Region Of Heat Shock Protein Research Articles

A dual-luciferase reporter system for characterization of small RNA target genes in both mammalian and insect cells.

MicroRNAs (miRNAs) are regulatory RNA molecules that bind to target messenger RNAs (mRNAs) and affect the stability or translational efficiency of the bound mRNAs. Single or dual-luciferase reporter systems have been successfully used to identify miRNA target genes in mammalian cells. These reporter systems, however, are not sensitive enough to verify miRNA-target gene relationships in insect cell lines because the promoters of the target luciferase (usually Renilla) used in these reporter systems are too weak to drive sufficient expression of the target luciferase in insect cells. In this study, we replaced the SV40 promoter in the psiCHECK-2 reporter vector, which is widely used with mammalian cell lines, with the HSV-TK or AC5.1 promoter to yield two new dual-luciferase reporter vectors, designated psiCHECK-2-TK and psiCHECK-2-AC5.1, respectively. Only psiCHECK-2 and psiCHECK-2-AC5.1 had suitable target (Renilla)/reference (firefly) luciferase activity ratios in mammalian (HeLa and HEK293) and insect (Sf9, S2, Helicoverpa zea fat body and ovary) cell lines, while psiCHECK-2-TK had suitable Renilla/firefly luciferase activity ratios regardless of the cell line. Moreover, psiCHECK-2-TK successfully detected the interaction between Helicoverpa armigera miRNA9a and its target, the 3'-untranslated region of heat shock protein 90, in both mammalian and H. zea cell lines, but psiCHECK-2 failed to do so in H. zea cell lines. Furthermore, psiCHECK-2-TK with the target sequence, HzMasc (H. zea Masculinizer), accurately differentiated between H. zea cell lines with or without the negative regulation factor (miRNA or piRNA) of HzMasc. These data demonstrate that psiCHECK-2-TK can be used to functionally characterize small RNA target genes in both mammalian and insect cells.

Varying Architecture of Heat Shock Elements Contributes to Distinct Magnitudes of Target Gene Expression and Diverged Biological Pathways in Heat Stress Response of Bread Wheat.

The heat shock transcription factor (HSF) binds to cis-regulatory motifs known as heat shock elements (HSEs) to mediate the transcriptional response of HSF target genes. However, the HSF–HSEs interaction is not clearly understood. Using the newly released genome reference sequence of bread wheat, we identified 39,478 HSEs (95.6% of which were non-canonical HSEs) and collapsed them into 30,604 wheat genes, accounting for 27.6% wheat genes. Using the intensively heat-responsive transcriptomes of wheat, we demonstrated that canonical HSEs have a higher propensity to induce a response in the closest downstream genes than non-canonical HSEs. However, the response magnitude induced by non-canonical HSEs was comparable to that induced by canonical HSEs. Significantly, some non-canonical HSEs that contain mismatched nucleotides at specific positions within HSEs had a larger response magnitude than that of canonical HSEs. Consistently, most of the HSEs identified in the promoter regions of heat shock proteins were non-canonical HSEs, suggesting an important role for these non-canonical HSEs. Lastly, distinct diverged biological processes were observed between genes containing different HSE types, suggesting that sequence variation in HSEs plays a key role in the evolution of heat responses and adaptation. Our results provide a new perspective to understand the regulatory network underlying heat responses.

Design and Development of Modified mRNA Encoding Core Antigen of Hepatitis C Virus: a Possible Application in Vaccine Production

Background:Hepatitis C virus (HCV) is a blood-borne pathogen, resulting in liver cirrhosis and liver cancer. Despite of many efforts in development of treatments for HCV, no vaccine has been licensed yet. The purpose of this study was to design and prepare a specific mRNA, without 5’ cap and poly (A) tail transcribed in vitro capable of coding core protein and also to determine its functionality.Methods:Candidate mRNA was prepared by in vitro transcription of the designed construct consisting of 5’ and 3’ untranslated regions of heat shock proteins 70 (hsp70) mRNA, T7 promoter, internal ribosome entry site (IRES) sequences of eIF4G related to human dendritic cells (DCs), and the Core gene of HCV. To design the modified mRNA, the 5’ cap and poly (A) tail structures were not considered. DCs were transfected by in vitro-transcribed messenger RNA (IVT-mRNA) and the expressions of green fluorescent protein (GFP), and Core genes were determined by microscopic examination and Western blotting assay.Results:Cell transfection results showed that despite the absence of 5’ cap and poly (A) tail, the structure of the mRNA was stable. Moreover, the successful expressions of GFP and Core genes were achieved.Conclusion:Our findings indicated the effectiveness of a designed IVT-mRNA harboring the Core gene of HCV in transfecting and expressing the antigens in DCs. Considering the simple and efficient protocol for the preparation of this IVT-mRNA and its effectiveness in expressing the gene that it carries, this IVT-mRNA could be suitable for development of an RNA vaccine against HCV.

