novel PRA-hsp65 patterns were observed with 11 isolates. Medical data were evaluated for inference of clinical relevance of these isolates.Micobactérias não-tuberculosas isoladas no Laboratório Central de Saúde Pública de Mato Grosso do Sul em 2003 e 2004 foram identificadas usando métodos fenotípicos convencionais (TI e PCR-Restriction Enzyme Analysis (PRA tendo o gene hsp65 como alvo (PRA-hsp65. Em 15 dos 32 isolados analisados os resultados obtidos com ambos métodos foram concordantes, sendo 8 Mycobacterium avium, 3 M. fortutium, 1 M. kansasii, 1 M. flavescens, 1 M. peregrinum e 1 Nocardia brasiliensis. TI de 12 isolados não foi conclusiva. Perfis não descritos de PRA-hsp65 foram observados com 11 isolados. Dados dos prontuários médicos foram avaliados para inferir a relevância clínica dos isolados.

A novel Hsp70 of the yeast ER lumen is required for the efficient translocation of a number of protein precursors.

OpenAIRE

Craven, R A; Egerton, M; Stirling, C J

1996-01-01

The yeast genome sequencing project predicts an open reading frame (YKL073) that would encode a novel member of the Hsp70 family of molecular chaperones. We report that this 881 codon reading frame represents a functional gene expressing a 113-119 kDa glycoprotein localized within the lumen of the endoplasmic reticulum (ER). We therefore propose to designate this gene LHS1 (Lumenal Hsp Seventy). Our studies indicate that LHS1 is regulated by the unfolded protein response pathway, as evidenced...

Sorafenib prevents liver fibrosis in a non-alcoholic steatohepatitis (NASH) rodent model

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Stefano, J.T.; Pereira, I.V.A.; Torres, M.M.; Bida, P.M. [Disciplina de Gastroenterologia Clínica (LIM-07), Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Coelho, A.M.M. [Disciplina de Transplante de Órgãos do Aparelho Digestivo (LIM-37), Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Xerfan, M.P. [Disciplina de Gastroenterologia Clínica (LIM-07), Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Cogliati, B. [Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP (Brazil); Barbeiro, D.F. [Disciplina de Emergências Clínicas (LIM-51), Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Mazo, D.F.C. [Disciplina de Gastroenterologia Clínica (LIM-07), Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Kubrusly, M.S.; D' Albuquerque, L.A.C. [Disciplina de Transplante de Órgãos do Aparelho Digestivo (LIM-37), Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Souza, H.P. [Disciplina de Emergências Clínicas (LIM-51), Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Carrilho, F.J.; Oliveira, C.P. [Disciplina de Gastroenterologia Clínica (LIM-07), Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil)

2015-02-24

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

Expression of HSP27, HSP72 and MRP proteins in in vitro co-culture ...

Indian Academy of Sciences (India)

We studied the expression of inducible heat shock protein (HSP27, HSP72) and multidrug-resistance protein (MRP) in co-cultures of human colon carcinoma cell spheroids obtained from different grades of tumour with normal human colon epithelium, myofibroblast and endothelial cell monolayers. We also measured the ...

Phylogenetic analysis of Mycobacterium massiliense strains having recombinant rpoB gene laterally transferred from Mycobacterium abscessus.

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Kim, Byoung-Jun; Kim, Ga-Na; Kim, Bo-Ram; Shim, Tae-Sun; Kook, Yoon-Hoh; Kim, Bum-Joon

2017-01-01

Recent multi locus sequence typing (MLST) and genome based studies indicate that lateral gene transfer (LGT) events in the rpoB gene are prevalent between Mycobacterium abscessus complex strains. To check the prevalence of the M. massiliense strains subject to rpoB LGT (Rec-mas), we applied rpoB typing (711 bp) to 106 Korean strains of M. massiliense infection that had already been identified by hsp65 sequence analysis (603 bp). The analysis indicated 6 smooth strains in M. massiliense Type I (10.0%, 6/60) genotypes but no strains in M. massiliense Type II genotypes (0%, 0/46), showing a discrepancy between the 2 typing methods. Further MLST analysis based on the partial sequencing of seven housekeeping genes, argH, cya, glpK, gnd, murC, pta and purH, as well as erm(41) PCR proved that these 6 Rec-mas strains consisted of two distinct genotypes belonging to M. massiliense and not M. abscessus. The complete rpoB sequencing analysis showed that these 6 Rec-mas strains have an identical hybrid rpoB gene, of which a 478 bp partial rpoB fragment may be laterally transferred from M. abscessus. Notably, five of the 6 Rec-mas strains showed complete identical sequences in a total of nine genes, including the seven MLST genes, hsp65, and rpoB, suggesting their clonal propagation in South Korea. In conclusion, we identified 6 M. massiliense smooth strains of 2 phylogenetically distinct genotypes with a specific hybrid rpoB gene laterally transferred from M. abscessus from Korean patients. Their clinical relevance and bacteriological traits remain to be elucidated.

Phylogenetic analysis of Mycobacterium massiliense strains having recombinant rpoB gene laterally transferred from Mycobacterium abscessus.

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Byoung-Jun Kim

Full Text Available Recent multi locus sequence typing (MLST and genome based studies indicate that lateral gene transfer (LGT events in the rpoB gene are prevalent between Mycobacterium abscessus complex strains. To check the prevalence of the M. massiliense strains subject to rpoB LGT (Rec-mas, we applied rpoB typing (711 bp to 106 Korean strains of M. massiliense infection that had already been identified by hsp65 sequence analysis (603 bp. The analysis indicated 6 smooth strains in M. massiliense Type I (10.0%, 6/60 genotypes but no strains in M. massiliense Type II genotypes (0%, 0/46, showing a discrepancy between the 2 typing methods. Further MLST analysis based on the partial sequencing of seven housekeeping genes, argH, cya, glpK, gnd, murC, pta and purH, as well as erm(41 PCR proved that these 6 Rec-mas strains consisted of two distinct genotypes belonging to M. massiliense and not M. abscessus. The complete rpoB sequencing analysis showed that these 6 Rec-mas strains have an identical hybrid rpoB gene, of which a 478 bp partial rpoB fragment may be laterally transferred from M. abscessus. Notably, five of the 6 Rec-mas strains showed complete identical sequences in a total of nine genes, including the seven MLST genes, hsp65, and rpoB, suggesting their clonal propagation in South Korea. In conclusion, we identified 6 M. massiliense smooth strains of 2 phylogenetically distinct genotypes with a specific hybrid rpoB gene laterally transferred from M. abscessus from Korean patients. Their clinical relevance and bacteriological traits remain to be elucidated.

DETECÇÃO DO COMPLEXO Mycobacterium tuberculosis NO LEITE PELA REAÇÃO EM CADEIA DA POLIMERASE SEGUIDA DE ANÁLISE DE RESTRIÇÃO DO FRAGMENTO AMPLIFICADO (PRA DETECTION OF Mycobacterium tuberculosis COMPLEX BY PCR-RESTRICTION FRAGMENT LENGTH POLYMORFISM ANALYSIS OF THE HSP65 GENE

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Joab Trajano Silva

2008-12-01

Full Text Available Mycobacterium bovis é membro do complexo Mycobacterium tuberculosis (MTBC, grupo este composto por espécies com grande homologia genética. É o agente etiológico da tuberculose bovina, importante zoonose transmissível ao homem, principalmente através da inalação do bacilo e/ou pelo consumo de leite e derivados não-pasteurizados provenientes de vacas tuberculosas. O objetivo deste estudo foi padronizar a identificação de micobactérias do complexo M. tuberculosis presentes no leite, por metodologia molecular. Fez-se a extração de DNA diretamente do leite contaminado e realizou-se a identificação molecular pela reação em cadeia da polimerase seguida de análise de restrição do fragmento amplificado (PRA. Utilizaram-se inhagens de referência e leite cru artificialmente contaminado com M. bovis IP. Um fragmento de 441pb do gene hsp65 foi amplificado, tratado com BstEII e HaeIII e empregou-se o perfil de restrição enzimática obtido para identificar o complexo M. tuberculosis no leite. Com a PRA foi possível detectar com especificidade e sensibilidade a presença de M. bovis em até 10 UFC/mL de leite. A metodologia padronizada poderá auxiliar os métodos microbiológicos e bioquímicos tradicionalmente usados na identificação do bacilo em alimentos suspeitos de contaminação, como, por exemplo, o leite proveniente de animais suspeitos de infecção por M. bovis.

Palavras-chaves: Análise de perfil de restrição enzimática (PRA, complexo Mycobacterium tuberculosis, leite, Mycobacterium bovis, limite de detecção (PCR. Mycobacterium bovis is a member of the M. tuberculosis complex, a group composed by species with high genetic homology. The pathogen is the etiological agent of bovine tuberculosis, an important zoonosis that is mainly transmitted by inhalation of infectious droplet nuclei or by ingestion of milk and crude milk derivative products from tuberculosis cows. The definitive identification of M. bovis

Hsp70/Hsp90 organising protein (hop): beyond interactions with chaperones and prion proteins.

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Baindur-Hudson, Swati; Edkins, Adrienne L; Blatch, Gregory L

2015-01-01

The Hsp70/Hsp90 organising protein (Hop), also known as stress-inducible protein 1 (STI1), has received considerable attention for diverse cellular functions in both healthy and diseased states. There is extensive evidence that intracellular Hop is a co-chaperone of the major chaperones Hsp70 and Hsp90, playing an important role in the productive folding of Hsp90 client proteins. Consequently, Hop is implicated in a number of key signalling pathways, including aberrant pathways leading to cancer. However, Hop is also secreted and it is now well established that Hop also serves as a receptor for the prion protein, PrP(C). The intracellular and extracellular forms of Hop most likely represent two different isoforms, although the molecular determinants of these divergent functions are yet to be identified. There is also a growing body of research that reports the involvement of Hop in cellular activities that appear independent of either chaperones or PrP(C). While Hop has been shown to have various cellular functions, its biological function remains elusive. However, recent knockout studies in mammals suggest that Hop has an important role in embryonic development. This review provides a critical overview of the latest molecular, cellular and biological research on Hop, critically evaluating its function in healthy systems and how this function is adapted in diseases states.

Heavy metals induce oxidative stress and trigger oxidative stress-mediated heat shock protein (hsp) modulation in the intertidal copepod Tigriopus japonicus.

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Kim, Bo-Mi; Rhee, Jae-Sung; Jeong, Chang-Bum; Seo, Jung Soo; Park, Gyung Soo; Lee, Young-Mi; Lee, Jae-Seong

2014-11-01

Heat shock proteins (hsps) are induced by a wide range of environmental stressors including heavy metals in aquatic organisms. However, the effect of heavy metals on zooplankton at the molecular level remains still unclear. In this study, we measured the intracellular reactive oxygen species (ROS) level and the antioxidant enzyme activities for 96 h after exposure to five heavy metals: arsenic (As), cadmium (Cd), copper (Cu), silver (Ag), and zinc (Zn) in the intertidal copepod Tigriopus japonicus. Activities of the antioxidant enzymes were highly elevated in metal-exposed copepods, indicating that heavy metals can induce oxidative stress by generating ROS, and stimulate the involvement of antioxidant enzymes as cellular defense mechanisms. Subsequently, transcriptional changes in hsp gene families were further investigated in the metal-exposed groups for 96 h. The ROS level and glutathione (GSH) content were significantly increased in Ag-, As-, and Cu-exposed copepods, while they were only slightly elevated in Cd- and Zn-exposed groups. Based on the numbers of significantly modulated hsp genes and their expression levels for 96 h, we measured the effect of heavy metals to stress genes of T. japonicus in the following order: Cu > Zn > Ag > As > Cd, implying that Cu acts as a stronger oxidative stress inducer than other heavy metals. Of them, the expression of hsp20 and hsp70 genes was substantially modulated by exposure to heavy metals, indicating that these genes would provide a sensitive molecular biomarker for aquatic monitoring of heavy metal pollution. Copyright © 2014 Elsevier Inc. All rights reserved.

The expression and correlation of Hsp 70 and Hsp 27 in serous middle ear effusion fluids of pediatric patients-a preliminary study.

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Min, Hyun Jin; Choe, Ji Won; Chang, Moon Young; Kim, Kyung Soo; Lee, Sei Young; Mun, Seog-Kyun

2017-10-01

Several cytokines and innate immune-associated molecules are present in middle ear effusions, but damage-associated molecular patterns (DAMPs) in middle ear effusion have not been studied. Therefore, we evaluated the role of heat shock proteins (Hsps) in the development of otitis media with effusion (OME). Serous middle ear effusions from 22 pediatric patients who were diagnosed with OME and underwent ventilation tube insertion from June 2015 to March 2017 were evaluated in our study. The levels of Hsp 90, 70, 27, IL-8, and TNF-α in effusion fluids were evaluated by enzyme-linked immunosorbent assays. The associations between the levels of these molecules and the degree of tympanic membrane inflammation were statistically evaluated. Finally, the relationships among these molecules were also evaluated. Hsp 70 and Hsp 27 were detected in all middle ear effusions, but Hsp 90 was detected in only five effusion fluid samples. IL-8 was also detected in all middle ear effusions, but TNF-α was detected in only four effusion fluid samples. When we compared the degree of tympanic membrane inflammation with the levels of Hsp 70, Hsp 27, and IL-8, which were detected in all effusion fluids, we could not find statistical significance. However, Hsp 70, Hsp 27, and IL-8 were significantly associated with each other (p effusions. Furthermore, the levels of Hsp 70 and Hsp 27 were positively correlated with each other, and were also positively associated with the neutrophil chemoattractant, IL-8. Our findings suggested that Hsp 70 and Hsp 27 might be involved in the pathophysiology of pediatric OME. Copyright © 2017 Elsevier B.V. All rights reserved.

Conserved effects of salinity acclimation on thermal tolerance and hsp70 expression in divergent populations of threespine stickleback (Gasterosteus aculeatus).

Science.gov (United States)

Metzger, David C H; Healy, Timothy M; Schulte, Patricia M

2016-10-01

In natural environments, organisms must cope with complex combinations of abiotic stressors. Here, we use threespine stickleback (Gasterosteus aculeatus) to examine how changes in salinity affect tolerance of high temperatures. Threespine stickleback inhabit a range of environments that vary in both salinity and thermal stability making this species an excellent system for investigating interacting stressors. We examined the effects of environmental salinity on maximum thermal tolerance (CTMax) and 70 kDa heat shock protein (hsp70) gene expression using divergent stickleback ecotypes from marine and freshwater habitats. In both ecotypes, the CTMax of fish acclimated to 20 ppt was significantly higher compared to fish acclimated to 2 ppt. The effect of salinity acclimation on the expression of hsp70-1 and hsp70-2 was similar in both the marine and freshwater stickleback ecotype. There were differences in the expression profiles of hsp70-1 and hsp70-2 during heat shock, with hsp70-2 being induced earlier and to a higher level compared to hsp70-1. These data suggest that the two hsp70 isoforms may have functionally different roles in the heat shock response. Lastly, acute salinity challenge coupled with heat shock revealed that the osmoregulatory demands experienced during the heat shock response have a larger effect on the hsp70 expression profile than does the acclimation salinity.

Activation of catalase activity by a peroxisome-localized small heat shock protein Hsp17.6CII.

Science.gov (United States)

Li, Guannan; Li, Jing; Hao, Rong; Guo, Yan

2017-08-20

Plant catalases are important antioxidant enzymes and are indispensable for plant to cope with adverse environmental stresses. However, little is known how catalase activity is regulated especially at an organelle level. In this study, we identified that small heat shock protein Hsp17.6CII (AT5G12020) interacts with and activates catalases in the peroxisome of Arabidopsis thaliana. Although Hsp17.6CII is classified into the cytosol-located small heat shock protein subfamily, we found that Hsp17.6CII is located in the peroxisome. Moreover, Hsp17.6CII contains a novel non-canonical peroxisome targeting signal 1 (PTS1), QKL, 16 amino acids upstream from the C-terminus. The QKL signal peptide can partially locate GFP to peroxisome, and mutations in the tripeptide lead to the abolishment of this activity. In vitro catalase activity assay and holdase activity assay showed that Hsp17.6CII increases CAT2 activity and prevents it from thermal aggregation. These results indicate that Hsp17.6CII is a peroxisome-localized catalase chaperone. Overexpression of Hsp17.6CII conferred enhanced catalase activity and tolerance to abiotic stresses in Arabidopsis. Interestingly, overexpression of Hsp17.6CII in catalase-deficient mutants, nca1-3 and cat2 cat3, failed to rescue their stress-sensitive phenotypes and catalase activity, suggesting that Hsp17.6CII-mediated stress response is dependent on NCA1 and catalase activity. Overall, we identified a novel peroxisome-located catalase chaperone that is involved in plant abiotic stress resistance by activating catalase activity. Copyright © 2017 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Imbalance of Hsp70 family variants fosters tau accumulation.

Science.gov (United States)

Jinwal, Umesh K; Akoury, Elias; Abisambra, Jose F; O'Leary, John C; Thompson, Andrea D; Blair, Laura J; Jin, Ying; Bacon, Justin; Nordhues, Bryce A; Cockman, Matthew; Zhang, Juan; Li, Pengfei; Zhang, Bo; Borysov, Sergiy; Uversky, Vladimir N; Biernat, Jacek; Mandelkow, Eckhard; Gestwicki, Jason E; Zweckstetter, Markus; Dickey, Chad A

2013-04-01

Dysfunctional tau accumulation is a major contributing factor in tauopathies, and the heat-shock protein 70 (Hsp70) seems to play an important role in this accumulation. Several reports suggest that Hsp70 proteins can cause tau degradation to be accelerated or slowed, but how these opposing activities are controlled is unclear. Here we demonstrate that highly homologous variants in the Hsp70 family can have opposing effects on tau clearance kinetics. When overexpressed in a tetracycline (Tet)-based protein chase model, constitutive heat shock cognate 70 (Hsc70) and inducible Hsp72 slowed or accelerated tau clearance, respectively. Tau synergized with Hsc70, but not Hsp72, to promote microtubule assembly at nearly twice the rate of either Hsp70 homologue in reconstituted, ATP-regenerating Xenopus extracts supplemented with rhodamine-labeled tubulin and human recombinant Hsp72 and Hsc70. Nuclear magnetic resonance spectroscopy with human recombinant protein revealed that Hsp72 had greater affinity for tau than Hsc70 (I/I0 ratio difference of 0.3), but Hsc70 was 30 times more abundant than Hsp72 in human and mouse brain tissue. This indicates that the predominant Hsp70 variant in the brain is Hsc70, suggesting that the brain environment primarily supports slower tau clearance. Despite its capacity to clear tau, Hsp72 was not induced in the Alzheimer's disease brain, suggesting a mechanism for age-associated onset of the disease. Through the use of chimeras that blended the domains of Hsp72 and Hsc70, we determined that the reason for these differences between Hsc70 and Hsp72 with regard to tau clearance kinetics lies within their C-terminal domains, which are essential for their interactions with substrates and cochaperones. Hsp72 but not Hsc70 in the presence of tau was able to recruit the cochaperone ubiquitin ligase CHIP, which is known to facilitate the ubiquitination of tau, describing a possible mechanism of how the C-termini of these homologous Hsp70 variants

Involvement of p27CIP/KIP in HSP25 or HSP70 Mediated Adaptive Response by Low Dose Radiation

International Nuclear Information System (INIS)

Seo, Hang Rhan; Lee, Yoon Jin; Lee, Su Jae; Bae, Sang woo; Lee, Yun Sil

2005-01-01

Adaptive responses that reduce the harmful effects of subsequent exposure to high-dose radiation have demonstrated in chromosome aberration, cell survival, sister chromatid exchanges, micronucleus induction, mutation and neoplastic transformation. The mechanisms and conditions for the adaptive response to radiation have not been clarified, although the continuous production of free radicals from radiation and other sources has stimulated cells to evolve a repair system for chromosome breaks. An alteration of the DNA molecule triggers the repair system, and frequent activation may increase the general repair capacity, irrespective of the cause of the damage. Besides, cell cycle regulation systems, antioxidant defense systems, molecular chaperone or stress-response systems. Our previous data showed that when cells were preirradiated with 1cGy, they showed the adaptive response. A reduction of apoptosis by low-dose preirradiation is another potential mechanism for this effect. We previously demonstrated that mouse RIF cells, which did not induce HSP25 and HSP70 did not exhibit a adaptive response after 1cGy preirradiation. whereas the thermoresistant TR cells, which expressed inducible HSP25 and HSP70 showed a response. Moreover, when HSP70 and HSP25 were transfected to RIF cells, the cells acquired adaptive response. In this study, to elucidate the mechanisms in induction of adaptiveresponse, we compared cell cycle distribution by low dose radiation after HSP25 or HSP70 transfected cells and p27CIP/KIP is responsible for the different induction of adaptive response

Epigallocatechin-3-gallate suppresses the expression of HSP70 and HSP90 and exhibits anti-tumor activity in vitro and in vivo

International Nuclear Information System (INIS)

Tran, Phan LCHB; Kim, Soo-A; Choi, Hong Seok; Yoon, Jung-Hoon; Ahn, Sang-Gun

2010-01-01

Epigallocatechin-3-gallate (EGCG), one of the major catechins in green tea, is a potential chemopreventive agent for various cancers. The aim of this study was to examine the effect of EGCG on the expression of heat shock proteins (HSPs) and tumor suppression. Cell colony formation was evaluated by a soft agar assay. Transcriptional activity of HSP70 and HSP90 was determined by luciferase reporter assay. An EGCG-HSPs complex was prepared using EGCG attached to the cyanogen bromide (CNBr)-activated Sepharose 4B. In vivo effect of EGCG on tumor growth was examined in a xenograft model. Treatment with EGCG decreased cell proliferation and colony formation of MCF-7 human breast cancer cells. EGCG specifically inhibited the expression of HSP70 and HSP90 by inhibiting the promoter activity of HSP70 and HSP90. Pretreatment with EGCG increased the stress sensitivity of MCF-7 cells upon heat shock (44°C for 1 h) or oxidative stress (H 2 O 2 , 500 μM for 24 h). Moreover, treatment with EGCG (10 mg/kg) in a xenograft model resulted in delayed tumor incidence and reduced tumor size, as well as the inhibition of HSP70 and HSP90 expression. Overall, these findings demonstrate that HSP70 and HSP90 are potent molecular targets of EGCG and suggest EGCG as a drug candidate for the treatment of human cancer

Crystal structures of the ATPase domains of four human Hsp70 isoforms: HSPA1L/Hsp70-hom, HSPA2/Hsp70-2, HSPA6/Hsp70B', and HSPA5/BiP/GRP78.

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Magdalena Wisniewska

2010-01-01

Full Text Available The 70-kDa heat shock proteins (Hsp70 are chaperones with central roles in processes that involve polypeptide remodeling events. Hsp70 proteins consist of two major functional domains: an N-terminal nucleotide binding domain (NBD with ATPase activity, and a C-terminal substrate binding domain (SBD. We present the first crystal structures of four human Hsp70 isoforms, those of the NBDs of HSPA1L, HSPA2, HSPA5 and HSPA6. As previously with Hsp70 family members, all four proteins crystallized in a closed cleft conformation, although a slight cleft opening through rotation of subdomain IIB was observed for the HSPA5-ADP complex. The structures presented here support the view that the NBDs of human Hsp70 function by conserved mechanisms and contribute little to isoform specificity, which instead is brought about by the SBDs and by accessory proteins.This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

Crystal structures of the ATPase domains of four human Hsp70 isoforms: HSPA1L/Hsp70-hom, HSPA2/Hsp70-2, HSPA6/Hsp70B', and HSPA5/BiP/GRP78.

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Wisniewska, Magdalena; Karlberg, Tobias; Lehtiö, Lari; Johansson, Ida; Kotenyova, Tetyana; Moche, Martin; Schüler, Herwig

2010-01-11

The 70-kDa heat shock proteins (Hsp70) are chaperones with central roles in processes that involve polypeptide remodeling events. Hsp70 proteins consist of two major functional domains: an N-terminal nucleotide binding domain (NBD) with ATPase activity, and a C-terminal substrate binding domain (SBD). We present the first crystal structures of four human Hsp70 isoforms, those of the NBDs of HSPA1L, HSPA2, HSPA5 and HSPA6. As previously with Hsp70 family members, all four proteins crystallized in a closed cleft conformation, although a slight cleft opening through rotation of subdomain IIB was observed for the HSPA5-ADP complex. The structures presented here support the view that the NBDs of human Hsp70 function by conserved mechanisms and contribute little to isoform specificity, which instead is brought about by the SBDs and by accessory proteins. This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

Systematic Proteomic Identification of the Heat Shock Proteins (Hsp) that Interact with Estrogen Receptor Alpha (ERα) and Biochemical Characterization of the ERα-Hsp70 Interaction.

