FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2019425933> ?p ?o ?g. }

• W2019425933 endingPage "23561" @default.
• W2019425933 startingPage "23554" @default.
• W2019425933 abstract "We describe here an experimental protocol for the resolution, detection, and quantitation of the reduced and oxidized conformers of human heat shock factor 1 (hHSF1) and report on the effects in vitro and in vivoof redox-active agents on the redox status, structure, and function of hHSF1. We showed that diamide, a reagent that promotes disulfide bond formation, caused a loss of immunorecognition of the monomeric hHSF1 protein in a standard Western blot detection procedure. Modification of the Western blot procedure to include dithiothreitol in the equilibration and transfer buffers after gel electrophoresis allowed for the detection of a compact, intramolecularly disulfide cross-linked oxidized hHSF1 (ox-hHSF1) in the diamide-treated sample. The effect of diamide was blocked by pretreatment with N-ethylmaleimide and was reversed by dithiothreitol added to the sample prior to gel electrophoresis. Incubation with nitrosoglutathione at 42 °C also promoted the conversion of HSF1 to ox-HSF1; at 25 °C, however, nitrosoglutathione was by itself without effect but blocked the formation of ox-hHSF1 in the presence of diamide. The disulfide cross-linked ox-hHSF1 was monomeric and resistant to the in vitro heat-induced trimerization and activation. The possibility that ox-HSF1 may occur in oxidatively stressed cells was evaluated. Treatment of HeLa cells with 2 mml-buthionine sulfoximine promoted the formation of ox-HSF1 and blocked the heat-induced activation of HSF DNA binding activity. Our result suggests that hHSF1 may have integrated redox chemistry of cysteine sulfhydryl into its functional responses. We describe here an experimental protocol for the resolution, detection, and quantitation of the reduced and oxidized conformers of human heat shock factor 1 (hHSF1) and report on the effects in vitro and in vivoof redox-active agents on the redox status, structure, and function of hHSF1. We showed that diamide, a reagent that promotes disulfide bond formation, caused a loss of immunorecognition of the monomeric hHSF1 protein in a standard Western blot detection procedure. Modification of the Western blot procedure to include dithiothreitol in the equilibration and transfer buffers after gel electrophoresis allowed for the detection of a compact, intramolecularly disulfide cross-linked oxidized hHSF1 (ox-hHSF1) in the diamide-treated sample. The effect of diamide was blocked by pretreatment with N-ethylmaleimide and was reversed by dithiothreitol added to the sample prior to gel electrophoresis. Incubation with nitrosoglutathione at 42 °C also promoted the conversion of HSF1 to ox-HSF1; at 25 °C, however, nitrosoglutathione was by itself without effect but blocked the formation of ox-hHSF1 in the presence of diamide. The disulfide cross-linked ox-hHSF1 was monomeric and resistant to the in vitro heat-induced trimerization and activation. The possibility that ox-HSF1 may occur in oxidatively stressed cells was evaluated. Treatment of HeLa cells with 2 mml-buthionine sulfoximine promoted the formation of ox-HSF1 and blocked the heat-induced activation of HSF DNA binding activity. Our result suggests that hHSF1 may have integrated redox chemistry of cysteine sulfhydryl into its functional responses. human heat shock factor 1 oxidized hHSF1 dithiothreitol N-ethylmaleimide S-nitrosothiols S-nitrosoglutathione S-nitrosoacetylpenicillamine sodium nitrosoprusside buthionine sulfoximine polyacrylamide gel electrophoresis phenylmethylsulfonyl fluoride heat shock element glutathione Cysteine, although not among the most common amino acid residues found in proteins, has unique chemical properties that confer upon it important and distinctive roles in protein structure and function. The free thiol group (S−, thiolate anion) of cysteine is a powerful nucleophile and is the most readily oxidized and nitrosylated of amino acid side chains (1Lunblad R.L. Techniques in Protein Modification. CRC Press, New York1995: 63-96Google Scholar, 2Stamler J.S. Hausladen A. Nat. Struct. Biol. 1998; 5: 247-249Crossref PubMed Scopus (247) Google Scholar). The disulfide-bonded cystine, by comparison, is relatively unreactive but provides a covalent link between different regions of a protein and confers stability to a specific conformation (3Brandon C. Tooze J. Introduction to Protein Structure. 