Design and Development of Modified mRNA Encoding Core Antigen of Hepatitis C Virus: a Possible Application in Vaccine Production

Hepatitis C virus (HCV) is a ‎blood-borne pathogen, resulting in liver cirrhosis and liver cancer. Despite of many efforts in development of treatments for HCV, no vaccine has been licensed yet. The purpose of this study was ‎to design and prepare a specific mRNA, without 5' cap and poly (A) tail transcribed in vitro capable of coding core protein and also to determine its functionality. Candidate mRNA was prepared by in vitro transcription of the designed construct consisting of ‎‎5ʹ and 3ʹ untranslated regions of heat shock proteins 70 (hsp70) mRNA, T7 promoter, internal ribosome entry site (IRES) sequences of eIF4G related to human dendritic cells (DCs), and the ‎Core gene of HCV. To design the modified mRNA, the ‎‎5' cap and poly (A) tail structures were not considered. DCs were transfected by in vitro-transcribed messenger RNA (IVT-mRNA) and the expressions of green fluorescent protein (GFP), and Core genes were determined by microscopic examination and Western blotting assay. Cell transfection results showed that despite the absence of ‎‎5' cap and poly (A) tail, the structure of the mRNA ‎was stable. Moreover, the successful expressions of GFP and Core genes were achieved. Our findings indicated the effectiveness of a designed IVT-mRNA harboring the Core gene of HCV in transfecting and expressing the antigens in DCs. Considering the simple and efficient protocol for the preparation of this IVT-mRNA and its effectiveness in expressing the gene that it carries, this IVT-mRNA could be suitable for development of an RNA vaccine against HCV.

Unexpected heat shock element binding ability and tumor-killing activity of the combinatorial function domain of apoptin.

Apoptin, derived from the chicken anemia virus, has been found to exert tumor-preferential apoptotic activity. It is a potential anticancer agent with direct clinical applications. However, if this viral protein were to be used as a new drug, it might also induce a strong immune response, causing toxic side effects. In a previous study, our group showed that TAT-apoptin downregulates the stress expression of heat shock protein 70 by competing with heat shock factor protein 1 in binding to the heat shock element (HSE) of the promoter region of heat shock protein 70, thus inducing specific apoptosis in HepG2 cells. In this study, we investigated the HSE-binding properties of the minimal functional region of apoptin. We showed that apoptin's nuclear localization signals 1 and nuclear localization signals 2 represented functional regions that could bind with HSE and that this binding capacity was increased by polymers formed through the introduction of a leucine-rich stretch. Our data also showed that truncated combinatorial apoptin peptide has greater tumor-specific cell-killing activity and could be a potential antitumor agent.

Domestic dogs as reservoir hosts for Leishmania donovani in the southernmost Western Ghats in India

The peripheral blood samples from domestic dogs (n=47) and wild rats (n=25) in the Kani Tribe settlements, located southernmost part of the Western Ghats, Thiruvananthapuram district, Kerala, India were examined for Leishmania infection. This area is known for cases of leishmaniasis with cutaneous manifestations and sandfly abundance. The tribes domesticate dogs to protect them from untoward activities of wild animals. Leishmania donovani parasite DNA was detected only from 6.4% (n=3) of the blood samples collected from the domestic dogs by amplification of the diagnostic kinetoplast mini-circle DNA and PCR-RFLP analysis of the UTR region of heat shock protein 70 (hsp70) gene. None of the blood samples collected from rats was positive. Through sequencing, L. donovani infection among dogs was confirmed. The DNA sequences generated for hsp70 were deposited with the GenBank. The GenBank accession numbers of these samples are KR905363, KR905364 and KR905365 for hsp70 genes. The results indicated that the DNA isolates from dog blood samples matched precisely with that of our earlier isolates from skin lesions of Kani tribes and also from P. argentipes vector. Thus, the role of dogs as reservoirs for L. donovani parasite in the Kani tribe settlements is confirmed.