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Dhamad, Ahmed E; Zhou, Zhenqi; Zhou, Jianhong; Du, Yuchun

2016-01-01

Heat shock proteins (Hsps) are known to associate with estrogen receptors (ER) and regulate ER-mediated cell proliferation. Historically, the studies in this area have focused on Hsp90. However, some critical aspects of the Hsp-ERα interactions remain unclear. For example, we do not know which Hsps are the major or minor ERα interactants and whether or not different Hsp isoforms associate equally with ERα. In the present study, through a quantitative proteomic method we found that 21 Hsps and 3 Hsp cochaperones were associated with ERα in human 293T cells that were cultured in a medium containing necessary elements for cell proliferation. Four Hsp70s (Hsp70-1, Hsc70, Grp75, and Grp78) were the most abundant Hsps identified to associate with ERα, followed by two Hsp90s (Hsp90α and Hsp90β) and three Hsp110s (Hsp105, HspA4, and HspA4L). Hsp90α was found to be 2-3 times more abundant than Hsp90β in the ERα-containing complexes. Among the reported Hsp cochaperones, we detected prostaglandin E synthase 3 (p23), peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP51), and E3 ubiquitin-protein ligase CHIP (CHIP). Studies with the two most abundant ERα-associated Hsps, Hsp70-1 and Hsc70, using human breast cancer MCF7 cells demonstrate that the two Hsps interacted with ERα in both the cytoplasm and nucleus when the cells were cultured in a medium supplemented with fetal bovine serum and phenol red. Interestingly, the ERα-Hsp70-1/Hsc70 interactions were detected only in the cytoplasm but not in the nucleus under hormone starvation conditions, and stimulation of the starved cells with 17β-estradiol (E2) did not change this. In addition, E2-treatment weakened the ERα-Hsc70 interaction but had no effect on the ERα-Hsp70-1 interaction. Further studies showed that significant portions of Hsp70-1 and Hsc70 were associated with transcriptionally active chromatin and inactive chromatin, and the two Hsps interacted with ERα in both forms of the chromatins in MCF7 cells.

ANTI-HSP60 and ANTI-HSP70 antibody levels and micro/ macrovascular complications in type 1 diabetes: the EURODIAB Study

DEFF Research Database (Denmark)

Gruden, G.; Bruno, G.; Chaturvedi, N.

2009-01-01

OBJECTIVES: The heat shock proteins 60 and 70 (HSP60, HSP70) play an important role in cytoprotection. Under stress conditions they are released into the circulation and elicit an immune response. Anti-HSP60 and anti-HSP70 antibody levels have been associated with cardiovascular disease. Type 1......-control study from the EURODIAB Study of 531 type 1 diabetic patients was performed. SUBJECTS: Cases (n = 363) were defined as those with one or more complications of diabetes; control subjects (n = 168) were all those with no evidence of any complication. We measured anti-HSP60 and anti-HSP70 antibody levels...... quartiles were associated with a 47% reduced odds ratio of micro/macrovascular complications, independently of conventional risk factors, markers of inflammation and endothelial dysfunction [odds ratio (OR) = 0.53, 95% confidence intervals (CI): 0.28-1.02]. CONCLUSIONS: In this large cohort of type 1...

Adaptive capability as indicated by behavioral and physiological responses, plasma HSP70 level, and PBMC HSP70 mRNA expression in Osmanabadi goats subjected to combined (heat and nutritional) stressors

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Shilja, Shaji; Sejian, V.; Bagath, M.; Mech, A.; David, C. G.; Kurien, E. K.; Varma, Girish; Bhatta, Raghavendra

2016-09-01

A study was conducted to assess the impact of heat and nutritional stress simultaneously on the adaptive capability as indicated by behavioral and physiological responses, plasma heat shock protein 70 (HSP70) level, and peripheral blood mononuclear cells (PBMC) HSP70 gene expression in goats. Twenty-four adult Osmanabadi bucks (average body weight (BW) 16.0 kg) were used in the present study. The bucks were divided into four groups viz., C ( n = 6; control), HS ( n = 6; heat stress), NS ( n = 6; nutritional stress), and CS ( n = 6; combined stress). The study was conducted for a period of 45 days. C and HS bucks had ad libitum access to their feed while NS and CS bucks were under restricted feed (30 % intake of C bucks) to induce nutritional stress. The HS and CS bucks were exposed to solar radiation for 6 h a day between 10:00 a.m. and 4:00 p.m. to induce heat stress. The data was analyzed using repeated measures analysis of variance. The standing time differed significantly ( P < 0.01) between ad libitum fed groups (C and HS) and restricted feeding groups (NS and CS). The highest ( P < 0.01) lying time was recorded in the CS group while the lowest in the C and HS groups. The highest ( P < 0.01) drinking frequency was also recorded in the CS group. Water intake recorded was significantly ( P < 0.01) higher in both the HS and CS groups. The highest respiration rate (RR), pulse rate (PR), and rectal temperature (RT) during the afternoon were also recorded in the CS group. Further, skin temperature of the head, flank, and scrotum during the afternoon was also higher ( P < 0.01) in the CS group. In addition, both plasma HSP70 concentration and PBMC HSP70 messenger RNA (mRNA) transcript expression were also significantly ( P < 0.01) higher in the CS group. It can be concluded from this study that when two stressors occur simultaneously, they may have severe impact on adaptive capabilities of Osmanabadi bucks as compared to that would occur individually. Further, the

Expression of hsp70, hsp90 and hsf1 in the reef coral Acropora digitifera under prospective acidified conditions over the next several decades

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Masako Nakamura

2012-02-01

Ocean acidification is an ongoing threat for marine organisms due to the increasing atmospheric CO2 concentration. Seawater acidification has a serious impact on physiologic processes in marine organisms at all life stages. On the other hand, potential tolerance to external pH changes has been reported in coral larvae. Information about the possible mechanisms underlying such tolerance responses, however, is scarce. In the present study, we examined the effects of acidified seawater on the larvae of Acropora digitifera at the molecular level. We targeted two heat shock proteins, Hsp70 and Hsp90, and a heat shock transcription factor, Hsf1, because of their importance in stress responses and in early life developmental stages. Coral larvae were maintained under the ambient and elevated CO2 conditions that are expected to occur within next 100 years, and then we evaluated the expression of hsps and hsf1 by quantitative real-time polymerase chain reaction (PCR. Expression levels of these molecules significantly differed among target genes, but they did not change significantly between CO2 conditions. These findings indicate that the expression of hsps is not changed due to external pH changes, and suggest that tolerance to acidified seawater in coral larvae may not be related to hsp expression.

Molecular mechanisms of canalization: Hsp90 and beyond

Indian Academy of Sciences (India)

2006-03-26

Mar 26, 2006 ... ... and regulatory circuits, accounting for the important role Hsp90 plays in ... by environmental stress, genetic or pharmaceutical targeting of Hsp90. The ... Here, we discuss the role of Hsp90 in canalization and organismal ...

A C-terminal HSP90 inhibitor restores glucocorticoid sensitivity and relieves a mouse allograft model of Cushing disease.

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Riebold, Mathias; Kozany, Christian; Freiburger, Lee; Sattler, Michael; Buchfelder, Michael; Hausch, Felix; Stalla, Günter K; Paez-Pereda, Marcelo

2015-03-01

One function of the glucocorticoid receptor (GR) in corticotroph cells is to suppress the transcription of the gene encoding proopiomelanocortin (POMC), the precursor of the stress hormone adrenocorticotropin (ACTH). Cushing disease is a neuroendocrine condition caused by partially glucocorticoid-resistant corticotroph adenomas that excessively secrete ACTH, which leads to hypercortisolism. Mutations that impair GR function explain glucocorticoid resistance only in sporadic cases. However, the proper folding of GR depends on direct interactions with the chaperone heat shock protein 90 (HSP90, refs. 7,8). We show here that corticotroph adenomas overexpress HSP90 compared to the normal pituitary. N- and C-terminal HSP90 inhibitors act at different steps of the HSP90 catalytic cycle to regulate corticotroph cell proliferation and GR transcriptional activity. C-terminal inhibitors cause the release of mature GR from HSP90, which promotes its exit from the chaperone cycle and potentiates its transcriptional activity in a corticotroph cell line and in primary cultures of human corticotroph adenomas. In an allograft mouse model, the C-terminal HSP90 inhibitor silibinin showed anti-tumorigenic effects, partially reverted hormonal alterations, and alleviated symptoms of Cushing disease. These results suggest that the pathogenesis of Cushing disease caused by overexpression of heat shock proteins and consequently misregulated GR sensitivity may be overcome pharmacologically with an appropriate HSP90 inhibitor.

MiR-29b inhibits collagen maturation in hepatic stellate cells through down-regulating the expression of HSP47 and lysyl oxidase

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Zhang, Yifei; Ghazwani, Mohammed; Li, Jiang [Center for Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Sun, Ming; Stolz, Donna B. [Department of Cell Biology and Physiology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261 (United States); He, Fengtian [Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Third Military Medical University, Chongqing 400038 (China); Fan, Jie [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Xie, Wen [Center for Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Li, Song, E-mail: sol4@pitt.edu [Center for Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261 (United States)

2014-04-18

Highlights: • Enhanced HSP47 and LOX expression is associated with decreased miR-29b level in liver fibrosis. • miR-29b down-regulates HSP47 and LOX expression. • The suppression of HSP47 and LOX by miR-29b is mediated by putative sites at their 3′-UTRs. • miR-29b inhibits extracellular LOX activity and collagen maturation. - Abstract: Altered expression of miR-29b is implicated in the pathogenesis and progression of liver fibrosis. We and others previously demonstrated that miR-29b down-regulates the expression of several extracellular-matrix (ECM) genes including Col 1A1, Col 3A1 and Elastin via directly targeting their 3′-UTRs. However, whether or not miR-29b plays a role in the post-translational regulation of ECM biosynthesis has not been reported. Heat shock protein 47 (HSP47) and lysyl oxidase (LOX) are known to be essential for ECM maturation. In this study we have demonstrated that expression of HSP47 and LOX was significantly up-regulated in culture-activated primary rat hepatic stellate cells (HSCs), TGF-β stimulated LX-2 cells and liver tissue of CCl{sub 4}-treated mice, which was accompanied by a decrease of miR-29b level. In addition, over-expression of miR-29b in LX-2 cells resulted in significant inhibition on HSP47 and LOX expression. Mechanistically, miR-29b inhibited the expression of a reporter gene that contains the respective full-length 3′-UTR from HSP47 and LOX gene, and this inhibitory effect was abolished by the deletion of a putative miR-29b targeting sequence from the 3′-UTRs. Transfection of LX-2 cells with miR-29b led to abnormal collagen structure as shown by electron-microscopy, presumably through down-regulation of the expression of molecules involved in ECM maturation including HSP47 and LOX. These results demonstrated that miR-29b is involved in regulating the post-translational processing of ECM and fibril formation.

MiR-29b inhibits collagen maturation in hepatic stellate cells through down-regulating the expression of HSP47 and lysyl oxidase

International Nuclear Information System (INIS)

Zhang, Yifei; Ghazwani, Mohammed; Li, Jiang; Sun, Ming; Stolz, Donna B.; He, Fengtian; Fan, Jie; Xie, Wen; Li, Song

2014-01-01

Highlights: • Enhanced HSP47 and LOX expression is associated with decreased miR-29b level in liver fibrosis. • miR-29b down-regulates HSP47 and LOX expression. • The suppression of HSP47 and LOX by miR-29b is mediated by putative sites at their 3′-UTRs. • miR-29b inhibits extracellular LOX activity and collagen maturation. - Abstract: Altered expression of miR-29b is implicated in the pathogenesis and progression of liver fibrosis. We and others previously demonstrated that miR-29b down-regulates the expression of several extracellular-matrix (ECM) genes including Col 1A1, Col 3A1 and Elastin via directly targeting their 3′-UTRs. However, whether or not miR-29b plays a role in the post-translational regulation of ECM biosynthesis has not been reported. Heat shock protein 47 (HSP47) and lysyl oxidase (LOX) are known to be essential for ECM maturation. In this study we have demonstrated that expression of HSP47 and LOX was significantly up-regulated in culture-activated primary rat hepatic stellate cells (HSCs), TGF-β stimulated LX-2 cells and liver tissue of CCl 4 -treated mice, which was accompanied by a decrease of miR-29b level. In addition, over-expression of miR-29b in LX-2 cells resulted in significant inhibition on HSP47 and LOX expression. Mechanistically, miR-29b inhibited the expression of a reporter gene that contains the respective full-length 3′-UTR from HSP47 and LOX gene, and this inhibitory effect was abolished by the deletion of a putative miR-29b targeting sequence from the 3′-UTRs. Transfection of LX-2 cells with miR-29b led to abnormal collagen structure as shown by electron-microscopy, presumably through down-regulation of the expression of molecules involved in ECM maturation including HSP47 and LOX. These results demonstrated that miR-29b is involved in regulating the post-translational processing of ECM and fibril formation

Substrate Discrimination by ClpB and Hsp104

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Danielle M. Johnston

2017-05-01

Full Text Available ClpB of E. coli and yeast Hsp104 are homologous molecular chaperones and members of the AAA+ (ATPases Associated with various cellular Activities superfamily of ATPases. They are required for thermotolerance and function in disaggregation and reactivation of aggregated proteins that form during severe stress conditions. ClpB and Hsp104 collaborate with the DnaK or Hsp70 chaperone system, respectively, to dissolve protein aggregates both in vivo and in vitro. In yeast, the propagation of prions depends upon Hsp104. Since protein aggregation and amyloid formation are associated with many diseases, including neurodegenerative diseases and cancer, understanding how disaggregases function is important. In this study, we have explored the innate substrate preferences of ClpB and Hsp104 in the absence of the DnaK and Hsp70 chaperone system. The results suggest that substrate specificity is determined by nucleotide binding domain-1.

Heterologous expression of three Camellia sinensis small heat shock protein genes confers temperature stress tolerance in yeast and Arabidopsis thaliana.

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Wang, Mingle; Zou, Zhongwei; Li, Qinghui; Xin, Huahong; Zhu, Xujun; Chen, Xuan; Li, Xinghui

2017-07-01

CsHSP17.7, CsHSP18.1, and CsHSP21.8 expressions are induced by heat and cold stresses, and CsHSP overexpression confers tolerance to heat and cold stresses in transgenic Pichia pastoris and Arabidopsis thaliana. Small heat shock proteins (sHSPs) are crucial for protecting plants against biotic and abiotic stresses, especially heat stress. However, knowledge concerning the functions of Camellia sinensis sHSP in heat and cold stresses remains poorly understood. In this study, three C. sinensis sHSP genes (i.e., CsHSP17.7, CsHSP18.1, and CsHSP21.8) were isolated and characterized using suppression subtractive hybridization (SSH) technology. The CsHSPs expression levels in C. sinensis leaves were significantly up-regulated by heat and cold stresses. Phylogenetic analyses revealed that CsHSP17.7, CsHSP18.1, and CsHSP21.8 belong to sHSP Classes I, II, and IV, respectively. Heterologous expression of the three CsHSP genes in Pichia pastoris cells enhanced heat and cold stress tolerance. When exposed to heat and cold treatments, transgenic Arabidopsis thaliana plants overexpressing CsHSP17.7, CsHSP18.1, and CsHSP21.8 had lower malondialdehyde contents, ion leakage, higher proline contents, and transcript levels of stress-related genes (e.g., AtPOD, AtAPX1, AtP5CS2, and AtProT1) compared with the control line. In addition, improved seed germination vigor was also observed in the CsHSP-overexpressing seeds under heat stress. Taken together, our results suggest that the three identified CsHSP genes play key roles in heat and cold tolerance.

Tumour eradication using synchronous thermal ablation and Hsp90 chemotherapy with protein engineered triblock biopolymer-geldanamycin conjugates.

Science.gov (United States)

Chen, Yizhe; Youn, Pilju; Pysher, Theodore J; Scaife, Courtney L; Furgeson, Darin Y

2014-12-01

Hepatocellular carcinoma (HCC) suffers high tumour recurrence rate after thermal ablation. Heat shock protein 90 (Hsp90) induced post-ablation is critical for tumour survival and progression. A combination therapy of thermal ablation and polymer conjugated Hsp90 chemotherapy was designed and evaluated for complete tumour eradication of HCC. A thermo-responsive, elastin-like polypeptide (ELP)-based tri-block biopolymer was developed and conjugated with a potent Hsp90 inhibitor, geldanamycin (GA). The anti-cancer efficacy of conjugates was evaluated in HCC cell cultures with and without hyperthermia (43 °C). The conjugates were also administered twice weekly in a murine HCC model as a single treatment or in combination with single electrocautery as the ablation method. ELP-GA conjugates displayed enhanced cytotoxicity in vitro and effective heat shock inhibition under hyperthermia. The conjugates alone significantly slowed the tumour growth without systemic toxicity. Four doses of thermo-responsive ELP-GA conjugates with concomitant simple electrocautery accomplished significant Hsp90 inhibition and sustained tumour suppression. Hsp90 inhibition plays a key role in preventing the recurrence of HCC, and the combination of ablation with targeted therapy holds great potential to improve prognosis and survival of HCC patients.

Plasma Hsp72 (HSPA1A) and Hsp27 (HSPB1) expression under heat stress: influence of exercise intensity.

Science.gov (United States)

Périard, Julien D; Ruell, Patricia; Caillaud, Corinne; Thompson, Martin W

2012-05-01

Extracellular heat-shock protein 72 (eHsp72) expression during exercise-heat stress is suggested to increase with the level of hyperthermia attained, independent of the rate of heat storage. This study examined the influence of exercise at various intensities to elucidate this relationship, and investigated the association between eHsp72 and eHsp27. Sixteen male subjects cycled to exhaustion at 60% and 75% of maximal oxygen uptake in hot conditions (40°C, 50% RH). Core temperature, heart rate, oxidative stress, and blood lactate and glucose levels were measured to determine the predictor variables associated with eHsp expression. At exhaustion, heart rate exceeded 96% of maximum in both conditions. Core temperature reached 39.7°C in the 60% trial (58.9 min) and 39.0°C in the 75% trial (27.2 min) (P exercise may relate to the duration (i.e., core temperature attained) and intensity (i.e., rate of increase in core temperature) of exercise. Thus, the immuno-inflammatory release of eHsp72 and eHsp27 in response to exercise in the heat may be duration and intensity dependent.

Variability within a pea core collection of LEAM and HSP22, two mitochondrial seed proteins involved in stress tolerance.

Science.gov (United States)

Avelange-Macherel, Marie-Hélène; Payet, Nicole; Lalanne, David; Neveu, Martine; Tolleter, Dimitri; Burstin, Judith; Macherel, David

2015-07-01

LEAM, a late embryogenesis abundant protein, and HSP22, a small heat shock protein, were shown to accumulate in the mitochondria during pea (Pisum sativum L.) seed development, where they are expected to contribute to desiccation tolerance. Here, their expression was examined in seeds of 89 pea genotypes by Western blot analysis. All genotypes expressed LEAM and HSP22 in similar amounts. In contrast with HSP22, LEAM displayed different isoforms according to apparent molecular mass. Each of the 89 genotypes harboured a single LEAM isoform. Genomic and RT-PCR analysis revealed four LEAM genes differing by a small variable indel in the coding region. These variations were consistent with the apparent molecular mass of each isoform. Indels, which occurred in repeated domains, did not alter the main properties of LEAM. Structural modelling indicated that the class A α-helix structure, which allows interactions with the mitochondrial inner membrane in the dry state, was preserved in all isoforms, suggesting functionality is maintained. The overall results point out the essential character of LEAM and HSP22 in pea seeds. LEAM variability is discussed in terms of pea breeding history as well as LEA gene evolution mechanisms. © 2014 John Wiley & Sons Ltd.

De novo characterisation of the greenlip abalone transcriptome (Haliotis laevigata) with a focus on the heat shock protein 70 (HSP70) family.

Science.gov (United States)

Shiel, Brett P; Hall, Nathan E; Cooke, Ira R; Robinson, Nicholas A; Strugnell, Jan M

2015-02-01

Abalone (Haliotis) are economically important molluscs for fisheries and aquaculture industries worldwide. Despite this, genomic resources for abalone and molluscs are still limited. Here we present a description and functional annotation of the greenlip abalone (Haliotis laevigata) transcriptome. We present a focused analysis on the heat shock protein 70 (HSP70) family of genes with putative functions affecting temperature stress and immunity. A total of ~38 million paired end Illumina reads were obtained, resulting in a Trinity assembly of 222,172 contigs with minimum length of 200 base pairs and maximum length of 33 kilobases. The 20,702 contigs were annotated with gene descriptions by BLAST. We created a program to maximise the number of functionally annotated genes, and over 10,000 contigs were assigned Gene ontologies (GO terms). By using CateGOrizer, immunity related GO terms for stressors such as heat, hypoxia, oxidative stress and wounding received the highest counts. Twenty-six contigs with homology to the HSP70 family of genes were identified. Ninety-one putative single-nucleotide polymorphisms were observed in the abalone HSP70 contigs. Eleven of these were considered non-synonymous. The annotated transcriptome described in this study will be a useful basis for future work investigating the genetic response of abalone to stress.

The Exported Chaperone PfHsp70x Is Dispensable for the Plasmodium falciparum Intraerythrocytic Life Cycle.

Science.gov (United States)

Cobb, David W; Florentin, Anat; Fierro, Manuel A; Krakowiak, Michelle; Moore, Julie M; Muralidharan, Vasant

2017-01-01

Export of parasite proteins into the host erythrocyte is essential for survival of Plasmodium falciparum during its asexual life cycle. While several studies described key factors within the parasite that are involved in protein export, the mechanisms employed to traffic exported proteins within the host cell are currently unknown. Members of the Hsp70 family of chaperones, together with their Hsp40 cochaperones, facilitate protein trafficking in other organisms, and are thus likely used by P. falciparum in the trafficking of its exported proteins. A large group of Hsp40 proteins is encoded by the parasite and exported to the host cell, but only one Hsp70, P. falciparum Hsp70x (PfHsp70x), is exported with them. PfHsp70x is absent in most Plasmodium species and is found only in P. falciparum and closely related species that infect apes. Herein, we have utilized clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing in P. falciparum to investigate the essentiality of PfHsp70x. We show that parasitic growth was unaffected by knockdown of PfHsp70x using both the dihydrofolate reductase (DHFR)-based destabilization domain and the glmS ribozyme system. Similarly, a complete gene knockout of PfHsp70x did not affect the ability of P. falciparum to proceed through its intraerythrocytic life cycle. The effect of PfHsp70x knockdown/knockout on the export of proteins to the host red blood cell (RBC), including the critical virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1), was tested, and we found that this process was unaffected. These data show that although PfHsp70x is the sole exported Hsp70, it is not essential for the asexual development of P. falciparum . IMPORTANCE Half of the world's population lives at risk for malaria. The intraerythrocytic life cycle of Plasmodium spp. is responsible for clinical manifestations of malaria; therefore, knowledge of the parasite's ability to survive within the erythrocyte is

A novel TaqMan® assay for Nosema ceranae quantification in honey bee, based on the protein coding gene Hsp70.

Science.gov (United States)

Cilia, Giovanni; Cabbri, Riccardo; Maiorana, Giacomo; Cardaio, Ilaria; Dall'Olio, Raffaele; Nanetti, Antonio

2018-04-01

Nosema ceranae is now a widespread honey bee pathogen with high incidence in apiculture. Rapid and reliable detection and quantification methods are a matter of concern for research community, nowadays mainly relying on the use of biomolecular techniques such as PCR, RT-PCR or HRMA. The aim of this technical paper is to provide a new qPCR assay, based on the highly-conserved protein coding gene Hsp70, to detect and quantify the microsporidian Nosema ceranae affecting the western honey bee Apis mellifera. The validation steps to assess efficiency, sensitivity, specificity and robustness of the assay are described also. Copyright © 2018 Elsevier GmbH. All rights reserved.

Effects of chronic thermal stress on growth performance, carcass traits, antioxidant indices and the expression of HSP70, growth hormone and superoxide dismutase genes in two broiler strains.