2nd Ed. Garland Publishing, New York1998: 353-356Google Scholar, 4Jaenicke R. Chadwick D.J. Widdows K. Protein Conformation. John Wiley and Sons, New York1991: 206-221Google Scholar). These considerations gave impetus to the suggestion that the redox-dependent thiol-disulfide exchange reaction can provide an important mechanism to regulate protein structure and function (5Gilbert H.F. Methods Enzymol. 1984; 107: 330-351Crossref PubMed Scopus (236) Google Scholar, 6Ziegler D.M. Annu. Rev. Biochem. 1985; 54: 305-329Crossref PubMed Scopus (692) Google Scholar). The notion that thiol-disulfide exchange may be involved in regulating transcription factor activity has gained much recent interest and support (7Ashlund F. Beckwith J. Cell. 1999; 96: 751-753Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). For example, activation of the prokaryotic OxyR transcription factor has been shown to be a two-step process involving first the oxidation of Cys-199 to a sulfenic acid followed by intramolecular disulfide cross-link of Cys-199 and Cys-208 (8Zheng M. Aslund F. Storz G. Science. 1998; 279: 1718-1721Crossref PubMed Scopus (970) Google Scholar). Importantly, OxyR can be activated independently by hydrogen peroxide or by a shift of the cellular redox state as in Escherichia coli mutants lacking components of the thioredoxin and glutaredoxin pathways (9Aslund F. Zheng M. Beckwith J. Storz G. Proc. Natl. Acad. Sci. 1999; 96: 6161-6165Crossref PubMed Scopus (434) Google Scholar). These observations provided the much needed information to relate changes in cellular redox status to the mechanism of regulation of OxyR and to the oxidative stress response in prokaryotes. Examples of eukaryotic transcription factor regulationin vitro through reversible redox-dependent modification of key cysteine-SH groups include AP-1 (10Abate C. Patel L. Rauscher III F.J. Curran T. Science. 1990; 249: 1157-1161Crossref PubMed Scopus (1374) Google Scholar), Rel/κB (11Mathews J.R. Wakasugi N. Virelizier J. Yodoi J. Hay R.T. Nucleic Acids Res. 1992; 20: 3821-3830Crossref PubMed Scopus (724) Google Scholar,12Kumar S. Rabson A.B. Gelinas C. Mol. Cell. Biol. 1992; 12: 3094-3106Crossref PubMed Google Scholar), Sp1 (13Wu X. Bishopric N.H. Discher D.J. Murphy B.J. Webster K.A. Mol. Cell. Biol. 1996; 16: 135-1046Crossref PubMed Scopus (145) Google Scholar), and Pax proteins (14Tell G. Scalon A. Pellizzari L. Formisano S. Pucillo C. Damante G. J. Biol. Chem. 1998; 273: 25062-25072Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Whereas in vivoevidence for a causal relationship of changes in cellular redox status and function of the candidate eukaryotic transcription factor(s) is lacking, the demonstration of redox-dependent regulation of OxyR in the living prokaryotic cells and the presence in eukaryotic cells of enzymes/proteins that catalyze thiol-disulfide exchange (e.g. thioredoxin, nucleoredoxin, and Ref-1 (15Hirota K. Matsui M Iwata S. Nishiyama A. Mori K. Yodoi J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3633-3638Crossref PubMed Scopus (720) Google Scholar)) would argue for conservation of this regulatory mechanism in evolution. We are interested in determining if redox may provide a mechanism of regulation of the structure and function of hHSF1,1 the transcription factor that mediates the heat shock transcriptional response to heat and other environmental stresses (16Morimoto R.I. Genes Dev. 1998; 12: 3788-3796Crossref PubMed Scopus (1533) Google Scholar, 17Wu C. Annu. Rev. Cell Dev. Biol. 1995; 11: 441-469Crossref PubMed Scopus (972) Google Scholar). This interest stems in part from our desire to understand the age-dependent dysfunction of HSF1 in a variety of model systems, including human diploid fibroblasts in culture (18Liu A.Y.-C. Lee Y.K. Manalo D. Huang L.E. Feige U. Morimoto R.I. Yahara I. Polla B.S. Stress-inducible Cellular Responses. Birkhauser/Springer, Basel, Boston, Berlin1996: 393-408Crossref Google Scholar, 19Lee Y.-K. Manalo D. Liu A.Y.-C. Biol. Signals. 