Abstract 2785: The role of heat shock factors for cancer stem cell regulation

Abstract Introduction: Heat shock factor family is a group of transcription factors that can bind specifically to heat shock elements (HSE) within the promoter region of heat shock protein (HSP). The HSF pathway activation helps cells survive under stress by preventing protein misfolding and aggregation. Three heat shock factors (HSF1, HSF2 and HSF4) have been identified in humans, among them, HSF1, which is believed to be the master regulator of cellular response to stress, is by far the best-characterized member of the HSF family. We first identified that high HSF1 is associated with high tumor grade and poor survival in multiple cancer models including breast, ovarian, lung and colon cancers. In addition, our recent data indicate that HSF1 drives a distinct transcription program that is unrelated to heat shock response in malignant tumors, and this HSF1 “non-heat shock related transcription program” correlates with poor outcome in breast cancer patients. Moreover, we confirmed that HSF1 drives the Cancer Stem Cell (CSC) phenotype in multiple breast cancer cell lines and facilitate chemo-treatment resistance by increasing CSC frequency in breast cancer cells. Previous studies have shown that HSF2 and HSF4 may play a role in cancer progression either on their own or through interaction with HSF1. HSF2 forms hetero-trimers with HSF1 and facilitate its function in response to heat shock. Inactivation of HSF4 in p53 null mouse models causes cell senescence and suppression of tumorigenesis, with great similarities to HSF1-/- p53 null mice. Results: In an attempt to investigate how HSF1 is activated to regulate CSC phenotype, we constructed phospho HSF1 at three different sites. We observed that there is no correlation of phospho activation of HSF1 with CSC phenotype. In follow-up work to the studies above, we found that both HSF2 and HSF4 are also significantly up-regulated in CSCs, suggesting that they may cooperate with HSF1 in regulating CSCs. Moreover, we discovered that HSF4 is over-expressed in breast cancers compared to normal mammary gland, and over-expression of HSF4 induces tumorsphere formation in breast cancer cells. Conclusion: These observations indicated that in order to gain a complete understanding of the role of the HSFs in cancer it is now necessary to study all three HSFs together. Citation Format: Ankita Thakkar, Bin Wang, Tan A. Ince. The role of heat shock factors for cancer stem cell regulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2785.

5′-Untranslated region of heat shock protein 70 mRNA drives translation under hypertonic conditions

In mammalian cells, adaptation to hypertonic conditions leads to the activation of an array of early (cell shrinkage, regulatory volume increase) and late (accumulation of compatible osmolytes) responses and increased level of HSPs (heat shock proteins). Protein synthesis is strongly inhibited few minutes after the hypertonic challenge as demonstrated in whole cells and as reproduced under controlled conditions in cell-free systems. Different mechanisms known to mediate the accumulation of HSP70, such as mRNA transcription and stabilization, require fully active protein synthesis. We show that the 5′-untranslated region of HSP70 messenger drives a hypertonicity-resistant translation (up to 0.425osmol/kg of water), whereas cap-dependent protein synthesis is almost totally blocked under the same conditions. The results, obtained in cell-free systems and in whole cells, might help to explain why HSP70 is accumulated in cells when total protein synthesis is impaired. We also observed that translation initiated by viral IRES (from Cricket paralysis virus) is highly efficient in cells exposed to hyperosmolarity, suggesting that the resistance to hypertonic conditions is a more general feature of cap-independent translation. The described mechanism may also play a role in the control of translation of other messengers encoding for proteins involved in the adaptation to hypertonicity.

Uncovering a Region of Heat Shock Protein 90 Important for Client Binding in E. coli and Chaperone Function in Yeast

The heat shock protein 90 (Hsp90) family of heat shock proteins is an abundantly expressed and highly conserved family of ATP-dependent molecular chaperones. Hsp90 facilitates remodeling and activation of hundreds of proteins. In this study, we developed a screen to identify Hsp90-defective mutants in E. coli. The mutations obtained define a region incorporating residues from the middle and C-terminal domains of E. coli Hsp90. The mutant proteins are defective in chaperone activity and client binding in vitro. We constructed homologous mutations in S. cerevisiae Hsp82 and identified several that caused defects in chaperone activity in vivo and in vitro. However, the Hsp82 mutant proteins were less severely defective in client binding to a model substrate than the corresponding E. coli mutant proteins. Our results identify a region in Hsp90 important for client binding in E. coli Hsp90 and suggest an evolutionary divergence in the mechanism of client interaction by bacterial and yeast Hsp90.