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Roushdy, Elshimaa M; Zaglool, Asmaa W; El-Tarabany, Mahmoud S

2018-05-01

The objective was to investigate the effects of genetic type and the duration of chronic thermal stress (36 °C) on the growing efficiency, carcass traits, antioxidant status, and the expression of liver heat shock protein 70 (HSP70), growth hormone (GH) and superoxide dismutase (SOD) genes. Two hundred and seventy one-day-old chicks (135 male chicks of each breed; Ross 308 and Cobb 500) were used in this work. On the 21st day of age, birds were allocated randomly into 3 equal groups till the 42 days of age (CON:raised in a thermoneutral condition; HS 1 and HS 2 groups were subjected to 4 and 6 h of daily thermal stress, respectively). Regardless of genetic type, thermal stress decreased the dressing percentage in broilers when compared with the thermoneutral conditions (p = 0.039). In both broiler strains, thermal stress for 6 h (HS 2 ) increased the heterophil to lymphocyte ratio (p = 0.036) and the serum albumin, cholesterol and triglyceride levels (p = 0.023, 0.012 and 0.005, respectively) compared with the thermoneutral group. Under the thermonuteral and heat stress conditions, the Ross broiler chickens showed a significant lower serum triiodothyronine level compared with the Cobb boilers (p = 0.042). It is interesting to note that the expression of HSP70 in the liver of heat-stressed Ross broilers, either 4 or 6 h, was significantly (p = 0.002) higher than that reported in the heat-stressed Cobb broilers. In both broiler strains, the thermal stress for 6 h up-regulate the expression of SOD gene (p = 0.001), but down-regulate the expression of GH gene (p = 0.021) when compared with the CON group. In conclusion, chronic thermal stress down-regulate the mRNA expression of liver GH, concomitantly with an increase in the expression of HSP70 and SOD genes in both broiler strains. This could be useful in the identification of molecular genetic markers to assist in selecting broilers that are more tolerant to heat stress

HSP20 phosphorylation and airway smooth muscle relaxation

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Mariam Ba

2009-06-01

Full Text Available Mariam Ba1, Cherie A Singer1, Manoj Tyagi2, Colleen Brophy3, Josh E Baker4, Christine Cremo4, Andrew Halayko5, William T Gerthoffer21Department of Pharmacology, University of Nevada School of Medicine, Reno, NV, USA; 2Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, AL, USA; 3Harrington Department of Biochemistry, Arizona State University, Tempe, AZ, USA; 4Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV, USA; 5Departments of Physiology and Internal Medicine, University of Manitoba, Winnipeg, MB, CanadaAbstract: HSP20 (HSPB6 is a small heat shock protein expressed in smooth muscles that is hypothesized to inhibit contraction when phosphorylated by cAMP-dependent protein kinase. To investigate this hypothesis in airway smooth muscle (ASM we showed that HSP20 was constitutively expressed as well as being inducible in cultured hASM cells by treatment with 1 µM isoproterenol or 10 µM salmeterol. In contrast, a mixture of proinflammatory mediators (interleukin-1β, tumor necrosis factor α, and interferon γ inhibited expression of HSP20 by about 50% in 48 hours. To determine whether phosphorylation of HSP20 is sufficient to induce relaxation, canine tracheal smooth muscle was treated with a cell permeant phosphopeptide that mimics the phosphorylation of HSP20. The HSP20 phosphopeptide antagonized carbacholinduced contraction by 60% with no change in myosin light chain phosphorylation. Recombinant full length HSP20 inhibited skeletal actin binding to smooth muscle myosin subfragment 1 (S1, and recombinant cell permeant TAT-HSP20 S16D mutant reduced F-actin filaments in cultured hASM cells. Carbachol stimulation of canine tracheal smooth muscle tissue caused redistribution of HSP20 from large macromolecular complexes (200–500 kDa to smaller complexes (<60 kDa. The results are consistent with HSP20 expression and macromolecular structure being dynamically regulated in airway

Durum wheat diversity for heat stress tolerance during inflorescence emergence is correlated to TdHSP101C expression in early developmental stages.

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Miguel Bento

Full Text Available The predicted world population increase along with climate changes threatens sustainable agricultural supply in the coming decades. It is therefore vital to understand crops diversity associated to abiotic stress response. Heat stress is considered one of the major constrains on crops productivity thus it is essential to develop new approaches for a precocious and rigorous evaluation of varietal diversity regarding heat tolerance. Plant cell membrane thermostability (CMS is a widely used method for wheat thermotolerance assessment although its limitations require complementary solutions. In this work we used CMS assay and explored TdHSP101C genes as an additional tool for durum wheat screening. Genomic and transcriptomic analyses of TdHSP101C genes were performed in varieties with contrasting CMS results and further correlated with heat stress tolerance during fertilization and seed development. Although the durum wheat varieties studied presented a very high homology on TdHSP101C genes (>99% the transcriptomic assessment allowed the discrimination between varieties with good CMS results and its correlation with differential impacts of heat treatment during inflorescence emergence and seed development on grain yield. The evidences here reported indicate that TdHSP101C transcription levels induced by heat stress in fully expanded leaves may be a promising complementary screening tool to discriminate between durum wheat varieties identified as thermotolerant through CMS.

Hsp90 inhibition differentially destabilises MAP kinase and TGF-beta signalling components in cancer cells revealed by kinase-targeted chemoproteomics

International Nuclear Information System (INIS)

Haupt, Armin; Dahl, Andreas; Lappe, Michael; Lehrach, Hans; Gonzalez, Cayetano; Drewes, Gerard; Lange, Bodo MH; Joberty, Gerard; Bantscheff, Marcus; Fröhlich, Holger; Stehr, Henning; Schweiger, Michal R; Fischer, Axel; Kerick, Martin; Boerno, Stefan T

2012-01-01

The heat shock protein 90 (Hsp90) is required for the stability of many signalling kinases. As a target for cancer therapy it allows the simultaneous inhibition of several signalling pathways. However, its inhibition in healthy cells could also lead to severe side effects. This is the first comprehensive analysis of the response to Hsp90 inhibition at the kinome level. We quantitatively profiled the effects of Hsp90 inhibition by geldanamycin on the kinome of one primary (Hs68) and three tumour cell lines (SW480, U2OS, A549) by affinity proteomics based on immobilized broad spectrum kinase inhibitors ('kinobeads'). To identify affected pathways we used the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway classification. We combined Hsp90 and proteasome inhibition to identify Hsp90 substrates in Hs68 and SW480 cells. The mutational status of kinases from the used cell lines was determined using next-generation sequencing. A mutation of Hsp90 candidate client RIPK2 was mapped onto its structure. We measured relative abundances of > 140 protein kinases from the four cell lines in response to geldanamycin treatment and identified many new potential Hsp90 substrates. These kinases represent diverse families and cellular functions, with a strong representation of pathways involved in tumour progression like the BMP, MAPK and TGF-beta signalling cascades. Co-treatment with the proteasome inhibitor MG132 enabled us to classify 64 kinases as true Hsp90 clients. Finally, mutations in 7 kinases correlate with an altered response to Hsp90 inhibition. Structural modelling of the candidate client RIPK2 suggests an impact of the mutation on a proposed Hsp90 binding domain. We propose a high confidence list of Hsp90 kinase clients, which provides new opportunities for targeted and combinatorial cancer treatment and diagnostic applications

Improved Metabolic Control in Diabetes, HSP60, and Proinflammatory Mediators

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Claudio Blasi

2012-01-01

Full Text Available The diabetes-atherosclerosis relationship remains to be fully defined. Repeated prolonged hyperglycemia, increased ROS production and endothelial dysfunction are important factors. One theory is that increased blood levels of heat shock protein (HSP60 are proinflammatory, through activation of innate immunity, and contribute to the progression of vascular disease. It was hypothesized that improvement of diabetes control in patients presenting with metabolic syndrome would lower HSP60, and anti-HSP60 antibody levels and decrease inflammatory markers. Paired sera of 17 Italian patients, before and after intensive treatment, were assayed for cytokines, HSP60 and anti-HSP60 antibodies. As expected, intensive treatment was associated with a decrease in HgbA1C (P<0.001 and BMI (P<0.001. After treatment, there was a significant decrease in IL-6 (P<0.05. HSP60 levels were before treatment −6.9+1.9, after treatment −7.1+2.0 ng/mL (P=ns. Overall HSP60 concentrations were lower than published reports. Anti-HSP60 antibody titers were high and did not decrease with treatment. In conclusion, improvement of diabetic control did not alter HSP60 concentrations or antiHSP60 antibody titers, but led to a reduction of IL-6 levels.

Systemic and mucosal immunization with Candida albicans hsp90 elicits hsp90-specific humoral response in vaginal mucosa which is further enhanced during experimental vaginal candidiasis.

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Raska, Milan; Belakova, Jana; Horynova, Milada; Krupka, Michal; Novotny, Jiri; Sebestova, Martina; Weigl, Evzen

2008-08-01

The Candida albicans heat shock protein 90 kDa (hsp90-CA) is an important target for protective antibodies in disseminated candidiasis of experimental mice and humans. Hsp90-CA is present in the cell wall of Candida pseudohyphae or hyphae--typical pathogenic morphotypes in both mucosal and systemic Candida infections. However, the potential protective effects of hsp90-CA-specific antibodies in vaginal candidiasis has not yet been reported. In the present study we used various vaccine formulations (recombinant hsp90-CA protein and hsp90-CA-encoding DNA vaccine) and routes of administration (intradermal, intranasal, and intravenous) to induce both hsp90-CA-specific systemic and vaginal mucosa immune responses in experimental BALB/c mice. The results showed that intradermal recombinant hsp90-CA protein priming, followed by intranasal or intradermal recombinant hsp90-CA protein boosting induced significant increases in both serum and vaginal hsp90-CA-specific IgG and IgA antibodies compared to the control group, as well as enhanced hsp90-CA-specific splenocyte responses in vitro. In the intradermally boosted group, subsequent experimental vaginal Candida infection induced additional increases in the hsp90-CA specific IgG isotype, suggesting that Candida has the ability to induce a local hsp90-specific antibody (IgG) response during vulvovaginal candidiasis. Further work is required to elucidate the importance of immunity to highly conserved antigens during infection of the human female reproductive tract where a balance between immunity to and tolerance for commonly antigens such as hsp90 is necessary for the maintenance of fertility.

[Antiarrhythmic effect of oligonucleotides accompanied by activation of HSP70 protein in the heart of rats].

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Kruglov, S V; Terekhina, O L; Smirnova, E A; Kashaeva, O V; Belkina, L M

2015-01-01

The mechanisms of the protective effect of oligonucleotides (OGN) during pathological processes are poorlyunderstood. The goal of this work was to study the effect of OGN on arrhythmias induced by myocardial ischemia and reperfusion, and the HSP70 level in the heart. As a source of OGN was used the drug "Derinat" ("Technomedservis", Russia). In male Wistar rats were pre-treated the drug for 7 days (i/m, 7.5 mg/kg).The intensity of the arrhythmias was assessed by ECG during 10 min occlusion of the left coronary artery and subsequent 5 min of reperfusion. Protein HSP70 determined in the left ventricle of the heart by Western-blot analysis. During ischemia, this drug reduced duration of extrasystolia by 13 times and the incidence of ventricular tachycardia by 1.5 times. During reperfusion the drug reduced the incidence of ventricular fibrillation, a more than 2-fold, as compared with the control (respectively 23% vs 56%) and by 5 times its duration (8,4 ± 2,3 48,1 ± sec vs 18 7 sec). "Derinat" increased the HSP70 level in the heart by 65% compared with control. These data support the fact that the activation of HSP70 synthesis, induced by OGN is one of the mechanisms that increases the heart resistance to the ischemic and reperfusion damages.

Structure, Function and Regulation of the Hsp90 Machinery

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Jing Li

2013-06-01

Full Text Available Heat shock protein 90 (Hsp90 is an ATP-dependent molecular chaperone which is essential in eukaryotes. It is required for the activation and stabilization of a wide variety of client proteins and many of them are involved in important cellular pathways. Since Hsp90 affects numerous physiological processes such as signal transduction, intracellular transport, and protein degradation, it became an interesting target for cancer therapy. Structurally, Hsp90 is a flexible dimeric protein composed of three different domains which adopt structurally distinct conformations. ATP binding triggers directionality in these conformational changes and leads to a more compact state. To achieve its function, Hsp90 works together with a large group of cofactors, termed co-chaperones. Co-chaperones form defined binary or ternary complexes with Hsp90, which facilitate the maturation of client proteins. In addition, posttranslational modifications of Hsp90, such as phosphorylation and acetylation, provide another level of regulation. They influence the conformational cycle, co-chaperone interaction, and inter-domain communications. In this review, we discuss the recent progress made in understanding the Hsp90 machinery.

The Complex Function of Hsp70 in Metastatic Cancer

Energy Technology Data Exchange (ETDEWEB)

Juhasz, Kata; Lipp, Anna-Maria; Nimmervoll, Benedikt; Sonnleitner, Alois; Hesse, Jan; Haselgruebler, Thomas; Balogi, Zsolt, E-mail: zsolt.balogi@cbl.at [Center for Advanced Bioanalysis GmbH, Gruberstr. 40-42, A-4020 Linz (Austria)

2013-12-20

Elevated expression of the inducible heat shock protein 70 (Hsp70) is known to correlate with poor prognosis in many cancers. Hsp70 confers survival advantage as well as resistance to chemotherapeutic agents, and promotes tumor cell invasion. At the same time, tumor-derived extracellular Hsp70 has been recognized as a “chaperokine”, activating antitumor immunity. In this review we discuss localization dependent functions of Hsp70 in the context of invasive cancer. Understanding the molecular principles of metastasis formation steps, as well as interactions of the tumor cells with the microenvironment and the immune system is essential for fighting metastatic cancer. Although Hsp70 has been implicated in different steps of the metastatic process, the exact mechanisms of its action remain to be explored. Known and potential functions of Hsp70 in controlling or modulating of invasion and metastasis are discussed.

Accumulation of hepatic Hsp70 and plasma cortisol in Oreochromis ...

African Journals Online (AJOL)

The hepatic isoforms Hsp70, Hsp74 and Hsp76 were identified and quantified from copper exposures. Long-term DDT exposure did not result in significant induction of hepatic Hsp70. An increase in plasma cortisol concentration was associated with a decrease in heat shock protein accumulation after cadmium exposure, ...

真蛸热休克蛋白90基因(HSP90)的克隆及表达%Molecular cloning and feature analysis of heat shock protein 90 ( HSP90 ) from Octopus vulgaris

Institute of Scientific and Technical Information of China (English)

孙田田; 苏永全; 洪婧妮; 邬阳; 张曼; 毛勇

2012-01-01

为深入了解真蛸热应激响应机制,实验以真蛸的应激蛋白为研究对象,借助同源扩增和cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术获得真蛸热休克蛋白90( HSP90)的cDNA全序列(2 709 bp),它包含119 bp的5′非编码区(untranslated regions,UTR)、454 bp的3′UTR和2 136 bp的开放阅读框(opening reading frame,ORF),ORF共编码71 1个氨基酸,推算其分子量为81.5 ku,理论等电点为5.09.同源分析显示,所推导的氨基酸序列高度保守,并且含有HSP90家族特有的5个保守信号序列区域.实时荧光定量PCR分析表明,该基因在所有选取的组织中均有表达,肝组织中表达量最高.%Octopus vulgaris,which owns a number of unique stress protection mechanisms is considered to be the most intelligent species of all invertebrates. A 2 709 bp full-length cDNA sequence of heat shock protein 90 ( HSP90 ) gene from O. vulgaris was obtained by homologous amplification and rapid amplification of cDNA ends (RACE). It consisted of a 119 bp 5'untranslated region (UTR) ,a2 136 bp open reading frame(ORF)and a 454 bp 3'UTR. The inferred amino acids sequence was composed of 711 amino acids, whose molecular weight and isoelectric point were 81. 5 ku and 5. 09, respectively. Homologous analysis showed the inferred amino acids sequence was highly conserved and contained five classical HSP90 signature sequences. Real-time quantitative PCR ( RT-PCR) results displayed HSP90 expressed in every tissues chosen, with the most in liver. This study which was the first time to obtain HSP90 gene from Cephalopod played an important role in studying stress mechanisms on the most intelligent invertebrate.

Preventative vaccine-loaded mannosylated chitosan nanoparticles intended for nasal mucosal delivery enhance immune responses and potent tumor immunity.

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Yao, Wenjun; Peng, Yixing; Du, Mingzhu; Luo, Juan; Zong, Li

2013-08-05

Chitosan (CS) has been extensively used as a protein drug and gene delivery carrier, but its delivery efficiency is unsatisfactory. In this study, a mannose ligand was used to modify CS, which could enhance the delivery efficiency of CS via mannose receptor-mediated endocytosis. A preventative anti-GRP DNA vaccine (pCR3.1-VS-HSP65-TP-GRP6-M2, pGRP) was condensed with mannosylated chitosan (MCS) to form MCS/pGRP nanoparticles. Nanoparticles were intranasally administered in a subcutaneous mice prostate carcinoma model to evaluate the efficacy on inhibition of the growth of tumor cells. The titers of anti-GRP IgG that lasted for 11 weeks were significantly higher than that for administration of CS/pGRP nanoparticles (p intramuscular administration of a pGRP solution (p nanoparticles could suppress the growth of tumor cells. The average tumor weight (0.79 ± 0.30 g) was significantly lower than that in the CS/pGRP nanoparticle group (1.69 ± 0.15 g) (p nanoparticles bound with C-type lectin receptors on macrophages. MCS was an efficient targeting gene delivery carrier and could be used in antitumor immunotherapy.

PREFERENTIAL SECRETION OF INDUCIBLE HSP70 BY VITILIGO MELANOCYTES UNDER STRESS

Science.gov (United States)

Mosenson, Jeffrey A.; Flood, Kelsey; Klarquist, Jared; Eby, Jonathan M.; Koshoffer, Amy; Boissy, Raymond E.; Overbeck, Andreas; C.Tung, Rebecca; Poole, I. Caroline Le

2014-01-01

SUMMARY Inducible HSP70 (HSP70i) chaperones peptides from stressed cells, protecting them from apoptosis. Upon extracellular release, HSP70i serves an adjuvant function, enhancing immune responses to bound peptides. We questioned whether HSP70i differentially protects control and vitiligo melanocytes from stress and subsequent immune responses. We compared expression of HSP70i in skin samples, evaluated the viability of primary vitiligo and control melanocytes exposed to bleaching phenols, and measured secreted HSP70i. We determined whether HSP70i traffics to melanosomes to contact immunogenic proteins by cell fractionation, western blotting, electron microscopy and confocal microscopy. Viability of vitiligo and control melanocytes was equally affected under stress. However, vitiligo melanocytes secreted increased amounts of HSP70i in response to MBEH, corroborating with aberrant HSP70i expression in patient skin. Intracellular HSP70i colocalized with melanosomes, and more so in response to MBEH in vitiligo melanocytes. Thus whereas either agent is cytotoxic to melanocytes, MBEH preferentially induces immune responses to melanocytes. PMID:24354861

Oligomeric structure and chaperone-like activity of Drosophila melanogaster mitochondrial small heat shock protein Hsp22 and arginine mutants in the alpha-crystallin domain.

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Dabbaghizadeh, Afrooz; Finet, Stéphanie; Morrow, Genevieve; Moutaoufik, Mohamed Taha; Tanguay, Robert M

2017-07-01

The structure and chaperone function of DmHsp22WT, a small Hsp of Drosophila melanogaster localized within mitochondria were examined. Mutations of conserved arginine mutants within the alpha-crystallin domain (ACD) domain (R105G, R109G, and R110G) were introduced, and their effects on oligomerization and chaperone function were assessed. Arginine to glycine mutations do not induce significant changes in tryptophan fluorescence, and the mutated proteins form oligomers that are of equal or smaller size than the wild-type protein. They all form oligomer with one single peak as determined by size exclusion chromatography. While all mutants demonstrate the same efficiency as the DmHsp22WT in a DTT-induced insulin aggregation assay, all are more efficient chaperones to prevent aggregation of malate dehydrogenase. Arginine mutants of DmHsp22 are efficient chaperones to retard aggregation of CS and Luc. In summary, this study shows that mutations of arginine to glycine in DmHsp22 ACD induce a number of structural changes, some of which differ from those described in mammalian sHsps. Interestingly, only the R110G-DmHsp22 mutant, and not the expected R109G equivalent to human R140-HspB1, R116-HspB4, and R120-HspB5, showed different structural properties compared with the DmHsp22WT.

A laser pointer driven microheater for precise local heating and conditional gene regulation in vivo. Microheater driven gene regulation in zebrafish

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Achermann Marc

2009-12-01

Full Text Available Abstract Background Tissue heating has been employed to study a variety of biological processes, including the study of genes that control embryonic development. Conditional regulation of gene expression is a particularly powerful approach for understanding gene function. One popular method for mis-expressing a gene of interest employs heat-inducible heat shock protein (hsp promoters. Global heat shock of hsp-promoter-containing transgenic animals induces gene expression throughout all tissues, but does not allow for spatial control. Local heating allows for spatial control of hsp-promoter-driven transgenes, but methods for local heating are cumbersome and variably effective. Results We describe a simple, highly controllable, and versatile apparatus for heating biological tissue and other materials on the micron-scale. This microheater employs micron-scale fiber optics and uses an inexpensive laser-pointer as a power source. Optical fibers can be pulled on a standard electrode puller to produce tips of varying sizes that can then be used to reliably heat 20-100 μm targets. We demonstrate precise spatiotemporal control of hsp70l:GFP transgene expression in a variety of tissue types in zebrafish embryos and larvae. We also show how this system can be employed as part of a new method for lineage tracing that would greatly facilitate the study of organogenesis and tissue regulation at any time in the life cycle. Conclusion This versatile and simple local heater has broad utility for the study of gene function and for lineage tracing. This system could be used to control hsp-driven gene expression in any organism simply by bringing the fiber optic tip in contact with the tissue of interest. Beyond these uses for the study of gene function, this device has wide-ranging utility in materials science and could easily be adapted for therapeutic purposes in humans.

Crystal structures of Hsp104 N-terminal domains from Saccharomyces cerevisiae and Candida albicans suggest the mechanism for the function of Hsp104 in dissolving prions

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Wang, Peng; Li, Jingzhi; Weaver, Clarissa; Lucius, Aaron; Sha, Bingdong

2017-03-31

Hsp104 is a yeast member of the Hsp100 family which functions as a molecular chaperone to disaggregate misfolded polypeptides. To understand the mechanism by which the Hsp104 N-terminal domain (NTD) interacts with its peptide substrates, crystal structures of the Hsp104 NTDs fromSaccharomyces cerevisiae(ScHsp104NTD) andCandida albicans(CaHsp104NTD) have been determined at high resolution. The structures of ScHsp104NTD and CaHsp104NTD reveal that the yeast Hsp104 NTD may utilize a conserved putative peptide-binding groove to interact with misfolded polypeptides. In the crystal structures ScHsp104NTD forms a homodimer, while CaHsp104NTD exists as a monomer. The consecutive residues Gln105, Gln106 and Lys107, and Lys141 around the putative peptide-binding groove mediate the monomer–monomer interactions within the ScHsp104NTD homodimer. Dimer formation by ScHsp104NTD suggests that the Hsp104 NTD may specifically interact with polyQ regions of prion-prone proteins. The data may reveal the mechanism by which Hsp104 NTD functions to suppress and/or dissolve prions.

Heat Shock Protein Genes Undergo Dynamic Alteration in Their Three-Dimensional Structure and Genome Organization in Response to Thermal Stress.

Science.gov (United States)

Chowdhary, Surabhi; Kainth, Amoldeep S; Gross, David S

2017-12-15

Three-dimensional (3D) chromatin organization is important for proper gene regulation, yet how the genome is remodeled in response to stress is largely unknown. Here, we use a highly sensitive version of chromosome conformation capture in combination with fluorescence microscopy to investigate Heat Shock Protein ( HSP ) gene conformation and 3D nuclear organization in budding yeast. In response to acute thermal stress, HSP genes undergo intense intragenic folding interactions that go well beyond 5'-3' gene looping previously described for RNA polymerase II genes. These interactions include looping between upstream activation sequence (UAS) and promoter elements, promoter and terminator regions, and regulatory and coding regions (gene "crumpling"). They are also dynamic, being prominent within 60 s, peaking within 2.5 min, and attenuating within 30 min, and correlate with HSP gene transcriptional activity. With similarly striking kinetics, activated HSP genes, both chromosomally linked and unlinked, coalesce into discrete intranuclear foci. Constitutively transcribed genes also loop and crumple yet fail to coalesce. Notably, a missense mutation in transcription factor TFIIB suppresses gene looping, yet neither crumpling nor HSP gene coalescence is affected. An inactivating promoter mutation, in contrast, obviates all three. Our results provide evidence for widespread, transcription-associated gene crumpling and demonstrate the de novo assembly and disassembly of HSP gene foci. Copyright © 2017 American Society for Microbiology.

Hsp70 facilitates trans-membrane transport of bacterial ADP-ribosylating toxins into the cytosol of mammalian cells.

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Ernst, Katharina; Schmid, Johannes; Beck, Matthias; Hägele, Marlen; Hohwieler, Meike; Hauff, Patricia; Ückert, Anna Katharina; Anastasia, Anna; Fauler, Michael; Jank, Thomas; Aktories, Klaus; Popoff, Michel R; Schiene-Fischer, Cordelia; Kleger, Alexander; Müller, Martin; Frick, Manfred; Barth, Holger

2017-06-02

Binary enterotoxins Clostridium (C.) botulinum C2 toxin, C. perfringens iota toxin and C. difficile toxin CDT are composed of a transport (B) and a separate non-linked enzyme (A) component. Their B-components mediate endocytic uptake into mammalian cells and subsequently transport of the A-components from acidic endosomes into the cytosol, where the latter ADP-ribosylate G-actin resulting in cell rounding and cell death causing clinical symptoms. Protein folding enzymes, including Hsp90 and peptidyl-prolyl cis/trans isomerases facilitate transport of the A-components across endosomal membranes. Here, we identified Hsp70 as a novel host cell factor specifically interacting with A-components of C2, iota and CDT toxins to facilitate their transport into the cell cytosol. Pharmacological Hsp70-inhibition specifically prevented pH-dependent trans-membrane transport of A-components into the cytosol thereby protecting living cells and stem cell-derived human miniguts from intoxication. Thus, Hsp70-inhibition might lead to development of novel therapeutic strategies to treat diseases associated with bacterial ADP-ribosylating toxins.

Regulation of the Hsp104 middle domain activity is critical for yeast prion propagation.

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Jennifer E Dulle

Full Text Available Molecular chaperones play a significant role in preventing protein misfolding and aggregation. Indeed, some protein conformational disorders have been linked to changes in the chaperone network. Curiously, in yeast, chaperones also play a role in promoting prion maintenance and propagation. While many amyloidogenic proteins are associated with disease in mammals, yeast prion proteins, and their ability to undergo conformational conversion into a prion state, are proposed to play a functional role in yeast biology. The chaperone Hsp104, a AAA+ ATPase, is essential for yeast prion propagation. Hsp104 fragments large prion aggregates to generate a population of smaller oligomers that can more readily convert soluble monomer and be transmitted to daughter cells. Here, we show that the middle (M domain of Hsp104, and its mobility, plays an integral part in prion propagation. We generated and characterized mutations in the M-domain of Hsp104 that are predicted to stabilize either a repressed or de-repressed conformation of the M-domain (by analogy to ClpB in bacteria. We show that the predicted stabilization of the repressed conformation inhibits general chaperone activity. Mutation to the de-repressed conformation, however, has differential effects on ATP hydrolysis and disaggregation, suggesting that the M-domain is involved in coupling these two activities. Interestingly, we show that changes in the M-domain differentially affect the propagation of different variants of the [PSI+] and [RNQ+] prions, which indicates that some prion variants are more sensitive to changes in the M-domain mobility than others. Thus, we provide evidence that regulation of the M-domain of Hsp104 is critical for efficient prion propagation. This shows the importance of elucidating the function of the M-domain in order to understand the role of Hsp104 in the propagation of different prions and prion variants.

Differential gene expressions in testes of L2 strain Taiwan country chicken in response to acute heat stress.

Science.gov (United States)

Wang, Shih-Han; Cheng, Chuen-Yu; Tang, Pin-Chi; Chen, Chih-Feng; Chen, Hsin-Hsin; Lee, Yen-Pai; Huang, San-Yuan

2013-01-15

Acute heat stress affects genes involved in spermatogenesis in mammals. However, there is apparently no elaborate research on the effects of acute heat stress on gene expression in avian testes. The purpose of this study was to investigate global gene expression in testes of the L2 strain of Taiwan country chicken after acute heat stress. Twelve roosters, 45 weeks old, were allocated into four groups, including control roosters kept at 25 °C, roosters subjected to 38 °C acute heat stress for 4 hours without recovery, with 2-hour recovery, and with 6-hour recovery, respectively. Testis samples were collected for RNA isolation and microarray analysis. Based on gene expression profiles, 169 genes were upregulated and 140 genes were downregulated after heat stress using a cutoff value of twofold or greater change. Based on gene ontology analysis, differentially expressed genes were mainly related to response to stress, transport, signal transduction, and metabolism. A functional network analysis displayed that heat shock protein genes and related chaperones were the major upregulated groups in chicken testes after acute heat stress. A quantitative real-time polymerase chain reaction analysis of mRNA expressions of HSP70, HSP90AA1, BAG3, SERPINB2, HSP25, DNAJA4, CYP3A80, CIRBP, and TAGLN confirmed the results of the microarray analysis. Because the HSP genes (HSP25, HSP70, and HSP90AA1) and the antiapoptotic BAG3 gene were dramatically altered in heat-stressed chicken testes, we concluded that these genes were important factors in the avian testes under acute heat stress. Whether these genes could be candidate genes for thermotolerance in roosters requires further investigation. Copyright © 2013 Elsevier Inc. All rights reserved.

Quercetin suppresses heat shock-induced nuclear translocation of Hsp72

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Antoni Gawron

2011-08-01

Full Text Available The effect of quercetin and heat shock on the Hsp72 level and distribution in HeLa cells was studied by Western blotting, indirect immunofluorescence and immunogold electron microscopy. In control cells and after quercetin treatment, Hsp72 was located both in the cytoplasm and in the nucleus in comparable amounts. After hyperthermia, the level of nuclear Hsp72 raised dramatically. Expression of Hsp72 in cytoplasm was also higher but not to such extent as that observed in the nucleus. Preincubation of heated cells with quercetin inhibited strong Hsp72 expression observed after hyperthermia and changed the intracellular Hsp72 distribution. The cytoplasmic level of protein exceeded the nuclear one, especially around the nucleus, where the coat of Hsp72 was noticed. Observations indicating that quercetin was present around and in the nuclear envelope suggested an involvement of this drug in the inhibition of nuclear translocation. Our results indicate that pro-apoptotic activity of quercetin may be correlated not only with the inhibition of Hsp72 expression but also with suppression of its migration to the nucleus.

Heat-Induced Changes in Heat Shock Protein Genes Expression in Crossbred and Baladi Pregnant Cows and Their Offspring

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Khalil, W.K.B.; Nessim, M.Z.; El- Masry, K.A.

2010-01-01

The experiment was carried out during August (hot climate) on twelve pregnant cows of six crossbred (50% native Baladi and 50% Brown Swiss) and six native Baladi pregnant cows in the same age and the second parity during their mid-pregnancy as detected by rectal palpation. The experiment was repeated during December (mild climate) on similar twelve pregnant cows. Blood sample was obtained from each cow at the end of August (first group) and at the end of December (second group) to obtain heat shock protein genes expression (HSP72, HSP70.01, HSP70, HSP47, k Dalton and Actin) in pregnant cows under mild and hot climate to find out, which breed is more tolerant to heat stress and to estimate offspring birth weight and their growth performances during suckling period. Comparison was made between hot climate cows group and mild climate cows group to estimate heat- induced changes in both breeds in expression level of the Hsp genes and to compare with their neonate birth weight and growth performances during suckling period. The results revealed that expression level of the Hsp genes (Hsp72, Hsp70.1, Hsp70 and Hsp 47) was higher (p<0.01) in hot season compared to that of mild season. Expression level of the Hsp genes (Hsp70.1, Hsp70 and Hsp 47) was higher (p<0.05) in crossbred cows than in Baldi cows under summer hot season. This indicates that crossbred cows are less heat tolerant than Baladi cows under heat stress climate. Heat induced decrease (p<0.01) in offspring birth weight in Baladi and crossbred by 18.1% and 25%, respectively, in weaning weight by 14.61% and 23.14%, respectively and in body weight gain by 14.61% and 21.18%, respectively

Recurrent nontuberculous mycobacterial endophthalmitis: a diagnostic conundrum

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Venkateswaran N

2014-05-01

infectious agent all along, and the organism was originally misidentified on the basis of less accurate tests.Conclusion: Recurrent atypical mycobacterial endophthalmitis requires expedient identification and management to prevent poor visual outcomes. Standard biochemical testing can identify the causative organism but is limited by the inability to distinguish between nontuberculous species reliably. We recommend the use of PCR in conjunction with sequencing of the hsp65 gene for reliable differentiation of M. chelonae and M. abscessus in atypical mycobacterial ocular infections. Minimum inhibitory concentration antibiotic susceptibility tests on cultured strains are the best guide to antibiotic selection, given the rapidly rising resistance to antimicrobials in atypical mycobacterial species.Keywords: atypical mycobacteria, herpes zoster ophthalmicus, hsp65, Mycobacterium chelonae, neurotrophic keratopathy, visual outcome

Critical analysis of eukaryotic phylogeny: a case study based on the HSP70 family.

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Germot, A; Philippe, H

1999-01-01

Trichomonads, together with diplomonads and microsporidia, emerge at the base of the eukaryotic tree, on the basis of the small subunit rRNA phylogeny. However, phylogenies based on protein sequences such as tubulin are markedly different with these protists emerging much later. We have investigated 70 kDa heat-shock protein (HSP70), which could be a reliable phylogenetic marker. In eukaryotes, HSP70s are found in cytosol, endoplasmic reticulum, and organelles (mitochondria and chloroplasts). In Trichomonas vaginalis we identified nine different HSP70-encoding genes and sequenced three nearly complete cDNAs corresponding to cytosolic, endoplasmic reticulum, and mitochondrial-type HSP70. Phylogenies of eukaryotes were reconstructed using the classical methods while varying the number of species and characters considered. Almost all the undoubtedly monophyletic groups, defined by ultrastructural characters, were recovered. However, due to the long branch attraction phenomenon, the evolutionary rates were the main factor determining the position of species, even with the use of a close outgroup, which is an important advantage of HSP70 with respect to many other markers. Numerous variable sites are peculiar to Trichomonas and probably generated the artefactual placement of this species at the base of the eukaryotes or as the sister group of fast-evolving species. The inter-phyla relationships were not well supported and were sensitive to the reconstruction method, the number of species; and the quantity of information used. This lack of resolution could be explained by the very rapid diversification of eukaryotes, likely after the mitochondrial endosymbiosis.

Hsp100/ClpB Chaperone Function and Mechanism

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Vierling, Elizabeth [Univ. of Massachusetts, Amherst, MA (United States). Dept. of Biochemistry and Molecular Biology

2015-01-27

The supported research investigated the mechanism of action of a unique class of molecular chaperones in higher plants, the Hsp100/ClpB proteins, with the ultimate goal of defining how these chaperones influence plant growth, development, stress tolerance and productivity. Molecular chaperones are essential effectors of cellular “protein quality control”, which comprises processes that ensure the proper folding, localization, activation and turnover of proteins. Hsp100/ClpB proteins are required for temperature acclimation in plants, optimal seed yield, and proper chloroplast development. The model plant Arabidopsis thaliana and genetic and molecular approaches were used to investigate two of the three members of the Hsp100/ClpB proteins in plants, cytosolic AtHsp101 and chloroplast-localized AtClpB-p. Investigating the chaperone activity of the Hsp100/ClpB proteins addresses DOE goals in that this activity impacts how “plants generate and assemble components” as well as “allowing for their self repair”. Additionally, Hsp100/ClpB protein function in plants is directly required for optimal “utilization of biological energy” and is involved in “mechanisms that control the architecture of energy transduction systems”.

Molecular Cloning and mRNA Expression of Heat Shock Protein Genes and Their Response to Cadmium Stress in the Grasshopper Oxya chinensis.

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Yuping Zhang

Full Text Available Heat shock proteins (Hsps are highly conserved molecular chaperones that are synthesized in response to stress. In this study, we cloned the full-length sequences of the Grp78 (glucose-regulated protein 78, Hsp70, Hsp90, and Hsp40 genes from the Chinese rice grasshopper Oxya chinensis. The full-length cDNA sequences of OcGrp78, OcHsp70, OcHsp90, and OcHsp40 contain open reading frames of 1947, 1920, 2172, and 1042 bp that encode proteins of 649, 640, 724, and 347 amino acids, respectively. Fluorescent real-time quantitative PCR (RT-qPCR was performed to quantify the relative transcript levels of these Hsp genes in different tissues and developmental stages. The mRNAs encoding these four Hsp genes were present at all developmental stages and in all tissues examined but were expressed at varying levels. Additionally, we investigated the mRNA expression profiles of these four Hsps in O. chinensis subjected to Cadmium (Cd stress. OcGrp78, OcHsp70, OcHsp90, and OcHsp40 mRNA expression was induced under acute Cd stress; the levels reached a maximum within a short time (6 h, were reduced significantly at 12 h, and were lowered to or below control levels by 48 h. Regarding induction efficiency, OcHsp70 was the most sensitive gene to acute Cd stress. Chronic Cd exposure showed that dietary Cd treatment induced increased OcGrp78, OcHsp90, and OcHsp40 expression. However, dietary Cd induced a significant reduction of OcHsp70 expression. In the period tested, no significant difference in the mortality of the grasshoppers was observed. Our results suggest that these four Hsps genes, especially OcHsp70, are sensitive to acute Cd stress and could be used as molecular markers for toxicology studies. However, our results also indicate that OcHsp70 is not suitable for use as a molecular marker of chronic Cd contamination.

Molecular Cloning and mRNA Expression of Heat Shock Protein Genes and Their Response to Cadmium Stress in the Grasshopper Oxya chinensis.

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Zhang, Yuping; Liu, Yaoming; Zhang, Jianzhen; Guo, Yaping; Ma, Enbo

2015-01-01

Heat shock proteins (Hsps) are highly conserved molecular chaperones that are synthesized in response to stress. In this study, we cloned the full-length sequences of the Grp78 (glucose-regulated protein 78), Hsp70, Hsp90, and Hsp40 genes from the Chinese rice grasshopper Oxya chinensis. The full-length cDNA sequences of OcGrp78, OcHsp70, OcHsp90, and OcHsp40 contain open reading frames of 1947, 1920, 2172, and 1042 bp that encode proteins of 649, 640, 724, and 347 amino acids, respectively. Fluorescent real-time quantitative PCR (RT-qPCR) was performed to quantify the relative transcript levels of these Hsp genes in different tissues and developmental stages. The mRNAs encoding these four Hsp genes were present at all developmental stages and in all tissues examined but were expressed at varying levels. Additionally, we investigated the mRNA expression profiles of these four Hsps in O. chinensis subjected to Cadmium (Cd) stress. OcGrp78, OcHsp70, OcHsp90, and OcHsp40 mRNA expression was induced under acute Cd stress; the levels reached a maximum within a short time (6 h), were reduced significantly at 12 h, and were lowered to or below control levels by 48 h. Regarding induction efficiency, OcHsp70 was the most sensitive gene to acute Cd stress. Chronic Cd exposure showed that dietary Cd treatment induced increased OcGrp78, OcHsp90, and OcHsp40 expression. However, dietary Cd induced a significant reduction of OcHsp70 expression. In the period tested, no significant difference in the mortality of the grasshoppers was observed. Our results suggest that these four Hsps genes, especially OcHsp70, are sensitive to acute Cd stress and could be used as molecular markers for toxicology studies. However, our results also indicate that OcHsp70 is not suitable for use as a molecular marker of chronic Cd contamination.

Neonatal Death and Heart Failure in Mouse with Transgenic HSP60 Expression

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Tsung-Hsien Chen

2015-01-01

Full Text Available Mitochondrial heat shock proteins, such as HSP60, are chaperones responsible for the folding, transport, and quality control of mitochondrial matrix proteins and are essential for maintaining life. Both prosurvival and proapoptotic roles have been proposed for HSP60, and HSP60 is reportedly involved in the initiation of autoimmune, metabolic, and cardiovascular diseases. The role of HSP60 in pathogenesis of these diseases remains unclear, partly because of the lack of mouse models expressing HSP60. In this study we generated HSP60 conditional transgenic mice suitable for investigating in vivo outcomes by expressing HSP60 at the targeted organ in disease models. Ubiquitous HSP60 induction in the embryonic stage caused neonatal death in mice at postnatal day 1. A high incidence of atrial septal defects was observed in HSP60-expressing mice, with increased apoptosis and myocyte degeneration that possibly contributed to massive hemorrhage and sponge-like cardiac muscles. Our results showed that neonatal heart failure through HSP60 induction likely involves developmental defects and excessive apoptosis. The conditional HSP60 mouse model is useful for studying crucial biological questions concerning HSP60.

Chemokine MCP1/CCL2 and RANTES/CCL5 gene polymorphisms influence Henoch–Schönlein purpura susceptibility and severity

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Hsin-Hui Yu

2015-04-01

Conclusion: Our results support the fact that chemokines play important roles in the pathogenesis of HSP. MCP1/CCL2 gene polymorphisms were associated with susceptibility for HSP. RANTES/CCL5 gene polymorphisms may be related to disease severity and HSP nephritis.

HDJC9, a novel human type C DnaJ/HSP40 member interacts with and cochaperones HSP70 through the J domain

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Han Chaofeng; Chen Taoyong; Li Nan; Yang Mingjin; Wan Tao; Cao Xuetao

2007-01-01

HSP40s are a subfamily of heat shock proteins (HSPs) and play important roles in regulation of cell proliferation, survival and apoptosis by serving as chaperones for HSP70s. Up to date hundreds of HSP40 proteins derived from various species ranging from Escherichia coli to homo sapiens have been identified. Here we report the cloning and characterization of a novel human type C DnaJ homologue, HDJC9, containing a typical N-terminal J domain. HDJC9 is upregulated at both mRNA and protein levels upon various stress and mitogenic stimulations. HDJC9 is mainly localized in cell nuclei under normal culture conditions while it is transported into cytoplasm and plasma membrane upon heat shock stress through a non-classical and lipid-dependent pathway. HDJC9 can interact with HSP70s and activate the ATPase activity of HSP70s, both of which are dependent on the J domain. Our data suggest that HDJC9 is a novel cochaperone for HSP70s

Exploring the Functional Complementation between Grp94 and Hsp90.

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Kevin A Maharaj

Full Text Available Grp94 and Hsp90 are the ER and cytoplasmic paralog members, respectively, of the hsp90 family of molecular chaperones. The structural and biochemical differences between Hsp90 and Grp94 that allow each paralog to efficiently chaperone its particular set of clients are poorly understood. The two paralogs exhibit a high degree of sequence similarity, yet also display significant differences in their quaternary conformations and ATPase activity. In order to identify the structural elements that distinguish Grp94 from Hsp90, we characterized the similarities and differences between the two proteins by testing the ability of Hsp90/Grp94 chimeras to functionally substitute for the wild-type chaperones in vivo. We show that the N-terminal domain or the combination of the second lobe of the Middle domain plus the C-terminal domain of Grp94 can functionally substitute for their yeast Hsp90 counterparts but that the equivalent Hsp90 domains cannot functionally replace their counterparts in Grp94. These results also identify the interface between the Middle and C-terminal domains as an important structural unit within the Hsp90 family.

Plant Hsp90 Proteins Interact with B-Cells and Stimulate Their Proliferation

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Corigliano, Mariana G.; Maglioco, Andrea; Laguía Becher, Melina; Goldman, Alejandra; Martín, Valentina; Angel, Sergio O.; Clemente, Marina

2011-01-01

Background The molecular chaperone heat shock protein 90 (Hsp90) plays an important role in folding stabilization and activation of client proteins. Besides, Hsp90 of mammals and mammalian pathogens displays immunostimulatory properties. Here, we investigated the role of plant-derived Hsp90s as B-cell mitogens by measuring their proliferative responses in vitro. Methodology Plant cytosolic Hsp90 isoforms from Arabidopsis thaliana (AtHsp81.2) and Nicotiana benthamiana (NbHsp90.3) were expressed in E. coli. Over-expression of recombinant plant Hsp90s (rpHsp90s) was confirmed by SDS-PAGE and western blot using and anti-AtHsp81.2 polyclonal anti-body. Both recombinant proteins were purified by Ni-NTA affinity chromatography and their identity confirmed by MALDI-TOF-TOF. Recombinant AtHsp81.2 and NbHsp90.3 proteins induced prominent proliferative responses in spleen cells form BALB/c mice. Polymyxin-B, a potent inhibitor of lipopolysaccharide (LPS), did not eliminate the rpHsp90-induced proliferation. In addition, in vitro incubation of spleen cells with rpHsp90 led to the expansion of CD19-bearing populations, suggesting a direct effect of these proteins on B lymphocytes. This effect was confirmed by immunofluorescence analysis, where a direct binding of rpHsp90 to B- but not to T-cells was observed in cells from BALB/c and C3H/HeN mice. Finally, we examined the involvement of Toll Like Receptor 4 (TLR4) molecules in the rpHsp90s induction of B-cell proliferation. Spleen cells from C3H/HeJ mice, which carry a point mutation in the cytoplasmic region of TLR4, responded poorly to prAtHsp90. However, the interaction between rpHsp90 and B-cells from C3H/HeJ mice was not altered, suggesting that the mutation on TLR4 would be affecting the signal cascade but not the rpHsp90-TLR4 receptor interaction. Conclusions Our results show for the first time that spleen cell proliferation can be stimulated by a non-pathogen-derived Hsp90. Furthermore, our data provide a new example of

Stress, and pathogen response gene expression in modeled microgravity

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Sundaresan, Alamelu; Pellis, Neal R.

2006-01-01

Purpose: Immune suppression in microgravity has been well documented. With the advent of human exploration and long-term space travel, the immune system of the astronaut must be optimally maintained. It is important to investigate the expression patterns of cytokine genes, because they are directly related to immune response. Heat shock proteins (HSPs), also called stress proteins, are a group of proteins that are present in the cells of every life form. These proteins are induced when a cell responds to stressors such as heat, cold and oxygen deprivation. Microgravity is another stressor that may regulate HSPs. Heat shock proteins trigger immune response through activities that occur both inside the cell (intracellular) and outside the cell (extracellular). Knowledge about these two gene groups could lead to establishment of a blueprint of the immune response and adaptation-related genes in the microgravity environment. Methods: Human peripheral blood cells were cultured in 1g (T flask) and modeled microgravity (MMG, rotating-wall vessel) for 24 and 72 hours. Cell samples were collected and subjected to gene array analysis using the Affymetrix HG_U95 array. Data was collected and subjected to a two-way analysis of variance. The genes related to immune and stress responses were analyzed. Results and Conclusions: HSP70 was up-regulated by more than two fold in microgravity culture, while HSP90 was significantly down-regulated. HSP70 is not typically expressed in all kinds of cells, but it is expressed at high levels in stress conditions. HSP70 participates in translation, protein translocation, proteolysis and protein folding, suppressing aggregation and reactivating denatured proteins. Increased serum HSP70 levels correlate with a better outcome for heat-stroke or severe trauma patients. At the same time, elevated serum levels of HSP70 have been detected in patients with peripheral or renal vascular disease. HSP90 has been identified in the cytosol, nucleus and

The MP65 gene is required for cell wall integrity, adherence to epithelial cells and biofilm formation in Candida albicans

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Girolamo Antonietta

2011-05-01

Full Text Available Abstract Background The MP65 gene of Candida albicans (orf19.1779 encodes a putative β-glucanase mannoprotein of 65 kDa, which plays a main role in a host-fungus relationship, morphogenesis and pathogenicity. In this study, we performed an extensive analysis of a mp65Δ mutant to assess the role of this protein in cell wall integrity, adherence to epithelial cells and biofilm formation. Results The mp65Δ mutant showed a high sensitivity to a range of cell wall-perturbing and degrading agents, especially Congo red, which induced morphological changes such as swelling, clumping and formation of hyphae. The mp65Δ mutant showed an activation of two MAPKs (Mkc1p and Cek1p, a high level of expression of two stress-related genes (DDR48 and SOD5, and a modulated expression of β-glucan epitopes, but no gross changes in cell wall polysaccharide composition. Interestingly, the mp65Δ mutant displayed a marked reduction in adhesion to BEC and Caco-2 cells and severe defects in biofilm formation when compared to the wild type. All of the mentioned properties were totally or partially recovered in a revertant strain, demonstrating the specificity of gene deletion. Conclusions We demonstrate that the MP65 gene of Candida albicans plays a significant role in maintaining cell wall integrity, as well as in adherence to epithelia and biofilm formation, which are major virulence attributes of this fungus.

Response of heat shock protein genes of the oriental fruit moth under diapause and thermal stress reveals multiple patterns dependent on the nature of stress exposure.