Differential effects of an expected actin-tropomyosin binding region of heat shock protein 20 on the relaxation in skinned carotid artery and taenia cecum from guinea pig

To explore the possible role of heat shock protein 20 (HSP20) -linked regulation of actin-myosin interaction in living vascular smooth muscle contraction, we studied the effects of HSP20p and TnIp, synthetic peptides originating from an actin tropomyosin binding region of human heat shock protein 20 [residues 110-121; GFVAREFHRRYR] and that of rabbit cardiac troponin I [residues 136-147; GKFKRPTLRRVR], respectively, on the active stress and phosphorylation level of myosin regulatory light chain (MLC(20)) during relaxation of skinned (cell membrane permeabilized) preparations from "tonic" carotid artery and "phasic" taenia cecum from guinea pig. Active stress of the skinned preparations, resulting from actin-myosin interaction, biphasically decayed following Ca(2+) removal (relaxation). Decay of MLC(20) phosphorylation level by Ca(2+) removal was much faster than active stress in an exponential manner. In skinned carotid artery, HSP20p did neither affect relaxation time course nor MLC(20) dephosphorylation, whereas, in skinned taenia cecum, the peptide slowed relaxation time course through inhibition of MLC(20) dephosphorylation and slowing "latch"-bridge dissociation. On the other hand, TnIp accelerated relaxation time course without affecting MLC(20) dephosphorylation in both skinned carotid artery and skinned taenia cecum. Our present results suggest that, HSP20p slows the relaxation processes through intracellular regulatory mechanisms such as Rho A/Rho-kinase mediated pathways, which are known to be dominant in "phasic" smooth muscles but to be recessive in "tonic" smooth muscles.

Polymorphisms of hsp 60 gene in Chinese Han people

To identify the single nucleotide polymorphisms (SNPs) in the regulatory and coding regions of heat shock protein 60 gene and search for its genetic makers in Chinese Han people. The 5' flank region, parts of the exons and introns of hsp60 gene were resequenced to identify the SNPs in Chinese Han people, and then the sequenced results to the Japanese, European and African's data in National Center for Biotechnology Information (NCBI) and HapMap databases were compared. One novel SNP was identified in exon 2 resulting in synonymous variant and the G allele frequency was 0.025. There were 11 reported SNPs in the sequenced region. The minor allele frequencies of rs1116734, rs3749095, rs1050347, rs8539 were 0.51, 0.30, 0.29, 0.49. The heterozygosity of the other 7 SNPs was 0. The distributions of rs1116734, rs1050347, rs8539, rs3749095 in Chinese Han people were similar to the Japanese's. The hsp60 rs3749095 which was not found in Japanese people was a high-frequency SNP in Chinese Han people; the distribution of rs8539 in Chinese Han people was quite different from European and African's (P < 0.01). The SNPs of hsp60 in Chinese Han people are different from the other peoples. The SNPs of hsp60 gene rs1116734, rs3749095, rs1050347, rs8539 are very common in Chinese Han people and might be used for candidate genetic markers of hsp60 gene.

Geldanamycin derivative inhibition of HGF/SF-mediated Met tyrosine kinase receptor-dependent urokinase-plasminogen activation

Ansamycins, including geldanamycin and the derivative 17-allylamino-17-demethoxygeldanamycin, and radicicol are known for their ability to tightly bind to the ATP-binding site of the amino-terminal domain region of heat shock protein 90. We have found that geldanamycin and some of its derivatives can inhibit hepatocyte growth factor/scatter factor-mediated Met tyrosine kinase receptor-dependent urokinase-plasminogen activation at femtomolar levels. Assessment is made of structural requirements for such an activity and evidence is given that distinguishes the target of such an activity from that of heat shock protein 90.

Major depression and heat shock protein 70-1 gene

Background: Heat shock protein (HSP) expression can be induced by any stress such as with adrenocorticotropic hormones and catecholamines. It has been reported that patients with major depression have a 162-base deletion in the 5′-flanking region of heat shock protein 70 (HSP70)-1 gene mRNA. Methods: To detect the HSP70-1 gene mRNA, total RNA was isolated and amplified by RT-PCR, and the sequence was confirmed in all five patients by DNA direct sequencing analysis. Results: RT-PCR produced was no deletions of 162 bp in the human heat shock protein 70-1 gene in any of the patients with major depression or the nine controls. Conclusion: This finding is inconsistent with previous reports. We suggest that the 162-base deletion in the 5′-flanking region of the HSP70-1 gene mRNA is not associated with major depression. Further studies are required to determine the amounts of HSP70 and its mRNA in stress disorders such as major depression.