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Zhang, Bo; Peng, Yu; Zheng, Jincheng; Liang, Lina; Hoffmann, Ary A; Ma, Chun-Sen

2016-07-01

Heat shock protein gene (Hsp) families are thought to be important in thermal adaptation, but their expression patterns under various thermal stresses have still been poorly characterized outside of model systems. We have therefore characterized Hsp genes and their stress responses in the oriental fruit moth (OFM), Grapholita molesta, a widespread global orchard pest, and compared patterns of expression in this species to that of other insects. Genes from four Hsp families showed variable expression levels among tissues and developmental stages. Members of the Hsp40, 70, and 90 families were highly expressed under short exposures to heat and cold. Expression of Hsp40, 70, and Hsc70 family members increased in OFM undergoing diapause, while Hsp90 was downregulated. We found that there was strong sequence conservation of members of large Hsp families (Hsp40, Hsp60, Hsp70, Hsc70) across taxa, but this was not always matched by conservation of expression patterns. When the large Hsps as well as small Hsps from OFM were compared under acute and ramping heat stress, two groups of sHsps expression patterns were apparent, depending on whether expression increased or decreased immediately after stress exposure. These results highlight potential differences in conservation of function as opposed to sequence in this gene family and also point to Hsp genes potentially useful as bioindicators of diapause and thermal stress in OFM.

Hsp90 selectively modulates phenotype in vertebrate development.

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Patricia L Yeyati

2007-03-01

Full Text Available Compromised heat shock protein 90 (Hsp90 function reveals cryptic phenotypes in flies and plants. These observations were interpreted to suggest that this molecular stress-response chaperone has a capacity to buffer underlying genetic variation. Conversely, the protective role of Hsp90 could account for the variable penetrance or severity of some heritable developmental malformations in vertebrates. Using zebrafish as a model, we defined Hsp90 inhibitor levels that did not induce a heat shock response or perturb phenotype in wild-type strains. Under these conditions the severity of the recessive eye phenotype in sunrise, caused by a pax6b mutation, was increased, while in dreumes, caused by a sufu mutation, it was decreased. In another strain, a previously unobserved spectrum of severe structural eye malformations, reminiscent of anophthalmia, microphthalmia, and nanophthalmia complex in humans, was uncovered by this limited inhibition of Hsp90 function. Inbreeding of offspring from selected unaffected carrier parents led to significantly elevated malformation frequencies and revealed the oligogenic nature of this phenotype. Unlike in Drosophila, Hsp90 inhibition can decrease developmental stability in zebrafish, as indicated by increased asymmetric presentation of anophthalmia, microphthalmia, and nanophthalmia and sunrise phenotypes. Analysis of the sunrise pax6b mutation suggests a molecular mechanism for the buffering of mutations by Hsp90. The zebrafish studies imply that mild perturbation of Hsp90 function at critical developmental stages may underpin the variable penetrance and expressivity of many developmental anomalies where the interaction between genotype and environment plays a major role.

Selection Transforms the Landscape of Genetic Variation Interacting with Hsp90.

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Geiler-Samerotte, Kerry A; Zhu, Yuan O; Goulet, Benjamin E; Hall, David W; Siegal, Mark L

2016-10-01

The protein-folding chaperone Hsp90 has been proposed to buffer the phenotypic effects of mutations. The potential for Hsp90 and other putative buffers to increase robustness to mutation has had major impact on disease models, quantitative genetics, and evolutionary theory. But Hsp90 sometimes contradicts expectations for a buffer by potentiating rapid phenotypic changes that would otherwise not occur. Here, we quantify Hsp90's ability to buffer or potentiate (i.e., diminish or enhance) the effects of genetic variation on single-cell morphological features in budding yeast. We corroborate reports that Hsp90 tends to buffer the effects of standing genetic variation in natural populations. However, we demonstrate that Hsp90 tends to have the opposite effect on genetic variation that has experienced reduced selection pressure. Specifically, Hsp90 tends to enhance, rather than diminish, the effects of spontaneous mutations and recombinations. This result implies that Hsp90 does not make phenotypes more robust to the effects of genetic perturbation. Instead, natural selection preferentially allows buffered alleles to persist and thereby creates the false impression that Hsp90 confers greater robustness.

Spatio-temporal regulation of Hsp90-ligand complex leads to immune activation.

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Yasuaki eTamura

2016-05-01

Full Text Available Hsp90 is the most abundant cytosolic HSP and is known to act as a molecular chaperone. We found that an Hsp90-cancer antigen peptide complex was efficiently cross-presented by human monocyte-derived dendritic cells and induced peptide-specific cytotoxic T lymphocytes. Furthermore, we observed that the internalized Hsp90-peptide complex was strictly sorted to the Rab5+, EEA1+ static early endosome and the Hsp90-chaperoned peptide was processed and bound to MHC class I molecules through a endosome-recycling pathway. We also found that extracellular Hsp90 complexed with CpG-A or self-DNA stimulates production of a large amount of IFN-α from pDCs via static early endosome targeting. Thus, extracellular Hsp90 can target the antigen or nucleic acid to a static early endosome by spatio-temporal regulation. Moreover, we showed that Hsp90 associates with and delivers TLR7/9 from the ER to early endosomes for ligand recognition. Hsp90 inhibitor, geldanamycin derivative inhibited the Hsp90 association with TLR7/9, resulting in inhibition IFN-α production, leading to improvement of SLE symptoms. Interstingly, we observed that serum Hsp90 is clearly increased in patients with active SLE compared with that in patients with inactive disease. Serum Hsp90 detected in SLE patients binds to self-DNA and/or anti-DNA Ab, thus leading to stimulation of pDCs to produce IFN-α. Thus, Hsp90 plays a crucial role in the pathogenesis of SLE and that an Hsp90 inhibitor will therefore provide a new therapeutic approach to SLE and other nucleic acid-related autoimmune diseases. We will discuss how spatio-temporal regulation of Hsp90-ligand complexes within antigen-presenting cells affects the innate immunity and adaptive immunity.

Crystallization and preliminary X-ray diffraction analysis of XAC1151, a small heat-shock protein from Xanthomonas axonopodis pv. citri belonging to the α-crystallin family

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Hilario, Eduardo; Teixeira, Elaine Cristina; Pedroso, Gisele Audrei; Bertolini, Maria Célia [Departamento de Bioquímica e Tecnologia Química, Instituto de Química, Universidade Estadual Paulista, Araraquara-SP (Brazil); Medrano, Francisco Javier, E-mail: fjmedrano@yahoo.com [Departamento de Cristalografia de Proteínas, Centro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Síncrotron, Caixa Postal 6192, CEP 13084-971, Campinas-SP (Brazil); Departamento de Bioquímica e Tecnologia Química, Instituto de Química, Universidade Estadual Paulista, Araraquara-SP (Brazil)

2006-05-01

XAC1151, a small heat-shock protein from X. axonopodis pv. citri belonging to the α-crystallin family, was crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium phosphate. X-ray diffraction data were collected to 1.65 Å resolution using a synchrotron-radiation source. The hspA gene (XAC1151) from Xanthomonas axonopodis pv. citri encodes a protein of 158 amino acids that belongs to the small heat-shock protein (sHSP) family of proteins. These proteins function as molecular chaperones by preventing protein aggregation. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium phosphate. X-ray diffraction data were collected to 1.65 Å resolution using a synchrotron-radiation source. The crystal belongs to the rhombohedral space group R3, with unit-cell parameters a = b = 128.7, c = 55.3 Å. The crystal structure was solved by molecular-replacement methods. Structure refinement is in progress.

Hsp70-Bag3 interactions regulate cancer-related signaling networks.

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Colvin, Teresa A; Gabai, Vladimir L; Gong, Jianlin; Calderwood, Stuart K; Li, Hu; Gummuluru, Suryaram; Matchuk, Olga N; Smirnova, Svetlana G; Orlova, Nina V; Zamulaeva, Irina A; Garcia-Marcos, Mikel; Li, Xiaokai; Young, Z T; Rauch, Jennifer N; Gestwicki, Jason E; Takayama, Shinichi; Sherman, Michael Y

2014-09-01

Bag3, a nucleotide exchange factor of the heat shock protein Hsp70, has been implicated in cell signaling. Here, we report that Bag3 interacts with the SH3 domain of Src, thereby mediating the effects of Hsp70 on Src signaling. Using several complementary approaches, we established that the Hsp70-Bag3 module is a broad-acting regulator of cancer cell signaling by modulating the activity of the transcription factors NF-κB, FoxM1, Hif1α, the translation regulator HuR, and the cell-cycle regulators p21 and survivin. We also identified a small-molecule inhibitor, YM-1, that disrupts the Hsp70-Bag3 interaction. YM-1 mirrored the effects of Hsp70 depletion on these signaling pathways, and in vivo administration of this drug was sufficient to suppress tumor growth in mice. Overall, our results defined Bag3 as a critical factor in Hsp70-modulated signaling and offered a preclinical proof-of-concept that the Hsp70-Bag3 complex may offer an appealing anticancer target. ©2014 American Association for Cancer Research.

Species- and Strain-Specific Adaptation of the HSP70 Super Family in Pathogenic Trypanosomatids

Czech Academy of Sciences Publication Activity Database

Drini, S.; Criscuolo, A.; Lechat, P.; Imamura, H.; Skalický, Tomáš; Rachidi, N.; Lukeš, Julius; Dujardin, J.-C.; Späth, G.F.

2016-01-01

Roč. 8, č. 6 (2016), s. 1980-1995 ISSN 1759-6653 R&D Projects: GA ČR(CZ) GA14-23986S Institutional support: RVO:60077344 Keywords : Leishmania * heat shock protein * HSP70 * evolution * phylogeny * synteny * copy number variation * gene lossgene loss Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.979, year: 2016

Predicción computacional de estructura terciaria de las proteínas humanas Hsp27, αB-cristalina y HspB8

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Homero Saenz-Suárez

2011-03-01

Full Text Available Objetivo. Realizar predicciones computacionales de estructura de las proteínas humanas Hsp27, αB cristalina y HspB8. Materiales y métodos. La predicción de la estructura secundaria se obtuvo mediante un consenso de los programas de predicción secundaria GOR 4,nnPred,Sspro, APSSP2, JPredict, Porter, Prof, SOPMA, HNN y Psi-Pred. Los modelos de estructura terciaria se elaboraron a partir de fragmentos homólogos de proteínas con estructura terciaria conocida que fueron obtenidos por múltiples alineamientos. Empleando la secuencia primaria se obtuvieron perfiles de antigenicidad de las proteínas nativas y fueron analizados los perfiles de hidrofobicidad, polaridad, flexibilidad, accesibilidad tanto de las proteínas nativas como de las mutadas. Resultados. Las predicciones de estructura secundaria y terciariade las proteínas estudiadas muestran que en los tres casos, más del 65% son regiones en coil, 20-25% en hoja plegada y menos del 10% en alfa hélice. Los análisis de estructura primaria muestran que al menos uno de los perfiles estudiados, en cada mutación está alterado. Conclusiones. Los análisis comparativos de estructura sugieren que las mutaciones afectan la solubilidad de las proteínas mutadas y con ello su función como chaperonas moleculares

Decreased Hsp90 expression in infiltrative lobular carcinoma: an immunohistochemical study

International Nuclear Information System (INIS)

Zagouri, Flora; Patsouris, Effstratios; Zografos, George; Sergentanis, Theodoros; Nonni, Afrodite; Papadimitriou, Christos; Pazaiti, Anastasia; Michalopoulos, Nikolaos V; Safioleas, Panagiotis; Lazaris, Andreas; Theodoropoulos, George

2010-01-01

Elevated Hsp90 expression has been documented in breast ductal carcinomas, whereas decreased Hsp90 expression has been reported in precursor lobular lesions. This study aims to assess Hsp90 expression in infiltrative lobular carcinomas of the breast. Tissue specimens were taken from 32 patients with infiltrative lobular carcinoma. Immunohistochemical assessment of Hsp90 was performed both in the lesion and the adjacent normal breast ducts and lobules; the latter serving as control. Concerning Hsp90 assessment: i) the percentage of positive cells and ii) the intensity were separately analyzed. Subsequently, the Allred score was adopted and calculated. The intensity was treated as an ordinal variable-score (0: negative, low: 1, moderate: 2, high: 3). Statistical analysis followed. All infiltrative lobular carcinoma foci mainly presented with a positive cytoplasmic immunoreaction for Hsp90. Compared to the adjacent normal ducts and lobules, infiltrative lobular carcinoma exhibited a statistically significant decrease in Hsp90 expression, both in terms of Hsp90 positive cells (%) and Allred score (74.2 ± 11.2 vs. 59.1 ± 14.2 p = 0.0001; 7.00 ± 0.95 vs. 6.22 ± 1.01, p = 0.007, Wilcoxon matched-pairs signed-ranks test). Concerning the intensity of Hsp90 immunostaining only a marginal decrease was noted (2.16 ± 0.68 vs. 1.84 ± 0.63, p = 0.087, Wilcoxon matched-pairs signed-ranks test). ILC lesions seem to exhibit decreased Hsp90 expression, a finding contrary to what might have been expected, given that high Hsp90 expression is a trait of invasive ductal carcinomas

A novel biomarker for marine environmental pollution of HSP90 from Mytilus coruscus

International Nuclear Information System (INIS)

Liu, Huihui; Wu, Jiong; Xu, Mengshan; He, Jianyu

2016-01-01

Heat shock protein 90 (HSP90) is a conserved molecular chaperone contributing to cell cycle control, organism development and the proper regulation of cytosolic proteins. The full-length HSP90 cDNA of Mytilus coruscus (McHSP90, KT946644) was 2420 bp, including an ORF of 2169 bp encoding a polypeptide of 722 amino acids with predicted pI/MW 4.89/83.22 kDa. BLASTp analysis and phylogenetic relationship strongly suggested McHSP90 was a member of HSP90 family, and it was highly conserved with other known HSP90, especially in the HSP90 family signatures, ATP/GTP-Binding sites and ‘EEVD’ motif. The mRNA of McHSP90 in haemolymph was upregulated in all treatments including Vibrio alginolyticus and Vibrio harveyi challenge, metals stresses (copper and cadmium) and 180 CST fuel exposure. All the results implied the expression of McHSP90 could be affected by Vibrio challenge and environmental stress, which might help us gain more insight into the molecular mechanism of HSP against adverse stresses in mollusca. - Highlights: • A novel HSP90 (McHSP90) was identified from Mytilus coruscus. • McHSP90 significantly affected by Vibrio challenge for immune defense. • McHSP90 mRNA was obviously up-regulated under stress of heavy metals and 180CST fuel. • McHSP90 might be an ideal marine pollution indicator.

Cloning, expression, purification, crystallization and preliminary X-ray analysis of the 31 kDa Vibrio cholerae heat-shock protein VcHsp31

International Nuclear Information System (INIS)

Das, Samir; Dey, Sanjay; Roy, Trina; Sen, Udayaditya

2011-01-01

A heat-shock protein from V. cholerae (VcHsp31) has been cloned, expressed, purified and crystallized. Crystals of VcHsp31 belonged to a monoclinic space group and diffracted to 1.9 Å resolution. The Gram-negative bacterium Vibrio cholerae, which is responsible for the diarrhoeal disease cholera in humans, induces the expression of numerous heat-shock genes. VcHsp31 is a 31 kDa putative heat-shock protein that belongs to the DJ-1/PfpI superfamily, functioning as both a chaperone and a protease. VcHsp31 has been cloned, overexpressed and purified by Ni 2+ –NTA affinity chromatography followed by gel filtration. Crystals of VcHsp31 were grown in the presence of PEG 6000 and MPD; they belonged to space group P2 1 and diffracted to 1.9 Å resolution. Assuming the presence of six molecules in the asymmetric unit, the Matthews coefficient was estimated to be 1.97 Å 3 Da −1 , corresponding to a solvent content of 37.4%

Association of endothelial nitric oxide synthase gene polymorphism with the risk of Henoch-Schönlein purpura/Henoch-Schönlein purpura nephritis.

Science.gov (United States)

Zhong, Weiqiang; Zhou, Tian-Biao; Jiang, Zongpei

2015-04-01

Association between endothelial nitric oxide synthase (eNOS) gene polymorphism and Henoch-Schönlein purpura (HSP)/Henoch-Schönlein purpura nephritis (HSPN) risk is still controversial. A meta-analysis was performed to evaluate the association between eNOS gene polymorphism and HSP/HSPN susceptibility. A predefined literature search and selection of eligible relevant studies were performed to collect data from electronic database. Three articles were identified for the analysis of association between eNOS gene polymorphism and HSPN/HSP risk. eNOS G894T gene polymorphism was not associated with HSPN susceptibility and the risk of patients with HSP developing into HSPN. Interestingly, eNOS G894T T allele and GG genotype were associated with HSP susceptibility, but not the TT genotype. eNOS T786C TT genotype was associated with HSPN susceptibility, but not C allele and CC genotype. Furthermore, eNOS T786C gene polymorphism was not associated with HSP risk and the risk of patients with HSP developing into HSPN. In conclusion, eNOS T786C TT genotype was associated with and eNOS G894T T allele and GG genotype were associated with HSP susceptibility. However, more studies should be performed in the future.

Localization of Heat Shock Protein 27 (Hsp27) in the Rat Gingiva and its Changes with Tooth Eruption

International Nuclear Information System (INIS)

Sasaki, Au; Yamada, Tohru; Inoue, Katsuyuki; Momoi, Tomoko; Tokunaga, Hiroshi; Sakiyama, Koji; Kanegae, Haruhide; Suda, Naoto; Amano, Osamu

2011-01-01

Heat shock protein 27 kDa (Hsp27) functions as a molecular chaperon to prevent apoptosis as well as to contribute to the regulation of cell proliferation and differentiation during development. In the present study, the localization of Hsp27 in the oral epithelium of rats and its expression change during formation of the gingiva with the tooth eruption were examined immunohistochemically to elucidate the roles of Hsp27 in the oral mucosa. In adult rats, Hsp27-immunoreactivity was localized in the prickle and granular layers but absent in the basal and horny layers of the oral epithelium. On the other hand, in the outer and sulcular epithelia of the free gingival, Hsp27-immunoreactivity was detected in the whole layers, while it was not found in the proliferation zone of the junctional epithelium immunoreactive for Ki67. In immature rats on 10th postnatal day, Hsp27-immunoreactivity was intense in the prickle and granular layers of the oral epithelium, but was not detected in its basal layer. In rats at the eruptive phase on 15th postnatal day, Hsp27-immunoreactivity was detected in sites of the basal layer adjacent to where the dental cusps penetrated through the oral epithelium. Although the immunoreactivity for Ki67 was found in the basal layer of the oral epithelium, it was not localized in the Hsp27-immunopositive sites of tooth-penetration in the basal layer. Just after the tooth-eruption on 20th postnatal day, Hsp27-immunoreactivity was not found in the stratified squamous epithelium at the dentogingival junction, whereas it was intense in a single layer of cuboidal epithelial cells attached to the tooth neck. Ki67-positive cells were scattered in the stratified squamous epithelium at the dentogingival junction, whereas no positive cells were found in the portion of a single layer of cuboidal epithelial cells. These findings suggest that the outer and sulcular epithelia of the free gingiva have a relatively slower rate of proliferation than other gingival and

Essential role of the NH2-terminal WD/EPF motif in the phosphorylation-activated protective function of mammalian Hsp27.

Science.gov (United States)

Thériault, Jimmy R; Lambert, Herman; Chávez-Zobel, Aura T; Charest, Gabriel; Lavigne, Pierre; Landry, Jacques

2004-05-28

Hsp27 is expressed at high levels after mild heat shock and contributes to making cells extremely resistant to subsequent treatments. The activity of the protein is regulated at the transcriptional level, but also by phosphorylation, which occurs rapidly during stress and is responsible for causing the dissociation of large 700-kDa Hsp27 oligomers into dimers. We investigated the mechanism by which phosphorylation and oligomerization modulate the protective activity of Chinese hamster Hsp27. In contrast to oligomer dissociation, which only required Ser90 phosphorylation, activation of Hsp27 thermoprotective activity required the phosphorylation of both Ser90 and Ser15. Replacement of Ser90 by Ala90, which prevented the dissociation of the oligomer upon stress, did cause a severe defect in the protective activity. Dissociation was, however, not a sufficient condition to activate the protein because replacement of Ser15 by Ala15, which caused little effect in the oligomeric organization of the protein, also yielded an inactive protein. Analyzes of mutants with short deletions in the NH2 terminus identified the Hsp27 WD/EPF or PF-rich domain as essential for protection, maintenance of the oligomeric structure, and in vitro chaperone activity of the protein. In light of a three-dimensional model of Hsp27 based on the crystallographic structure of wheat Hsp16.9, we propose that the conserved WD/EPF motif of mammalian Hsp27 mediates important intramolecular interactions with hydrophic surfaces of the alpha-crystallin domain of the protein. These interactions are destabilized by Ser90 phosphorylation, making the motif free to interact with heterologous molecular targets upon the additional phosphorylation of the nearby Ser15.

Decreased Hsp90 expression in infiltrative lobular carcinoma: an immunohistochemical study

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Zagouri Flora

2010-08-01

Full Text Available Abstract Background Elevated Hsp90 expression has been documented in breast ductal carcinomas, whereas decreased Hsp90 expression has been reported in precursor lobular lesions. This study aims to assess Hsp90 expression in infiltrative lobular carcinomas of the breast. Methods Tissue specimens were taken from 32 patients with infiltrative lobular carcinoma. Immunohistochemical assessment of Hsp90 was performed both in the lesion and the adjacent normal breast ducts and lobules; the latter serving as control. Concerning Hsp90 assessment: i the percentage of positive cells and ii the intensity were separately analyzed. Subsequently, the Allred score was adopted and calculated. The intensity was treated as an ordinal variable-score (0: negative, low: 1, moderate: 2, high: 3. Statistical analysis followed. Results All infiltrative lobular carcinoma foci mainly presented with a positive cytoplasmic immunoreaction for Hsp90. Compared to the adjacent normal ducts and lobules, infiltrative lobular carcinoma exhibited a statistically significant decrease in Hsp90 expression, both in terms of Hsp90 positive cells (% and Allred score (74.2 ± 11.2 vs. 59.1 ± 14.2 p = 0.0001; 7.00 ± 0.95 vs. 6.22 ± 1.01, p = 0.007, Wilcoxon matched-pairs signed-ranks test. Concerning the intensity of Hsp90 immunostaining only a marginal decrease was noted (2.16 ± 0.68 vs. 1.84 ± 0.63, p = 0.087, Wilcoxon matched-pairs signed-ranks test. Conclusion ILC lesions seem to exhibit decreased Hsp90 expression, a finding contrary to what might have been expected, given that high Hsp90 expression is a trait of invasive ductal carcinomas.

Stability of the Human Hsp90-p50Cdc37 Chaperone Complex against Nucleotides and Hsp90 Inhibitors, and the Influence of Phosphorylation by Casein Kinase 2

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Sanne H. Olesen

2015-01-01

Full Text Available The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein–protein interaction (PPI inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2 did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM, while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands.

HSP72 protects cells from ER stress-induced apoptosis via enhancement of IRE1alpha-XBP1 signaling through a physical interaction.

LENUS (Irish Health Repository)

Gupta, Sanjeev

2010-01-01

Endoplasmic reticulum (ER) stress is a feature of secretory cells and of many diseases including cancer, neurodegeneration, and diabetes. Adaptation to ER stress depends on the activation of a signal transduction pathway known as the unfolded protein response (UPR). Enhanced expression of Hsp72 has been shown to reduce tissue injury in response to stress stimuli and improve cell survival in experimental models of stroke, sepsis, renal failure, and myocardial ischemia. Hsp72 inhibits several features of the intrinsic apoptotic pathway. However, the molecular mechanisms by which Hsp72 expression inhibits ER stress-induced apoptosis are not clearly understood. Here we show that Hsp72 enhances cell survival under ER stress conditions. The UPR signals through the sensor IRE1alpha, which controls the splicing of the mRNA encoding the transcription factor XBP1. We show that Hsp72 enhances XBP1 mRNA splicing and expression of its target genes, associated with attenuated apoptosis under ER stress conditions. Inhibition of XBP1 mRNA splicing either by dominant negative IRE1alpha or by knocking down XBP1 specifically abrogated the inhibition of ER stress-induced apoptosis by Hsp72. Regulation of the UPR was associated with the formation of a stable protein complex between Hsp72 and the cytosolic domain of IRE1alpha. Finally, Hsp72 enhanced the RNase activity of recombinant IRE1alpha in vitro, suggesting a direct regulation. Our data show that binding of Hsp72 to IRE1alpha enhances IRE1alpha\\/XBP1 signaling at the ER and inhibits ER stress-induced apoptosis. These results provide a physical connection between cytosolic chaperones and the ER stress response.