Effects of single nucleotide polymorphisms in the 5′-flanking region of heat shock protein 70.2 gene on semen quality in boars

This study aims to elucidate the effects of single nucleotide polymorphisms (SNPs) in the 5′-flanking region of porcine heat shock protein 70.2 gene ( HSP70.2) on semen quality in boars. Genomic DNA isolated from 55 boars (41 Duroc, nine Landrace, and five Yorkshire) was subjected to PCR amplification of the 5′-flanking region of HSP70.2. The nucleotide sequences were determined by automated sequencing. Five SNPs (sites 44, 232, 250, 345, and 393) were detected in this region. Semen quality was evaluated in terms of sperm motility, percentage of normal sperm, percentage of sperm with proximal plasma droplet, percentage of abnormal sperm, sperm concentration, semen volume per ejaculate and total sperm number per ejaculate. The effect of the SNPs on semen quality was evaluated based on breed-corrected data within a season. During the cool season, the sperm motility of boars with AA genotype at the 232 site was significantly higher than that of boars with CC genotype ( P<0.05). Meanwhile, boars with AC genotype at the 232 site had higher total sperm number per ejaculate than did those with CC genotype. In the hot season, heterozygotes at both the 232 and 250 sites had significantly higher total sperm number of per ejaculate than AA homozygotes ( P<0.05). Semen volume of boars with TT and TC genotypes at the 345 site was significantly larger than that of those with CC genotype ( P<0.05). Meanwhile, semen quality for boars with TT genotype at the 345 site was significantly higher than that of boars with TC or CC genotype ( P<0.05), that is the semen contained higher percentages of normal sperm and lower percentages of abnormal sperm or sperm with proximal plasma droplets. Results herein suggest that the SNPs in the 5′-flanking region of porcine HSP70.2 are associated with semen quality traits in the hot season.

Characterization of the Hydrophobic Region of Heat Shock Protein 90

The modes of binding of heat shock protein 90 with phenyl-Sepharose, myristoylated AE-cellulose, and monomyristoylated lysozyme were studied to characterize a hydrophobic region(s) on the surface of the heat shock protein 90 molecule and the following results were obtained. (1) The binding of heat shock protein 90 with phenyl-Sepharose was inhibited by the addition of 30% ethylene glycol. This indicates that the binding involves a hydrophobic interaction. (2) The binding was strengthened by the addition of 10 mM Mg2+, Ca2+, Sr2+, and Ba2+ ions, but not by K+ or Na+ ions. (3) The binding of hsp 90 with phenyl-Sepharose decreased initially and then increased as the temperature was increased from 0 to 50 degrees C, with a minimum at around 35 degrees C. (4) Lowering the pH stimulated the binding of hsp 90 with phenyl-Sepharose. (5) Heat shock protein 90 bound to myristoylated AE-cellulose, which has aliphatic hydrophobic residues, but not to acetylated AE-cellulose. (6) Heat shock protein 90 bound to monomyristoylated lysozyme, but not to control unmodified lysozyme. Based on these results, the possible function of the hydrophobic region(s) of heat shock protein 90 in the interaction with hydrophobic proteins is discussed.

Purification of heat shock protein 90 from calf uterus and rat liver and characterization of the highly hydrophobic region

Heat shock protein 90 was purified from calf uterus and rat liver. Both heat shock protein 90s had similar molecular weights, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, of M r 87 000 and 88 000, isoelectric points of 5.2, and Stokes radii of 6.7 and 6.5 nm, respectively. Heat shock protein 90 bound to phenyl-Sepharose CL-4B even at low ionic strength, and also bound to butyl-Toyopearl at high ionic strength. Heat shock protein 90 bound to phenyl-Sepharose could be eluted with a buffer containing organic solvents or detergents such as 2-propanol, dioxane, dimethylformamide, methyl cellosolve, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate or Triton X-100, but not with ionic salts such as 1 M KCl. These results suggest that heat shock protein 90 possesses a significant hydrophobic region on the surface of the molecule. Hydrophobicities of heat shock protein 90 and 4S calf uterine estrogen receptor were both decreased by formation of a 8S estrogen receptor complex. The role of the hydrophobic region of heat shock protein 90 in the interaction with estrogen receptor and other proteins is discussed.