Proteomics shows Hsp70 does not bind peptide sequences indiscriminately in vivo

International Nuclear Information System (INIS)

Grossmann, Michael E.; Madden, Benjamin J.; Gao, Fan; Pang, Yuan-Ping; Carpenter, John E.; McCormick, Daniel; Young, Charles Y.F.

2004-01-01

Heat shock protein 70 (Hsp70) binds peptide and has several functions that include protein folding, protein trafficking, and involvement with immune function. However, endogenous Hsp70-binding peptides had not previously been identified. Therefore, we eluted and identified several hundred endogenously bound peptides from Hsp70 using liquid chromatography ion trap mass spectrophotometry (LC-ITMS). Our work shows that the peptides are capable of binding Hsp70 as previously described. They are generally 8-26 amino acids in length and correspond to specific regions of many proteins. Through computationally assisted analysis of peptides eluted from Hsp70 we determined variable amino acid sequences, including a 5 amino acid core sequence that Hsp70 favorably binds. We also developed a computer algorithm that predicts Hsp70 binding within proteins. This work helps to define what peptides are bound by Hsp70 in vivo and suggests that Hsp70 facilitates peptide selection by aiding a funneling mechanism that is flexible but allows only a limited number of peptides to be processed

Tumor imaging and targeting potential of an Hsp70-derived 14-mer peptide.

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Mathias Gehrmann

Full Text Available We have previously used a unique mouse monoclonal antibody cmHsp70.1 to demonstrate the selective presence of a membrane-bound form of Hsp70 (memHsp70 on a variety of leukemia cells and on single cell suspensions derived from solid tumors of different entities, but not on non-transformed cells or cells from corresponding 'healthy' tissue. This antibody can be used to image tumors in vivo and target them for antibody-dependent cellular cytotoxicity. Tumor-specific expression of memHsp70 therefore has the potential to be exploited for theranostic purposes. Given the advantages of peptides as imaging and targeting agents, this study assessed whether a 14-mer tumor penetrating peptide (TPP; TKDNNLLGRFELSG, the sequence of which is derived from the oligomerization domain of Hsp70 which is expressed on the cell surface of tumor cells, can also be used for targeting membrane Hsp70 positive (memHsp70+ tumor cells, in vitro.The specificity of carboxy-fluorescein (CF- labeled TPP (TPP to Hsp70 was proven in an Hsp70 knockout mammary tumor cell system. TPP specifically binds to different memHsp70+ mouse and human tumor cell lines and is rapidly taken up via endosomes. Two to four-fold higher levels of CF-labeled TPP were detected in MCF7 (82% memHsp70+ and MDA-MB-231 (75% memHsp70+ cells compared to T47D cells (29% memHsp70+ that exhibit a lower Hsp70 membrane positivity. After 90 min incubation, TPP co-localized with mitochondrial membranes in memHsp70+ tumors. Although there was no evidence that any given vesicle population was specifically localized, fluorophore-labeled cmHsp70.1 antibody and TPP preferentially accumulated in the proximity of the adherent surface of cultured cells. These findings suggest a potential association between membrane Hsp70 expression and cytoskeletal elements that are involved in adherence, the establishment of intercellular synapses and/or membrane reorganization.This study demonstrates the specific binding and rapid

Recombinant heat shock protein 27 (HSP27/HSPB1) protects against cadmium-induced oxidative stress and toxicity in human cervical cancer cells.

Science.gov (United States)

Alvarez-Olmedo, Daiana G; Biaggio, Veronica S; Koumbadinga, Geremy A; Gómez, Nidia N; Shi, Chunhua; Ciocca, Daniel R; Batulan, Zarah; Fanelli, Mariel A; O'Brien, Edward R

2017-05-01

Cadmium (Cd) is a carcinogen with several well-described toxicological effects in humans, but its molecular mechanisms are still not fully understood. Overexpression of heat shock protein 27 (HSP27/HSPB1)-a multifunctional protein chaperone-has been shown to protect cells from oxidative damage and apoptosis triggered by Cd exposure. The aims of this work were to investigate the potential use of extracellular recombinant HSP27 to prevent/counteract Cd-induced cellular toxicity and to evaluate if peroxynitrite was involved in the development of Cd-induced toxicity. Here, we report that the harmful effects of Cd correlated with changes in oxidative stress markers: upregulation of reactive oxygen species, reduction in nitric oxide (NO) bioavailability, increment in lipid peroxidation, peroxynitrite (PN), and protein nitration; intracellular HSP27 was reduced. Treatments with Cd (100 μM) for 24 h or with the peroxynitrite donor, SIN-1, decreased HSP27 levels (~50%), suggesting that PN formation is responsible for the reduction of HSP27. Pre-treatments of the cells either with Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) (a pharmacological inhibitor of NO synthase) or with recombinant HSP27 (rHSP27) attenuated the disruption of the cellular metabolism induced by Cd, increasing in a 55 and 52%, respectively, the cell viability measured by CCK-8. Cd induced necrotic cell death pathways, although apoptosis was also activated; pre-treatment with L-NAME or rHSP27 mitigated cell death. Our findings show for the first time a direct relationship between Cd-induced toxicity and PN production and a role for rHSP27 as a potential therapeutic agent that may counteract Cd toxicity.

Candidate genes and molecular markers associated with heat tolerance in colonial Bentgrass.

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Jespersen, David; Belanger, Faith C; Huang, Bingru

2017-01-01

Elevated temperature is a major abiotic stress limiting the growth of cool-season grasses during the summer months. The objectives of this study were to determine the genetic variation in the expression patterns of selected genes involved in several major metabolic pathways regulating heat tolerance for two genotypes contrasting in heat tolerance to confirm their status as potential candidate genes, and to identify PCR-based markers associated with candidate genes related to heat tolerance in a colonial (Agrostis capillaris L.) x creeping bentgrass (Agrostis stolonifera L.) hybrid backcross population. Plants were subjected to heat stress in controlled-environmental growth chambers for phenotypic evaluation and determination of genetic variation in candidate gene expression. Molecular markers were developed for genes involved in protein degradation (cysteine protease), antioxidant defense (catalase and glutathione-S-transferase), energy metabolism (glyceraldehyde-3-phosphate dehydrogenase), cell expansion (expansin), and stress protection (heat shock proteins HSP26, HSP70, and HSP101). Kruskal-Wallis analysis, a commonly used non-parametric test used to compare population individuals with or without the gene marker, found the physiological traits of chlorophyll content, electrolyte leakage, normalized difference vegetative index, and turf quality were associated with all candidate gene markers with the exception of HSP101. Differential gene expression was frequently found for the tested candidate genes. The development of candidate gene markers for important heat tolerance genes may allow for the development of new cultivars with increased abiotic stress tolerance using marker-assisted selection.

Candidate genes and molecular markers associated with heat tolerance in colonial Bentgrass.

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David Jespersen

Full Text Available Elevated temperature is a major abiotic stress limiting the growth of cool-season grasses during the summer months. The objectives of this study were to determine the genetic variation in the expression patterns of selected genes involved in several major metabolic pathways regulating heat tolerance for two genotypes contrasting in heat tolerance to confirm their status as potential candidate genes, and to identify PCR-based markers associated with candidate genes related to heat tolerance in a colonial (Agrostis capillaris L. x creeping bentgrass (Agrostis stolonifera L. hybrid backcross population. Plants were subjected to heat stress in controlled-environmental growth chambers for phenotypic evaluation and determination of genetic variation in candidate gene expression. Molecular markers were developed for genes involved in protein degradation (cysteine protease, antioxidant defense (catalase and glutathione-S-transferase, energy metabolism (glyceraldehyde-3-phosphate dehydrogenase, cell expansion (expansin, and stress protection (heat shock proteins HSP26, HSP70, and HSP101. Kruskal-Wallis analysis, a commonly used non-parametric test used to compare population individuals with or without the gene marker, found the physiological traits of chlorophyll content, electrolyte leakage, normalized difference vegetative index, and turf quality were associated with all candidate gene markers with the exception of HSP101. Differential gene expression was frequently found for the tested candidate genes. The development of candidate gene markers for important heat tolerance genes may allow for the development of new cultivars with increased abiotic stress tolerance using marker-assisted selection.

Transfection of Chinese hamster ovary DHFR/sup -/ cells with the gene coding for heat shock protein 70 from drosophila melanogaster

International Nuclear Information System (INIS)

Duffy, J.J.; Carper, S.W.; Gerner, E.W.

1987-01-01

Chinese hamster ovary DHFR/sup -/ cells (CHO-DHFR/sup -/) were transfected with the plasmid pSV2-dhfr expressing the mouse gene coding for dhfr or with the same plasmid containing the gene coding for the Drosophila melanogaster heat shock protein 70 (hsp70), pSVd-hsp70. Three subcloned cell lines selected for expression of the dhfr gene were shown to contain either the vector sequence (G cells) or varying copies of pSVd-hsp70 (H cells). One line of H cells was shown to contain > 30 copies of the D. melanogaster hsp70 gene and to express the hsp70 RNA at significant levels. No difference between G and H cells was observed in the rate of growth, in the development of thermotolerance, or in the sensitivity of actin microfilament bundles to heat shock. However, H cells containing the transfected hsp70 gene had an altered morphology when compared to the G cells and the parental CHO-DHFR/sup -/ cells being more fibroblastic. The adhesion properties of the H cells was also decreased when compared to the G cells. These results show that insertion of the D. melanogaster gene into CHO cells does not effect growth rates or heat shock responses but may alter cell morphology and adhesion

Extrapolation of Inter Domain Communications and Substrate Binding Cavity of Camel HSP70 1A: A Molecular Modeling and Dynamics Simulation Study.

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Saurabh Gupta

Full Text Available Heat shock protein 70 (HSP70 is an important chaperone, involved in protein folding, refolding, translocation and complex remodeling reactions under normal as well as stress conditions. However, expression of HSPA1A gene in heat and cold stress conditions associates with other chaperons and perform its function. Experimental structure for Camel HSP70 protein (cHSP70 has not been reported so far. Hence, we constructed 3D models of cHSP70 through multi- template comparative modeling with HSP110 protein of S. cerevisiae (open state and with HSP70 protein of E. coli 70kDa DnaK (close state and relaxed them for 100 nanoseconds (ns using all-atom Molecular Dynamics (MD Simulation. Two stable conformations of cHSP70 with Substrate Binding Domain (SBD in open and close states were obtained. The collective mode analysis of different transitions of open state to close state and vice versa was examined via Principal Component Analysis (PCA and Minimum Distance Matrix (MDM. The results provide mechanistic representation of the communication between Nucleotide Binding Domain (NBD and SBD to identify the role of sub domains in conformational change mechanism, which leads the chaperone cycle of cHSP70. Further, residues present in the chaperon functioning site were also identified through protein-peptide docking. This study provides an overall insight into the inter domain communication mechanism and identification of the chaperon binding cavity, which explains the underlying mechanism involved during heat and cold stress conditions in camel.

Regulation of Hsp27 and Hsp70 expression in human and mouse skin construct models by caveolae following exposure to the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide

International Nuclear Information System (INIS)

Black, Adrienne T.; Hayden, Patrick J.; Casillas, Robert P.; Heck, Diane E.; Gerecke, Donald R.; Sinko, Patrick J.; Laskin, Debra L.; Laskin, Jeffrey D.

2011-01-01

Dermal exposure to the vesicant sulfur mustard causes marked inflammation and tissue damage. Basal keratinocytes appear to be a major target of sulfur mustard. In the present studies, mechanisms mediating skin toxicity were examined using a mouse skin construct model and a full-thickness human skin equivalent (EpiDerm-FT TM ). In both systems, administration of the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide (CEES, 100-1000 μM) at the air surface induced mRNA and protein expression of heat shock proteins 27 and 70 (Hsp27 and Hsp70). CEES treatment also resulted in increased expression of caveolin-1, the major structural component of caveolae. Immunohistochemistry revealed that Hsp27, Hsp70 and caveolin-1 were localized in basal and suprabasal layers of the epidermis. Caveolin-1 was also detected in fibroblasts in the dermal component of the full thickness human skin equivalent. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that Hsp27 and Hsp70 were localized in caveolae. Treatment of mouse keratinocytes with filipin III or methyl-β-cyclodextrin, which disrupt caveolar structure, markedly suppressed CEES-induced Hsp27 and Hsp70 mRNA and protein expression. CEES treatment is known to activate JNK and p38 MAP kinases; in mouse keratinocytes, inhibition of these enzymes suppressed CEES-induced expression of Hsp27 and Hsp70. These data suggest that MAP kinases regulate Hsp 27 and Hsp70; moreover, caveolae-mediated regulation of heat shock protein expression may be important in the pathophysiology of vesicant-induced skin toxicity.

Molecular mechanisms of canalization: Hsp90 and beyond

Indian Academy of Sciences (India)

Madhu Sudhan

2007-03-26

Mar 26, 2007 ... In plants, Hsp90 function remained little characterized until recent .... unknown if Hsp90 and chromatin remodeling factors act independently or in ..... protein 90 in plant disease resistance; EMBO J. 22 5690–5699. Maloof J N ...

Hormone replacement therapy increases levels of antibodies against heat shock protein 65 and certain species of oxidized low density lipoprotein

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Uint L.

2003-01-01

Full Text Available Hormone replacement therapy (HRT reduces cardiovascular risks, although the initiation of therapy may be associated with transient adverse ischemic and thrombotic events. Antibodies against heat shock protein (Hsp and oxidized low density lipoprotein (LDL have been found in atherosclerotic lesions and plasma of patients with coronary artery disease and may play an important role in the pathogenesis of atherosclerosis. The aim of the present study was to assess the effects of HRT on the immune response by measuring plasma levels of antibodies against Hsp 65 and LDL with a low and high degree of copper-mediated oxidative modification of 20 postmenopausal women before and 90 days after receiving orally 0.625 mg equine conjugate estrogen plus 2.5 mg medroxyprogesterone acetate per day. HRT significantly increased antibodies against Hsp 65 (0.316 ± 0.03 vs 0.558 ± 0.11 and against LDL with a low degree of oxidative modification (0.100 ± 0.01 vs 0.217 ± 0.02 (P<0.05 and P<0.001, respectively, ANOVA. The hormone-mediated immune response may trigger an inflammatory response within the vessel wall and potentially increase plaque burden. Whether or not this immune response is temporary or sustained and deleterious requires further investigation.

The Type II Hsp40 Sis1 cooperates with Hsp70 and the E3 ligase Ubr1 to promote degradation of terminally misfolded cytosolic protein.

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Daniel W Summers

Full Text Available Mechanisms for cooperation between the cytosolic Hsp70 system and the ubiquitin proteasome system during protein triage are not clear. Herein, we identify new mechanisms for selection of misfolded cytosolic proteins for degradation via defining functional interactions between specific cytosolic Hsp70/Hsp40 pairs and quality control ubiquitin ligases. These studies revolved around the use of S. cerevisiae to elucidate the degradation pathway of a terminally misfolded reporter protein, short-lived GFP (slGFP. The Type I Hsp40 Ydj1 acts with Hsp70 to suppress slGFP aggregation. In contrast, the Type II Hsp40 Sis1 is required for proteasomal degradation of slGFP. Sis1 and Hsp70 operate sequentially with the quality control E3 ubiquitin ligase Ubr1 to target slGFP for degradation. Compromise of Sis1 or Ubr1 function leads slGFP to accumulate in a Triton X-100-soluble state with slGFP degradation intermediates being concentrated into perinuclear and peripheral puncta. Interestingly, when Sis1 activity is low the slGFP that is concentrated into puncta can be liberated from puncta and subsequently degraded. Conversely, in the absence of Ubr1, slGFP and the puncta that contain slGFP are relatively stable. Ubr1 mediates proteasomal degradation of slGFP that is released from cytosolic protein handling centers. Pathways for proteasomal degradation of misfolded cytosolic proteins involve functional interplay between Type II Hsp40/Hsp70 chaperone pairs, PQC E3 ligases, and storage depots for misfolded proteins.

A primate specific extra domain in the molecular chaperone Hsp90.

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Vishwadeepak Tripathi

Full Text Available Hsp90 (heat shock protein 90 is an essential molecular chaperone that mediates folding and quality control of client proteins. Many of them such as protein kinases, steroid receptors and transcription factors are involved in cellular signaling processes. Hsp90 undergoes an ATP hydrolysis dependent conformational cycle to assist folding of the client protein. The canonical Hsp90 shows a typical composition of three distinct domains and interacts with individual cochaperone partners such as Hop, Cdc37 and Aha1 (activator of Hsp90 ATPase that regulate the reaction cycle of the molecular chaperone. A bioinformatic survey identified an additional domain of 122 amino acids in front of the canonical Hsp90 sequence. This extra domain (E domain is specific to the Catarrhini or drooping nose monkeys, a subdivision of the higher primates that includes man, the great apes and the old world monkeys but is absent from all other species. Our biochemical analysis reveals that Hsp103 associates with cochaperone proteins such as Hop, Cdc37 and Aha1 similar to Hsp90. However, the extra domain reduces the ATP hydrolysis rate to about half when compared to Hsp90 thereby acting as a negative regulator of the molecular chaperonés intrinsic ATPase activity.

Cell stress promotes the association of phosphorylated HspB1 with F-actin.

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Joseph P Clarke

Full Text Available Previous studies have suggested that the small heat shock protein, HspB1, has a direct influence on the dynamics of cytoskeletal elements, in particular, filamentous actin (F-actin polymerization. In this study we have assessed the influence of HspB1 phosphorylation on its interaction(s with F-actin. We first determined the distribution of endogenous non-phosphorylated HspB1, phosphorylated HspB1 and F-actin in neuroendocrine PC12 cells by immunocytochemistry and confocal microscopy. We then investigated a potential direct interaction between HspB1 with F-actin by precipitating F-actin directly with biotinylated phalloidin followed by Western analyses; the reverse immunoprecipitation of HspB1 was also carried out. The phosphorylation influence of HspB1 in this interaction was investigated by using pharmacologic inhibition of p38 MAPK. In control cells, HspB1 interacts with F-actin as a predominantly non-phosphorylated protein, but subsequent to stress there is a redistribution of HspB1 to the cytoskeletal fraction and a significantly increased association of pHspB1 with F-actin. Our data demonstrate HspB1 is found in a complex with F-actin both in phosphorylated and non-phosphorylated forms, with an increased association of pHspB1 with F-actin after heat stress. Overall, our study combines both cellular and biochemical approaches to show cellular localization and direct demonstration of an interaction between endogenous HspB1 and F-actin using methodolgy that specifically isolates F-actin.

Characterize and Gene Expression of Heat Shock Protein 90 in Marine Crab Charybdis japonica following Bisphenol A and 4-Nonylphenol Exposures.

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Park, Kiyun; Kwak, Ihn-Sil

2014-01-01

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone important in the maturation of a broad spectrum of protein. In this study, an HSP90 gene was isolated from Asian paddle crab, Charybdis japonica, as a bio-indicator to monitor the marine ecosystem. This work reports the responses of C. japonica HSP90 mRNA expression to cellular stress by endocrine disrupting chemicals (EDCs), such as bisphenol A (BPA) and 4-nonylphenol (NP) using real-time. reverse transcription polymerase chain reaction. The deduced amino acid sequence of HSP90 from C. japonica shared a high degree of homology with their homologues in other species. In a phylogenetic analysis, C. japonica HSP90 is evolutionally related with an ortholog of the other crustacean species. The expression of HSP90 gene was almost distributed in all the examined tissues of the C. japonica crab but expression levels varied among the different body parts of the crabs. We examined HSP90 mRNA expression pattern in C. japonica crabs exposed to EDCs for various exposure times. The expression of HSP90 transcripts was significantly increased in C. japonica crabs exposed to BPA and NP at different concentrations for 12, 24, 48 and 96 hours. The mRNA expression of HSP90 gene was significantly induced in a concentration- and time-dependent manner after BPA or NP exposures for 96 hours. Taken together, expression analysis of Asian paddle crab HSP90 gene provided useful molecular information about crab responses in stress conditions and potential ways to monitor the EDCs stressors in marine environments.

Quercetin suppresses drug-resistant spheres via the p38 MAPK-Hsp27 apoptotic pathway in oral cancer cells.

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Su-Feng Chen

Full Text Available BACKGROUND: Treatment failure in oral squamous cell carcinoma (OSCC leading to local recurrence(s and metastases is mainly due to drug resistance. Cancer stem cells (CSCs are thought be responsible for the development of drug resistance. However, the correlations between CSCs, drug resistance, and new strategy against drug resistance in OSCC remain elusive. METHODS: A drug-resistant sphere (DRSP model was generated by using a nonadhesive culture system to induce drug-resistant cells from SCC25 oral cancer cells. A comparative analysis was performed between the parent control cells and DRSPs with a related treatment strategy focusing on the expression of epithelial-mesenchymal transition (EMT-associated markers, drug-resistance-related genes, and CSC properties in vitro, as well as tumorigenicity and the regimen for tumor regression in vivo. RESULTS: Our data show the presence of a phenomenon of EMT with gradual cellular transition from an epithelioid to mesenchymal-like spheroid morphology during induction of drug resistance. The characterization of DRSPs revealed the upregulation of the drug-resistance-related genes ABCG2 and MDR-1 and of CSC-representative markers, suggesting that DRSPs have greater resistance to cisplatin (Cis and stronger CSC properties compared with the control. Moreover, overexpression of phosphorylated heat-shock protein 27 (p-Hsp27 via the activation of p38 MAPK signaling was observed in DRSPs. Knockdown of Hsp27 decreased Cis resistance and induced apoptosis in DRSPs. Furthermore, an inhibitor of Hsp27, quercetin (Qu, suppressed p-Hsp27 expression, with alterations of the EMT signature, leading to the promotion of apoptosis in DRSPs. A xenographic study also confirmed the increase of tumorigenicity in DRSPs. The combination of Qu and Cis can reduce tumor growth and decrease drug resistance in OSCC. CONCLUSIONS: The p38 MAPK-Hsp27 axis plays an important role in CSCs-mediated drug resistance in OSCC. Targeting this axis

HSP72 protects cells from ER stress-induced apoptosis via enhancement of IRE1alpha-XBP1 signaling through a physical interaction.

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Sanjeev Gupta

2010-07-01

Full Text Available Endoplasmic reticulum (ER stress is a feature of secretory cells and of many diseases including cancer, neurodegeneration, and diabetes. Adaptation to ER stress depends on the activation of a signal transduction pathway known as the unfolded protein response (UPR. Enhanced expression of Hsp72 has been shown to reduce tissue injury in response to stress stimuli and improve cell survival in experimental models of stroke, sepsis, renal failure, and myocardial ischemia. Hsp72 inhibits several features of the intrinsic apoptotic pathway. However, the molecular mechanisms by which Hsp72 expression inhibits ER stress-induced apoptosis are not clearly understood. Here we show that Hsp72 enhances cell survival under ER stress conditions. The UPR signals through the sensor IRE1alpha, which controls the splicing of the mRNA encoding the transcription factor XBP1. We show that Hsp72 enhances XBP1 mRNA splicing and expression of its target genes, associated with attenuated apoptosis under ER stress conditions. Inhibition of XBP1 mRNA splicing either by dominant negative IRE1alpha or by knocking down XBP1 specifically abrogated the inhibition of ER stress-induced apoptosis by Hsp72. Regulation of the UPR was associated with the formation of a stable protein complex between Hsp72 and the cytosolic domain of IRE1alpha. Finally, Hsp72 enhanced the RNase activity of recombinant IRE1alpha in vitro, suggesting a direct regulation. Our data show that binding of Hsp72 to IRE1alpha enhances IRE1alpha/XBP1 signaling at the ER and inhibits ER stress-induced apoptosis. These results provide a physical connection between cytosolic chaperones and the ER stress response.

[Environmental investigation of ground water contamination at Wright- Patterson Air Force Base, Ohio]. Volume 4, Health and Safety Plan (HSP); Phase 1, Task 4 Field Investigation report: Draft

Energy Technology Data Exchange (ETDEWEB)

1991-10-01

This Health and Safety Plan (HSP) was developed for the Environmental Investigation of Ground-water Contamination Investigation at Wright-Patterson Air Force Base near Dayton, Ohio, based on the projected scope of work for the Phase 1, Task 4 Field Investigation. The HSP describes hazards that may be encountered during the investigation, assesses the hazards, and indicates what type of personal protective equipment is to be used for each task performed. The HSP also addresses the medical monitoring program, decontamination procedures, air monitoring, training, site control, accident prevention, and emergency response.

A Toolbox for Quantitative Gene Expression in Varroa destructor: RNA Degradation in Field Samples and Systematic Analysis of Reference Gene Stability.

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Ewan M Campbell

Full Text Available Varroa destructor is the major pest of Apis mellifera and contributes to the global honey bee health crisis threatening food security. Developing new control strategies to combat Varroa will require the application of molecular biology, including gene expression studies by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR. Both high quality RNA samples and suitable stable internal reference genes are required for accurate gene expression studies. In this study, ten candidate genes (succinate dehydrogenase (SDHA, NADH dehydrogenase (NADH, large ribsosmal subunit, TATA-binding protein, glyceraldehyde-3-phosphate dehydrogenase, 18S rRNA (18S, heat-shock protein 90 (HSP90, cyclophilin, α-tubulin, actin, were evaluated for their suitability as normalization genes using the geNorm, Normfinder, BestKeeper, and comparative ΔCq algorithims. Our study proposes the use of no more than two of the four most stable reference genes (NADH, 18S, SDHA and HSP90 in Varroa gene expression studies. These four genes remain stable in phoretic and reproductive stage Varroa and are unaffected by Deformed wing virus load. When used for determining changes in vitellogenin gene expression, the signal-to-noise ratio (SNR for the relatively unstable genes actin and α-tubulin was much lower than for the stable gene combinations (NADH + HSP90 +18S; NADH + HSP90; or NADH. Using both electropherograms and RT-qPCR for short and long amplicons as quality controls, we demonstrate that high quality RNA can be recovered from Varroa up to 10 days later stored at ambient temperature if collected into RNAlater and provided the body is pierced. This protocol allows the exchange of Varroa samples between international collaborators and field sample collectors without requiring frozen collection or shipping. Our results make important contributions to gene expression studies in Varroa by proposing a validated sampling protocol to obtain high quality Varroa

Cell-surface expression of Hsp70 on hematopoietic cancer cells after inhibition of HDAC activity

DEFF Research Database (Denmark)

Jensen, Helle; Andresen, Lars; Hansen, Karen Aagaard

Heat shock proteins (HSPs) are highly conserved molecules, which support folding of proteins under physiological conditions and mediate protection against lethal damage after various stress stimuli. Five HSP families exist defined by their molecular size (i.e. HSP100, HSP90, HSP70, HSP60, and the......Heat shock proteins (HSPs) are highly conserved molecules, which support folding of proteins under physiological conditions and mediate protection against lethal damage after various stress stimuli. Five HSP families exist defined by their molecular size (i.e. HSP100, HSP90, HSP70, HSP60...... clinically applied reagents, such as alkyl-lysophospholipides, chemotherapeutic agents, and anti-inflammatory reagents, have been found to enhance Hsp70 surface expression on cancer cells. We have found that inhibition of histone deacetylase (HDAC) activity leads to surface expression of Hsp70 on various...... hematopoietic cancer cells, an occurance that was not observed on naïve or activated peripheral blood cells. HDAC-inhibitor mediated Hsp70 surface expression was confined to the apoptotic Annexin V positive cells and blocked by inhibition of apoptosis. Other chemotherapeutic inducers of apoptosis...

Hsp27, Hsp70 and mismatch repair proteins hMLH1 and hMSH2 expression in peripheral blood lymphocytes from healthy subjects and cancer patients.

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Nadin, Silvina Beatriz; Vargas-Roig, Laura M; Drago, Gisela; Ibarra, Jorge; Ciocca, Daniel R

2007-07-08

Mismatch repair (MMR) deficiency and higher expression levels of heat shock proteins (Hsps) have been implicated with drug resistance to topoisomerase II poisons (doxorubicin) and to platinum compounds (cisplatin). This study was designed to determine individual influences of doxorubicin and cisplatin treatment on the expression of Hsp27, Hsp70, hMLH1 and hMSH2 proteins and in the DNA damage status in peripheral blood lymphocytes (PBLs). In addition, we studied whether these proteins and the DNA damage correlated with the survival of cancer patients. PBLs from 10 healthy donors and 25 cancer patients (before and after three cycles of chemotherapy) were exposed to in vitro treatments: C (control), HS (heat shock at 42 degrees C), Do or Pt (doxorubicin or cisplatin alone), and HS+Do or HS+Pt (heat shock+doxorubicin or heat shock+cisplatin). PBLs were collected at time 0 (T0: immediately after drug treatment) and after 24h of repair (T24). Hsp27, Hsp70, hMLH1 and hMSH2 were studied by immunocytochemistry and the DNA damage by alkaline comet assay. Immunofluorescence studies and confocal microscopy revealed that hMLH1 and hMSH2 colocalized with Hsp27 and Hsp72 (inducible form of Hsp70). hMLH1 and hMSH2 were significantly induced by Pt and HS+Pt at T24 in cancer patients, but only modestly influenced by Do. Cancer patients presented higher basal expression of total and nuclear Hsp27 and Hsp70 than controls, and these proteins were also increased by HS, Do and HS+Do. The Hsp70 induction by Pt and HS+Pt was noted in cancer patients, especially nuclear Hsp70. In cancer patients, basal DNA damage was slightly higher than in healthy persons; and after Pt and HS+Pt treatments, DNA migration and number of apoptotic cells were higher than controls. Hsps accomplished a cytoprotective function in pre-chemotherapy PBLs (HS before Do or Pt), but not in post-chemotherapy samples. In Pt-treated patients the ratio N/C (nuclear/cytoplasmic) of Hsp27 was related to disease free survival

Short-hairpin RNA-mediated Heat shock protein 90 gene silencing inhibits human breast cancer cell growth in vitro and in vivo

International Nuclear Information System (INIS)

Zuo, Keqiang; Li, Dan; Pulli, Benjamin; Yu, Fei; Cai, Haidong; Yuan, Xueyu; Zhang, Xiaoping; Lv, Zhongwei

2012-01-01

Highlights: ► Hsp90 is over-expressed in human breast cancer. ► The shRNA-mediated gene silencing of Hsp90 resulted in inhibition of cell growth. ► Akt and NF-kB were down-regulation after transfection due to Hsp90 silencing. ► The tumor growth ratio was decline due to Hsp90 silencing. ► The PCNA expression was down-regulation due to Hsp90 silencing. -- Abstract: Hsp90 interacts with proteins that mediate signaling pathways involved in the regulation of essential processes such as proliferation, cell cycle control, angiogenesis and apoptosis. Hsp90 inhibition is therefore an attractive strategy for blocking abnormal pathways that are crucial for cancer cell growth. In the present study, the role of Hsp90 in human breast cancer MCF-7 cells was examined by stably silencing Hsp90 gene expression with an Hsp90-silencing vector (Hsp90-shRNA). RT-PCR and Western blot analyses showed that Hsp90-shRNA specifically and markedly down-regulated Hsp90 mRNA and protein expression. NF-kB and Akt protein levels were down-regulated in Hsp90-shRNA transfected cells, indicating that Hsp90 knockout caused a reduction of survival factors and induced apoptosis. Treatment with Hsp90-shRNA significantly increased apoptotic cell death and caused cell cycle arrest in the G1/S phase in MCF-7 cells, as shown by flow cytometry. Silencing of Hsp90 also reduced cell viability, as determined by MTT assay. In vivo experiments showed that MCF-7 cells stably transfected with Hsp90-shRNA grew slowly in nude mice as compared with control groups. In summary, the Hsp90-shRNA specifically silenced the Hsp90 gene, and inhibited MCF-7 cell growth in vitro and in vivo. Possible molecular mechanisms underlying the effects of Hsp90-shRNA include the degradation of Hsp90 breast cancer-related client proteins, the inhibition of survival signals and the upregulation of apoptotic pathways. shRNA-mediated interference may have potential therapeutic utility in human breast cancer.

HSP60 mediates the neuroprotective effects of curcumin by suppressing microglial activation.

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Ding, Feijia; Li, Fan; Li, Yunhong; Hou, Xiaolin; Ma, Yi; Zhang, Nan; Ma, Jiao; Zhang, Rui; Lang, Bing; Wang, Hongyan; Wang, Yin

2016-08-01

Curcumin has anti-inflammatory and antioxidant properties and has been widely used to treat or prevent neurodegenerative diseases. However, the mechanisms underlying the neuroprotective effects of curcumin are not well known. In the present study, the effect of curcumin on lipopolysaccharide (LPS)-stimulated BV2 mouse microglia cells was investigated using enzyme-linked immunosorbent assays of the culture medium and western blotting of cell lysates. The results showed that curcumin significantly inhibited the LPS-induced expression and release of heat shock protein 60 (HSP60) in the BV2 cells. The level of heat shock factor (HSF)-1 was upregulated in LPS-activated BV2 microglia, indicating that the increased expression of HSP60 was driven by HSF-1 activation. However, the increased HSF-1 level was downregulated by curcumin. Extracellular HSP60 is a ligand of Toll-like receptor 4 (TLR-4), and the level of the latter was increased in the LPS-activated BV2 microglia and inhibited by curcumin. The activation of TLR-4 is known to be associated with the activation of myeloid differentiation primary response 88 (MyD88) and nuclear factor (NF)-κB, with the subsequent production of proinflammatory and neurotoxic factors. In the present study, curcumin demonstrated marked suppression of the LPS-induced expression of MyD88, NF-κB, caspase-3, inducible nitric oxide synthase, tumor necrosis factor-α, interleukin (IL)-1β and IL-6 in the microglia. These results indicate that curcumin may exert its neuroprotective and anti-inflammatory effects by inhibiting microglial activation through the HSP60/TLR-4/MyD88/NF-κB signaling wpathway. Therefore, curcumin may be useful for the treatment of neurodegenerative diseases that are associated with microglial activation.

Sulfate Reducing Bacteria and Mycobacteria Dominate the Biofilm Communities in a Chloraminated Drinking Water Distribution System.

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Gomez-Smith, C Kimloi; LaPara, Timothy M; Hozalski, Raymond M

2015-07-21

The quantity and composition of bacterial biofilms growing on 10 water mains from a full-scale chloraminated water distribution system were analyzed using real-time PCR targeting the 16S rRNA gene and next-generation, high-throughput Illumina sequencing. Water mains with corrosion tubercles supported the greatest amount of bacterial biomass (n = 25; geometric mean = 2.5 × 10(7) copies cm(-2)), which was significantly higher (P = 0.04) than cement-lined cast-iron mains (n = 6; geometric mean = 2.0 × 10(6) copies cm(-2)). Despite spatial variation of community composition and bacterial abundance in water main biofilms, the communities on the interior main surfaces were surprisingly similar, containing a core group of operational taxonomic units (OTUs) assigned to only 17 different genera. Bacteria from the genus Mycobacterium dominated all communities at the main wall-bulk water interface (25-78% of the community), regardless of main age, estimated water age, main material, and the presence of corrosion products. Further sequencing of the mycobacterial heat shock protein gene (hsp65) provided species-level taxonomic resolution of mycobacteria. The two dominant Mycobacteria present, M. frederiksbergense (arithmetic mean = 85.7% of hsp65 sequences) and M. aurum (arithmetic mean = 6.5% of hsp65 sequences), are generally considered to be nonpathogenic. Two opportunistic pathogens, however, were detected at low numbers: M. hemophilum (arithmetic mean = 1.5% of hsp65 sequences) and M. abscessus (arithmetic mean = 0.006% of hsp65 sequences). Sulfate-reducing bacteria from the genus Desulfovibrio, which have been implicated in microbially influenced corrosion, dominated all communities located underneath corrosion tubercules (arithmetic mean = 67.5% of the community). This research provides novel insights into the quantity and composition of biofilms in full-scale drinking water distribution systems, which is critical for assessing the risks to public health and to the

Expression of Hsp27 and Hsp70 and vacuolization in the pituitary glands in cases of fatal hypothermia.

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Doberentz, Elke; Markwerth, Philipp; Wagner, Rebecca; Madea, Burkhard

2017-09-01

Hypothermia causes systemic cellular stress. The pituitary gland is an endocrine gland and plays an important role in thermoregulation. When the core body temperature drops, the pituitary gland is activated by stimulation of hypothalamic hormones. In this study, we investigated morphological alterations of the pituitary gland in cases of fatal hypothermia. Several morphological alterations of the anterior lobe of the pituitary gland, such as hemorrhage, vacuolization, and hyperemia, have been previously described in fatal hypothermia. However, the diagnostic value of these findings is controversial. We compared 11 cases of fatal hypothermia with 10 cases lacking antemortem hypothermic influences. In the presence of thermal cellular stress, the expression of heat shock proteins increases to protect cellular structures. Therefore, we immunohistochemically analyzed Hsp27 and Hsp70. Hsp27 expression was detected in 27.3% of the cases of fatal hypothermia and in 10.0% of the control cases, whereas Hsp70 expression was not detected in any case. Additionally, Sudan staining was performed to quantify fatty degeneration. A positive reaction was found in 45.5% of the study group and in 10.0% of the control group. This indicates that fatty degeneration might be a valuable marker when other macroscopic signs of hypothermia are absent.

Reactive oxygen species-dependent HSP90 protein cleavage participates in arsenical As+3- and MMA+3-induced apoptosis through inhibition of telomerase activity via JNK activation

International Nuclear Information System (INIS)

Shen, S.-C.; Yang, L.-Y.; Lin, H.-Y.; Wu, C.-Y.; Su, T.-H.; Chen, Y.-C.

2008-01-01

The effects of six arsenic compounds including As +3 , MMA +3 , DMA +3 , As +5 , MMA +5 , and DMA +5 on the viability of NIH3T3 cells were examined. As +3 and MMA +3 , but not the others, exhibited significant cytotoxic effects in NIH3T3 cells through apoptosis induction. The apoptotic events such as DNA fragmentation and chromosome condensation induced by As +3 and MMA +3 were prevented by the addition of NAC and CAT, and induction of HO-1 gene expression in accordance with cleavage of the HSP90 protein, and suppression of telomerase activity were observed in NIH3T3 cells under As +3 and MMA +3 treatments. An increase in the intracellular peroxide level was examined in As +3 - and MMA +3 -treated NIH3T3 cells, and As +3 - and MMA +3 -induced apoptotic events were blocked by NAC, CAT, and DPI addition. HSP90 inhibitors, GA and RD, significantly attenuated the telomerase activity in NIH3T3 cells with an enhancement of As +3 - and MMA +3 -induced cytotoxicity. Suppression of JNKs significantly inhibited As +3 - and MMA +3 -induced apoptosis by blocking HSP90 protein cleavage and telomerase reduction in NIH3T3 cells. Furthermore, Hb, SnPP, and dexferosamine showed no effect against As +3 - and MMA +3 -induced apoptosis, and overexpression of HO-1 protein or inhibition of HO-1 protein expression did not affect the apoptosis induced by As +3 or MMA +3 . These data provide the first evidence to indicate that apoptosis induced by As +3 and MMA +3 is mediated by an ROS-dependent degradation of HSP90 protein and reduction of telomerase via JNK activation, and HO-1 induction might not be involved

Cat exposure induces both intra- and extracellular Hsp72: the role of adrenal hormones.

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Fleshner, Monika; Campisi, Jay; Amiri, Leila; Diamond, David M

2004-10-01

Heat-shock proteins (Hsp) play an important role in stress physiology. Exposure to a variety of stressors will induce intracellular Hsp72, and this induction is believed to be beneficial for cell survival. In contrast, Hsp72 released during stress (extracellular Hsp72; eHsp72) activates pro-inflammatory responses. Clearly, physical stressors such as heat, cold, H(2)O(2), intense exercise and tail shock will induce both intra- and extracellular Hsp72. The current study tested whether a psychological stressor, cat exposure, would also trigger this response. In addition, the potential role of adrenal hormones in the Hsp72 response was examined. Adult, male Sprague Dawley rats were either adrenalectomized (ADX) or sham operated. Ten days post-recovery, rats were exposed to either a cat with no physical contact or control procedures (n = 5-6/group) for 2 h. Levels of intracellular Hsp72 were measured in the brain (frontal cortex, hippocampus, hypothalamus, dorsal vagal complex) and pituitary (ELISA). Levels of eHsp72 (ELISA) and corticosterone (RIA) were measured from serum obtained at the end of the 2-h stress period. Rats that were exposed to a cat had elevated intracellular Hsp72 in hypothalamus and dorsal vagal complex, and elevated eHsp72 and corticosterone in serum. Both the intra- and extracellular Hsp72 responses were blocked or attenuated by ADX. This study demonstrates that cat exposure can stimulate the Hsp72 response and that adrenal hormones contribute to this response.

Hsp70 Expression Profile in Preeclampsia Model of Pregnant Rat (Rattus norvegicus) after Giving the EVOO

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Irianti, E.; ilyas, S.; Rosidah; Hutahaean, S.

2017-03-01

Heat shock protein (Hsp) has long been known to protect cells from oxidative stress. In this case an increased expression is found on several cases of preeclampsia. One of the efforts to prevent preeclampsia is by giving antioxidants such as Extra Virgin Olive Oil (EVOO) or it’s better known as olive oil (Oleoa europaea), in the form of extra virgin known for its rich antioxidant content of tocopherols (vitamin E). The purpose of this study is to determine the expression levels of Hsp70 serum on pregnant white rat model of preeclampsia after being given EVOO. This type of research is true experiment; the subjects were female white rats and male virgin with Sprague Dawley, ± 8-11 weeks old, 180g BB s / d 200g, healthy and didn’t show any physical defects. Samples were 25 animals, divided into 5 groups, which consisted of different control and treatment given to T2 (rat model of preeclampsia), T3 (rat model of preeclampsia + EVOO 0.45g/bw/day), T4 (rat model of preeclampsia + EVOO 0.9g/bw/day) and T5 (rat model of preeclampsia + EVOO 1.8g/bw/day). The determination of each group was done by simple random sampling. Result on serum levels of Hsp70 that were tested by Elisa test in rats showed the average control was 14.64 mg / ml, group T2: 22:51 mg/ml, T3: 13.62 mg/ml, T4: 15.92 mg/ml, T5: 16:09 mg/ml. ANOVA test showed the P value was 0.001 <0.005, which meant there were significant differences on serum Hsp70 levels in the control and treatment pregnant rats group. It was known that there was a significant difference level of Hsp70 serum in group of control rats with T2 (P value <0.001) after LSD test was conducted, but not so with the group T3, T4, and T5, where the difference was not significant. There was a significant difference in the levels of Hsp70 serum on group T2 and T3 (P value 0.000), T4 (0004), T5 (0000). The gift of EVOO in the treatment group which was given EVOO with even low doses was able to control the induction of Hsp70 serum levels, which

Noradrenaline increases the expression and release of Hsp72 by human neutrophils.

Science.gov (United States)

Giraldo, E; Multhoff, G; Ortega, E

2010-05-01

The blood concentration of extracellular 72kDa heat shock protein (eHsp72) increases under conditions of stress, including intense exercise. However, the signal(s), source(s), and secretory pathways in its release into the bloodstream have yet to be clarified. The aim of the present study was to evaluate the role of noradrenaline (NA) as a stress signal on the expression and release of Hsp72 by circulating neutrophils (as a source), all within a context of the immunophysiological regulation during exercise-induced stress in sedentary and healthy young (21-26years) women. The expression of Hsp72 on the surface of isolated neutrophils was determined by flow cytometry, and its release by cultured isolated neutrophils was determined by ELISA. Incubation with cmHsp70-FITC showed that neutrophils express Hsp72 on their surface under basal conditions. In addition, cultured isolated neutrophils (37 degrees C and 5% CO(2)) also released Hsp72 under basal conditions, with this release increasing from 10min to 24h in the absence of cell damage. NA at 10(-9)-10(-5)M doubled the percentage of neutrophils expressing Hsp72 after 60min and 24h incubation. NA also stimulated (by about 20%) the release of Hsp72 after 10min of incubation. (1) Hsp72 is expressed on the surface of isolated neutrophils under basal conditions, and this expression is augmented by NA. (2) Isolated neutrophils can also release Hsp72 under cultured basal conditions in the absence of cell death, and NA can increase this release. These results may contribute to confirming the hypothesis that NA can act as a "stress signal" for the increased eHsp72 in the context of exercise stress, with a role for neutrophils as a source for the expression and, to a lesser degree, the release of Hsp72 after activation by NA. Copyright 2010 Elsevier Inc. All rights reserved.

A member of the HSP90 family from ovine Babesia in China: molecular characterization, phylogenetic analysis and antigenicity.

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Guan, Guiquan; Liu, Junlong; Liu, Aihong; Li, Youquan; Niu, Qingli; Gao, Jinliang; Luo, Jianxun; Chauvin, Alain; Yin, Hong; Moreau, Emmanuelle

2015-09-01

Heat shock protein 90 (HSP90) is a key component of the molecular chaperone complex essential for activating many signalling proteins involved in the development and progression of pathogenic cellular transformation. A Hsp90 gene (BQHsp90) was cloned and characterized from Babesia sp. BQ1 (Lintan), an ovine Babesia isolate belonging to Babesia motasi-like group, by screening a cDNA expression library and performing rapid amplification of cDNA ends. The full-length cDNA of BQHsp90 is 2399 bp with an open reading frame of 2154 bp encoding a predicted 83 kDa polypeptide with 717 amino acid residues. It shows significant homology and similar structural characteristics to Hsp90 of other apicomplex organisms. Phylogenetic analysis, based on the HSP90 amino acid sequences, showed that the Babesia genus is clearly separated from other apicomplexa genera. Five Chinese ovine Babesia isolates were divided into 2 phylogenetic clusters, namely Babesia sp. Xinjiang (previously designated a new species) cluster and B. motasi-like cluster which could be further divided into 2 subclusters (Babesia sp. BQ1 (Lintan)/Babesia sp. Tianzhu and Babesia sp. BQ1 (Ningxian)/Babesia sp. Hebei). Finally, the antigenicity of rBQHSP90 protein from prokaryotic expression was also evaluated using western blot and enzyme-linked immunosorbent assay (ELISA).

Health screenings for women over age 65

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Health maintenance visit - women - over age 65; Physical exam - women - over age 65; Yearly exam - women - over age 65; Checkup - women - over age 65; Women's health - over age 65; Preventive care exam - women - over ...

Health screenings for men over age 65

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Health maintenance visit - men - over age 65; Physical exam - men - over age 65; Yearly exam - men - over age 65; Checkup - men - over age 65; Men's health - over age 65; Preventive care exam - men - over ...

Schiff Base Metal Derivatives Enhance the Expression of HSP70 and Suppress BAX Proteins in Prevention of Acute Gastric Lesion

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Shahram Golbabapour

2013-01-01

Full Text Available Schiff base complexes have appeared to be promising in the treatment of different diseases and disorders and have drawn a lot of attention to their biological activities. This study was conducted to evaluate the regulatory effect of Schiff base metal derivatives on the expression of heat shock proteins (HSP 70 and BAX in protection against acute haemorrhagic gastric ulcer in rats. Rats were assigned to 6 groups of 6 rats: the normal control (Tween 20 5% v/v, 5 mL/kg, the positive control (Tween 20 5% v/v, 5 mL/kg, and four Schiff base derivative groups named Schiff_1, Schiff_2, Schiff_3, and Schiff_4 (25 mg/kg. After 1 h, all of the groups received ethanol 95% (5 mL/kg but the normal control received Tween 20 (Tween 20 5% v/v, 5 mL/kg. The animals were euthanized after 60 min and the stomachs were dissected for histology (H&E, immunohistochemistry, and western blot analysis against HSP70 and BAX proteins. The results showed that the Schiff base metal derivatives enhanced the expression of HSP70 and suppressed the expression of BAX proteins during their gastroprotection against ethanol-induced gastric lesion in rats.

Schiff base metal derivatives enhance the expression of HSP70 and suppress BAX proteins in prevention of acute gastric lesion.

Science.gov (United States)

Golbabapour, Shahram; Gwaram, Nura Suleiman; Al-Obaidi, Mazen M Jamil; Soleimani, A F; Ali, Hapipah Mohd; Abdul Majid, Nazia

2013-01-01

Schiff base complexes have appeared to be promising in the treatment of different diseases and disorders and have drawn a lot of attention to their biological activities. This study was conducted to evaluate the regulatory effect of Schiff base metal derivatives on the expression of heat shock proteins (HSP) 70 and BAX in protection against acute haemorrhagic gastric ulcer in rats. Rats were assigned to 6 groups of 6 rats: the normal control (Tween 20 5% v/v, 5 mL/kg), the positive control (Tween 20 5% v/v, 5 mL/kg), and four Schiff base derivative groups named Schiff_1, Schiff_2, Schiff_3, and Schiff_4 (25 mg/kg). After 1 h, all of the groups received ethanol 95% (5 mL/kg) but the normal control received Tween 20 (Tween 20 5% v/v, 5 mL/kg). The animals were euthanized after 60 min and the stomachs were dissected for histology (H&E), immunohistochemistry, and western blot analysis against HSP70 and BAX proteins. The results showed that the Schiff base metal derivatives enhanced the expression of HSP70 and suppressed the expression of BAX proteins during their gastroprotection against ethanol-induced gastric lesion in rats.

Identification of Mycobacterium tuberculosis in Clinical Specimens of Patients Suspected of Having Extrapulmonary Tuberculosis by Application of Nested PCR on Five Different Genes.

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Khosravi, Azar D; Alami, Ameneh; Meghdadi, Hossein; Hosseini, Atta A

2017-01-01

Definitive and rapid diagnosis of extrapulmonary tuberculosis (EPTB) is challenging since conventional techniques have limitations due to the paucibacillary nature of the disease. To increase the sensitivity of detection of Mycobacterium tuberculosis (MTB) in EPTB specimens, we performed a nested PCR assay targeting several genes of MTB on EPTB specimens. A total of 100 clinical specimens from suspected cases of EPTB were processed. Standard staining for acid fast bacilli (AFB) was performed as the preliminary screening test. Extracted DNAs from specimens were subjected to Nested PCR technique for the detection of five different MTB target genes of IS6110, IS1081, hsp65kd, mbp64 , and mtp40 . On performing AFB staining, only 13% of specimens were positive, of which ascites fluid (33.3%), followed by pleural effusion (30.8%) showed the greatest AFB positivity rate. We demonstrated slight improvement in yields in lymph node which comprised the majority of specimens in this study, by employing PCR targeted to IS6110 - and hsp65-genes in comparison to AFB staining. However, the yields in ascites fluid and pleural effusion were not substantially improved by PCR, but those from bone and wound were, as in nested PCR employing either gene, the same positivity rate were obtained for ascites fluid (33.3%), while for pleural effusion specimens only IS1081 based PCR showed identical positivity rate with AFB stain (30.8%). The results for bone and wound specimens, however, demonstrated an improved yield mainly by employing IS1081 gene. Here, we report higher detection rate of EPTB in clinical specimens using five different targeted MTB genes. This nested PCR approach facilitates the comparison and the selection of the most frequently detected genes. Of course this study demonstrated the priority of IS1081 followed by mtp40 and IS6110 , among the five tested genes and indicates the effectiveness of any of the three genes in the design of an efficient nested-PCR test that

Hsp90 orchestrates transcriptional regulation by Hsf1 and cell wall remodelling by MAPK signalling during thermal adaptation in a pathogenic yeast.

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Michelle D Leach

2012-12-01

Full Text Available Thermal adaptation is essential in all organisms. In yeasts, the heat shock response is commanded by the heat shock transcription factor Hsf1. Here we have integrated unbiased genetic screens with directed molecular dissection to demonstrate that multiple signalling cascades contribute to thermal adaptation in the pathogenic yeast Candida albicans. We show that the molecular chaperone heat shock protein 90 (Hsp90 interacts with and down-regulates Hsf1 thereby modulating short term thermal adaptation. In the longer term, thermal adaptation depends on key MAP kinase signalling pathways that are associated with cell wall remodelling: the Hog1, Mkc1 and Cek1 pathways. We demonstrate that these pathways are differentially activated and display cross talk during heat shock. As a result ambient temperature significantly affects the resistance of C. albicans cells to cell wall stresses (Calcofluor White and Congo Red, but not osmotic stress (NaCl. We also show that the inactivation of MAP kinase signalling disrupts this cross talk between thermal and cell wall adaptation. Critically, Hsp90 coordinates this cross talk. Genetic and pharmacological inhibition of Hsp90 disrupts the Hsf1-Hsp90 regulatory circuit thereby disturbing HSP gene regulation and reducing the resistance of C. albicans to proteotoxic stresses. Hsp90 depletion also affects cell wall biogenesis by impairing the activation of its client proteins Mkc1 and Hog1, as well as Cek1, which we implicate as a new Hsp90 client in this study. Therefore Hsp90 modulates the short term Hsf1-mediated activation of the classic heat shock response, coordinating this response with long term thermal adaptation via Mkc1- Hog1- and Cek1-mediated cell wall remodelling.

Expression analysis of nine small heat shock protein genes from Tamarix hispida in response to different abiotic stresses and abscisic acid treatment.

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Yang, Guiyan; Wang, Yucheng; Zhang, Kaimin; Gao, Caiqiu

2014-03-01

Heat shock proteins (HSPs) play important roles in protecting plants against environmental stresses. Furthermore, small heat shock proteins (sHSPs) are the most ubiquitous HSP subgroup with molecular weights ranging from 15 to 42 kDa. In this study, nine sHSP genes (designated as ThsHSP1-9) were cloned from Tamarix hispida. Their expression patterns in response to cold, heat shock, NaCl, PEG and abscisic acid (ABA) treatments were investigated in the roots and leaves of T. hispida by real-time RT-PCR analysis. The results showed that most of the nine ThsHSP genes were expressed at higher levels in roots than in leaves under normal growth condition. All of ThsHSP genes were highly induced under conditions of cold (4 °C) and different heat shocks (36, 40, 44, 48 and 52 °C). Under NaCl stress, all nine ThsHSPs genes were up-regulated at least one stress time-point in both roots and leaves. Under PEG and ABA treatments, the nine ThsHSPs showed various expression patterns, indicating a complex regulation pathway among these genes. This study represents an important basis for the elucidation of ThsHSP gene function and provides essential information that can be used for stress tolerance genetic engineering in future studies.

Fluoride exposure changed the structure and the expressions of HSP related genes in testes of pubertal rats.

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Zhao, Yangfei; Zhao, Jun; Wang, Jinming; Wang, Jundong

2017-10-01

Previous studies have indicated that fluoride exposure damaged the male reproductive function; however, the cellular mechanism of fluoride-induced testicular toxicity is still unclear. In this study, twenty-two female pregnant Wistar rats were allotted randomly to two groups: control (deionized water) and sodium fluoride (NaF, contain F - : 67.86 mg/L) groups. After delivery, the dosage was continued for 15 weeks for puppies. Twelve rats in each group were tested at 6 and 9 (pubertal); 12 and 15 (mature) weeks of age. Our results suggested that organ coefficient of epididymis was significantly decreased in the mature (12 and 15 week-old) rats. Epididymal sperm abnormality and femur fluoride concentration were increased with the concomitant decrease in sperm motility and concentration in these experimental periods. Compared to the control, in the NaF group, the seminiferous tubules of each age were reduced in terms of diameter and thickness. The sperm cells were lost and shedding and finally disappeared after 9 weeks. mRNA and protein levels of HSP27 and 90 were decreased with a concomitant increase in HSP70 and HSF mRNA and protein levels in NaF exposed rats. The mRNA and protein levels of HSP27 and HSF (only mRNA) were significantly increased in NaF treated rats at 9 and 15 weeks of age, respectively. In summary, these results emphasize that NaF induces testicular and sperm abnormalities through the involvement of HSPs especially during the pubertal period. Copyright © 2017 Elsevier Ltd. All rights reserved.

Heat Shock Protein 90 (Hsp90 Expression and Breast Cancer

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Christos A. Papadimitriou

2012-09-01

Full Text Available Hsp90 is an abundant protein in mammalian cells. It forms several discrete complexes, each containing distinct groups of co-chaperones that assist protein folding and refolding during stress, protein transport and degradation. It interacts with a variety of proteins that play key roles in breast neoplasia including estrogen receptors, tumor suppressor p53 protein, angiogenesis transcription factor HIF-1alpha, antiapoptotic kinase Akt, Raf-1 MAP kinase and a variety of receptor tyrosine kinases of the erbB family. Elevated Hsp90 expression has been documented in breast ductal carcinomas contributing to the proliferative activity of breast cancer cells; whilst a significantly decreased Hsp90 expression has been shown in infiltrative lobular carcinomas and lobular neoplasia. Hsp90 overexpression has been proposed as a component of a mechanism through which breast cancer cells become resistant to various stress stimuli. Therefore, pharmacological inhibition of HSPs can provide therapeutic opportunities in the field of cancer treatment. 17-allylamino,17-demethoxygeldanamycin is the first Hsp90 inhibitor that has clinically been investigated in phase II trial, yielding promising results in patients with HER2-overexpressing metastatic breast cancer, whilst other Hsp90 inhibitors (retaspimycin HCL, NVP-AUY922, NVP-BEP800, CNF2024/BIIB021, SNX-5422, STA-9090, etc. are currently under evaluation.

Heat Shock Protein HSP27 Secretion by Ovarian Cancer Cells Is Linked to Intracellular Expression Levels, Occurs Independently of the Endoplasmic Reticulum Pathway and HSP27’s Phosphorylation Status, and Is Mediated by Exosome Liberation

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Matthias B. Stope

2017-01-01

Full Text Available The heat shock protein HSP27 has been correlated in ovarian cancer (OC patients with aggressiveness and chemoresistance and, therefore, represents a promising potential biomarker for OC diagnosis, prognosis, and treatment response. Notably, secretion of soluble HSP27 has been described by a few cell types and may take place as well in OC cells. Therefore, we studied HSP27 secretion mechanisms under diverse cellular conditions in an OC cell model system. Secretion of HSP27 was characterized after overexpression of HSP27 by transfected plasmids and after heat shock. Intra- and extracellular HSP27 amounts were assessed by Western blotting and ELISA. Protein secretion was blocked by brefeldin A and the impact of the HSP27 phosphorylation status was analyzed overexpressing HSP27 phosphomutants. The present study demonstrated that HSP27 secretion by OVCAR-3 and SK-OV-3 cells depends on intracellular HSP27 concentrations. Moreover, HSP27 secretion is independent of the endoplasmic reticulum secretory pathway and HSP27 phosphorylation. Notably, analysis of OC cell-born exosomes not only confirmed the concentration-dependent correlation of HSP27 expression and secretion but also demonstrated a concentration-dependent incorporation of HSP27 protein into exosomes. Thus, secreted HSP27 may become more important as an extracellular factor which controls the tumor microenvironment and might be a noninvasive biomarker.

Calcium/calmodulin kinase1 and its relation to thermotolerance and HSP90 in Sporothrix schenckii: an RNAi and yeast two-hybrid study

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Gonzalez-Mendez Ricardo

2011-07-01

Full Text Available Abstract Background Sporothrix schenckii is a pathogenic dimorphic fungus of worldwide distribution. It grows in the saprophytic form with hyaline, regularly septated hyphae and pyriform conidia at 25°C and as the yeast or parasitic form at 35°C. Previously, we characterized a calcium/calmodulin kinase in this fungus. Inhibitors of this kinase were observed to inhibit the yeast cell cycle in S. schenckii. Results The presence of RNA interference (RNAi mechanism in this fungus was confirmed by the identification of a Dicer-1 homologue in S. schenckii DNA. RNAi technology was used to corroborate the role of calcium/calmodulin kinase I in S. schenckii dimorphism. Yeast cells were transformed with the pSilent-Dual2G (pSD2G plasmid w/wo inserts of the coding region of the calcium/calmodulin kinase I (sscmk1 gene. Transformants were selected at 35°C using resistance to geneticin. Following transfer to liquid medium at 35°C, RNAi transformants developed as abnormal mycelium clumps and not as yeast cells as would be expected. The level of sscmk1 gene expression in RNAi transformants at 35°C was less than that of cells transformed with the empty pSD2G at this same temperature. Yeast two-hybrid analysis of proteins that interact with SSCMK1 identified a homologue of heat shock protein 90 (HSP90 as interacting with this kinase. Growth of the fungus similar to that of the RNAi transformants was observed in medium with geldanamycin (GdA, 10 μM, an inhibitor of HSP90. Conclusions Using the RNAi technology we silenced the expression of sscmk1 gene in this fungus. RNAi transformants were unable to grow as yeast cells at 35°C showing decreased tolerance to this temperature. The interaction of SSCMK1 with HSP90, observed using the yeast two-hybrid assay suggests that this kinase is involved in thermotolerance through its interaction with HSP90. SSCMK1 interacted with the C terminal domain of HSP90 where effector proteins and co-chaperones interact. These

Prevalence of H63D, S65C, and C282Y hereditary hemochromatosis gene variants in Madeira Island (Portugal).

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Spínola, Carla; Brehm, António; Spínola, Hélder

2011-01-01

Hereditary HFE Hemochromatosis is an inherited disorder of iron metabolism that results from mutations in the HFE gene. Almost all patients with hereditary hemochromatosis show a C282Y mutation in homozygosity or in compound heterozygosity with H63D. Also, the mutation S65C has been shown to be associated to a milder iron overload. Since allele and genotype frequencies of these three variants of the HFE gene vary between populations, the determination of their prevalence in Madeira Island will clarify the population susceptibility to hereditary hemochromatosis. One hundred and fifty-four samples from Madeira Island were genotyped for the three most common HFE gene mutations, H63D, C282Y, and S65C, by polymerase chain reaction followed by restriction fragment length polymorphism analysis. Results have shown a prevalence of 20.5%, 0.33%, and 1% for H63D, C282Y, and S65C, respectively. Accordingly to our estimates, both genotypes associated to hereditary hemochromatosis, C282Y homozygotes and C282/H63D compound heterozygotes, could be present in Madeira Island population in 1,648 individuals, which represents 0.65% of the total population.

Hsp90 governs echinocandin resistance in the pathogenic yeast Candida albicans via calcineurin.

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Sheena D Singh

2009-07-01

Full Text Available Candida albicans is the leading fungal pathogen of humans, causing life-threatening disease in immunocompromised individuals. Treatment of candidiasis is hampered by the limited number of antifungal drugs whose efficacy is compromised by host toxicity, fungistatic activity, and the emergence of drug resistance. We previously established that the molecular chaperone Hsp90, which regulates the form and function of diverse client proteins, potentiates resistance to the azoles in C. albicans and in the model yeast Saccharomyces cerevisiae. Genetic studies in S. cerevisiae revealed that Hsp90's role in azole resistance is to enable crucial cellular responses to the membrane stress exerted by azoles via the client protein calcineurin. Here, we demonstrate that Hsp90 governs cellular circuitry required for resistance to the only new class of antifungals to reach the clinic in decades, the echinocandins, which inhibit biosynthesis of a critical component of the fungal cell wall. Pharmacological or genetic impairment of Hsp90 function reduced tolerance of C. albicans laboratory strains and resistance of clinical isolates to the echinocandins and created a fungicidal combination. Compromising calcineurin function phenocopied compromising Hsp90 function. We established that calcineurin is an Hsp90 client protein in C. albicans: reciprocal co-immunoprecipitation validated physical interaction; Hsp90 inhibition blocked calcineurin activation; and calcineurin levels were depleted upon genetic reduction of Hsp90. The downstream effector of calcineurin, Crz1, played a partial role in mediating calcineurin-dependent stress responses activated by echinocandins. Hsp90's role in echinocandin resistance has therapeutic potential given that genetic compromise of C. albicans HSP90 expression enhanced the efficacy of an echinocandin in a murine model of disseminated candidiasis. Our results identify the first Hsp90 client protein in C. albicans, establish an entirely

Sequence characterization of heat shock protein gene of Cyclospora cayetanensis isolates from Nepal, Mexico, and Peru.

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Sulaiman, Irshad M; Torres, Patricia; Simpson, Steven; Kerdahi, Khalil; Ortega, Ynes

2013-04-01

We have described the development of a 2-step nested PCR protocol based on the characterization of the 70-kDa heat shock protein (HSP70) gene for rapid detection of the human-pathogenic Cyclospora cayetanensis parasite. We tested and validated these newly designed primer sets by PCR amplification followed by nucleotide sequencing of PCR-amplified HSP70 fragments belonging to 16 human C. cayetanensis isolates from 3 different endemic regions that include Nepal, Mexico, and Peru. No genetic polymorphism was observed among the isolates at the characterized regions of the HSP70 locus. This newly developed HSP70 gene-based nested PCR protocol provides another useful genetic marker for the rapid detection of C. cayetanensis in the future.

Characterization of Small HSPs from Anemonia viridis Reveals Insights into Molecular Evolution of Alpha Crystallin Genes among Cnidarians

OpenAIRE

Nicosia, Aldo; Maggio, Teresa; Mazzola, Salvatore; Gianguzza, Fabrizio; Cuttitta, Angela; Costa, Salvatore

2014-01-01

Gene family encoding small Heat-Shock Proteins (sHSPs containing α-crystallin domain) are found both in prokaryotic and eukaryotic organisms; however, there is limited knowledge of their evolution. In this study, two small HSP genes termed AvHSP28.6 and AvHSP27, both organized in one intron and two exons, were characterised in the Mediterranean snakelocks anemone Anemonia viridis. The release of the genome sequence of Hydra magnipapillata and Nematostella vectensis enabled a comprehensive stu...

Evidence for loss of mitochondria in Microsporidia from a mitochondrial-type HSP70 in Nosema locustae.

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Germot, A; Philippe, H; Le Guyader, H

1997-08-01

In molecular phylogenies based on ribosomal RNA, three amitochondriate protist lineages, Microsporidia, Metamonada (including diplomonads) and Parabasala (including trichomonads), are the earliest offshoots of the eukaryotic tree. As an explantation for the lack of mitochondria in these organisms, the hypothesis that they have diverged before the mitochondrial endosymbiosis is preferred to the less parsimonious hypothesis of several independent losses of the organelle. Nevertheless, if they had descended from mitochondrion-containing ancestors, it may be possible to find in their nuclear DNA genes that derive from the endosymbiont which gave rise to mitochondria. Based on similar evidence, secondary losses of mitochondria have recently been suggested for Entamoeba histolytica and for Trichomonas vaginalis. In this study, we have isolated a gene encoding a chaperone protein (HSP70, 70 kDa heat shock protein) from the microspordian Nosema locustae. In phylogenetic trees, this HSP70 was located within a group of sequences that in other lineages is targetted to the mitochondrial compartment, itself included in the proteobacterial clade. In addition, the N. locustae protein contained the GDAW(V) motif shared by mitochondrial and proteobacterial sequences, with only one conservative substitution. Moreover, microsporidia, a phylum which was assumed to emerge close to the base of the eukaryotic tree, appears as the sister-group of fungi in the HSP70 phylogeny, in agreement with some ultrastructural characters and phylogenies based on alpha- and beta-tubulins. Loss of mitochondria, now demonstrated for several amitochondriate groups, indicates that the common ancestor of all the extant eukaryotic species could have been a mitochondriate eukaryote.

Repercussion of mitochondria deformity induced by anti-Hsp90 drug 17AAG in human tumor cells

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Vishal, Chaturvedi

2011-06-07

Inhibiting Hsp90 chaperone roles using 17AAG induces cytostasis or apoptosis in tumor cells through destabilization of several mutated cancer promoting proteins. Although mitochondria are central in deciding the fate of cells, 17AAG induced effects on tumor cell mitochondria were largely unknown. Here, we show that Hsp90 inhibition with 17AAG first affects mitochondrial integrity in different human tumor cells, neuroblastoma, cervical cancer and glial cells. Using human neuroblastoma tumor cells, we found the early effects associated with a change in mitochondrial membrane potential, elongation and engorgement of mitochondria because of an increased matrix vacuolization. These effects are specific to Hsp90 inhibition as other chemotherapeutic drugs did not induce similar mitochondrial deformity. Further, the effects are independent of oxidative damage and cytoarchitecture destabilization since cytoskeletal disruptors and mitochondrial metabolic inhibitors also do not induce similar deformity induced by 17AAG. The 1D PAGE LC MS/ MS mitochondrial proteome analysis of 17AAG treated human neuroblastoma cells showed a loss of 61% proteins from membrane, metabolic, chaperone and ribonucleoprotein families. About 31 unmapped protein IDs were identified from proteolytic processing map using Swiss-Prot accession number, and converted to the matching gene name searching the ExPASy proteomics server. Our studies display that Hsp90 inhibition effects at first embark on mitochondria of tumor cells and compromise mitochondrial integrity. the author(s), publisher and licensee Libertas Academica Ltd.

Effects of HSP27 chaperone on THP-1 tumor cell apoptosis.

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Kaigorodova, E V; Ryazantseva, N V; Novitskii, V V; Maroshkina, A N; Belkina, M V

2012-11-01

The role of Hsp27 (heat shock protein 27) chaperone in regulation of THP-1 tumor cell apoptosis was studied. Realization of tumor cell apoptosis under conditions of in vitro culturing with Hsp27 specific inhibitor (KRIBB3) was evaluated by fluorescent microscopy with FITC-labeled annexin V and propidium iodide. Measurements of Bcl-2 family proteins (Bcl-2, Bax, Bad) in tumor cells incubated with Hsp27 inhibitor were carried out by Western blotting. Chaperone Hsp27 acted as apoptosis inhibitor in THP-1 tumor cells modulating the proportion of antiapoptotic (Bcl-2) and proapoptotic (Bax and Bad) proteins.

The Hsp70 homolog Ssb and the 14-3-3 protein Bmh1 jointly regulate transcription of glucose repressed genes in Saccharomyces cerevisiae.

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Hübscher, Volker; Mudholkar, Kaivalya; Chiabudini, Marco; Fitzke, Edith; Wölfle, Tina; Pfeifer, Dietmar; Drepper, Friedel; Warscheid, Bettina; Rospert, Sabine

2016-07-08

Chaperones of the Hsp70 family interact with a multitude of newly synthesized polypeptides and prevent their aggregation. Saccharomyces cerevisiae cells lacking the Hsp70 homolog Ssb suffer from pleiotropic defects, among others a defect in glucose-repression. The highly conserved heterotrimeric kinase SNF1/AMPK (AMP-activated protein kinase) is required for the release from glucose-repression in yeast and is a key regulator of energy balance also in mammalian cells. When glucose is available the phosphatase Glc7 keeps SNF1 in its inactive, dephosphorylated state. Dephosphorylation depends on Reg1, which mediates targeting of Glc7 to its substrate SNF1. Here we show that the defect in glucose-repression in the absence of Ssb is due to the ability of the chaperone to bridge between the SNF1 and Glc7 complexes. Ssb performs this post-translational function in concert with the 14-3-3 protein Bmh, to which Ssb binds via its very C-terminus. Raising the intracellular concentration of Ssb or Bmh enabled Glc7 to dephosphorylate SNF1 even in the absence of Reg1. By that Ssb and Bmh efficiently suppressed transcriptional deregulation of Δreg1 cells. The findings reveal that Ssb and Bmh comprise a new chaperone module, which is involved in the fine tuning of a phosphorylation-dependent switch between respiration and fermentation. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Review: The HSP90 molecular chaperone-an enigmatic ATPase.

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Pearl, Laurence H

2016-08-01

The HSP90 molecular chaperone is involved in the activation and cellular stabilization of a range of 'client' proteins, of which oncogenic protein kinases and nuclear steroid hormone receptors are of particular biomedical significance. Work over the last two decades has revealed a conformational cycle critical to the biological function of HSP90, coupled to an inherent ATPase activity that is regulated and manipulated by many of the co-chaperones proteins with which it collaborates. Pharmacological inhibition of HSP90 ATPase activity results in degradation of client proteins in vivo, and is a promising target for development of new cancer therapeutics. Despite this, the actual function that HSP90s conformationally-coupled ATPase activity provides in its biological role as a molecular chaperone remains obscure. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 594-607, 2016. © 2016 The Authors. Biopolymers Published by Wiley Periodicals, Inc.

Increased levels of IgG antibodies against human HSP60 in patients with spondyloarthritis

DEFF Research Database (Denmark)

Hjelholt, Astrid; Carlsen, Thomas Gelsing; Deleuran, Bent Winding

2013-01-01

Introduction: Spondyloarthritis (SpA) comprises a heterogeneous group of inflammatory diseases, with strong association to human leukocyte antigen (HLA)-B27. SpA is suggested triggered by bacterial infection, and bacterial heat shock protein (HSP) seems to be a strong T cell antigen. Since...... against human HSP60, but not antibodies against bacterial HSP60, were elevated in the SpA group compared with the control group. Association between IgG3 antibodies against human HSP60 and BASMI was shown in HLA-B27+ patients. Only weak correlation between antibodies against bacterial and human HSP60...... was seen, and there was no indication of cross-reaction. Conclusion: These results suggest that antibodies against human HSP60 is associated with SpA, however, the theory that antibodies against human HSP60 is a specific part of the aetiology, through cross-reaction to bacterial HSP60, cannot be supported...