1996; 5: 180-191Crossref PubMed Scopus (50) Google Scholar), and is based on the generally acknowledged importance of oxidation and oxidative damage of proteins as contributing causes of aging (20Berlett B.S. Stadtman E.R. J. Biol. Chem. 1997; 272: 20313-20316Abstract Full Text Full Text PDF PubMed Scopus (2783) Google Scholar). As a first step toward this goal, we developed the necessary reagents and experimental protocols for the resolution, detection, and quantitation of redox conformers of the human heat shock factor 1, and we evaluated the effects in vitro and in vivo of various redox-active compounds on the redox status, structure, and function of hHSF1. The polyclonal rabbit anti-hHSF1 antibody was prepared by immunizing a rabbit with histidine-tagged hHSF1 produced inE. coli and affinity-purified using Ni2+-nitrilotriacetic acid resin from Qiagen, Valencia, CA. The specificity of this antibody was very similar to the antibody previously provided to us (21Huang L.E. Zhang H. Bae S.W. Liu A.Y.-C. J. Biol. Chem. 1994; 48: 30718-30725Abstract Full Text PDF Google Scholar) from the laboratory of Dr. C. Wu at the NCI, National Institutes of Health (22Rabindran S.K. Haroun R.I. Clos J. Wisnieski J. Wu C. Science. 1993; 259: 230-234Crossref PubMed Scopus (386) Google Scholar). The enhanced chemiluminescence Western blot detection kit was from Amersham Pharmacia Biotech. The plasmid, pJC20(HSF1) was from the laboratory of Dr. Carl Wu (23Rabindran D.K. Giorgi F. Clos J. Wu C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6906-6910Crossref PubMed Scopus (376) Google Scholar). Restriction enzymes were from New England Biolabs. Inc., Beverly, MA. Transcription in vitro was done using mMessage-mMachine T7-Transcripton kit (Ambion, Inc., Austin, TX), and translationin vitro was done using rabbit reticulocyte lysate fromPromega, Inc., Madison, WI. Consensus oligonucleotides of Oct-1, TATA-1, AP-1, NF-κB, and SP-1 were from Promega Inc., Madison, WI. Diamide (diazenedicarboxylic acid bis(N,N-dimethylamide)), buthionine sulfoximine (BSO), sodium nitrate, sodium nitroprusside (sodium nitroferricyanide, Na2Fe(CN)5NO), and other SH-directed reagents were from Sigma or Pierce. Other chemicals were of molecular biology grade or reagent grade. HeLa cells were grown as monolayer cultures at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 50 units/ml penicillin plus 50 μg/ml streptomycin. The growth medium was replenished every 3 days until cells reach confluency. Cells were harvested at the end of an experiment by first removing the medium, rinsing the monolayer twice with ice-cold phosphate-buffered saline (150 mm NaCl, 10 mm sodium phosphate, pH 7.4), scraping the cells off with a plastic scraper, and pelleting by centrifugation at 1,800 × g for 4 min. The primary objective of this study was to determine the effectsin vitro of diamide and nitrosoglutathione on the redox status and function of HSF1. For this, the S100 cell extract of control HeLa cells containing the latent HSF1 protein was used. The method of preparation of the S100 extract was as described previously (24Baler R. Gerhard D. Voellmy R. Mol. Cell. Biol. 1993; 13: 2486-2496Crossref PubMed Scopus (393) Google Scholar). Briefly, the harvested cell pellet was resuspended by light vortexing in 4× packed cell volume of Buffer A (15 mm HEPES (pH 7.9), 10 mm KCl, 1.5 mm MgCl2, 0.5 mm DTT, 0.5 mm PMSF, 1 μg/ml each of leupeptin and pepstatin, 0.01 units/ml aprotinin), repelleted by centrifugation, and resuspended in 2× packed cell volume of Buffer A. After a 15-min incubation on ice, the swollen cells were Dounce-homogenized with a B-type pestle (15 strokes). The cell homogenate was then centrifuged at 10,000 × g for 8 min, and the pellet was saved for nuclear extract preparation described below. The supernatant was transferred to a new Eppendorf tube, mixed with 0.11× volume of Buffer B (180 mm HEPES (pH 7.9), 20% glycerol, 1.0 m NaCl, 13.5 mmMgCl2, 0.2 mm EDTA, 0.5 mm DTT, 0.5 mm PMSF, 1 μg/ml each of leupeptin and pepstatin, and 0.01 units/ml aprotinin), and centrifuged at 100,000 ×g for 60 min. The supernatant was carefully removed and dialyzed against 50× volume of Buffer D (25 mm HEPES (pH 7.9), 22% glycerol, 100 mm KCl, 0.2 mm EDTA, 0.5 mm PMSF, and 0.5 mm DTT) for 4 h at 4 °C. The S100 cell extract thus obtained was aliquoted and kept frozen at −70 °C until use. For studies of the effects in vivo of glutathione depletion on the redox status and function of HSF1, HeLa cells were incubated with 1 mm BSO, a specific inhibitor of γ-glutamylcysteine synthetase, for 24 h at 37 °C, followed by an additional 2-h incubation under control (37 °C) or heat shock (42 °C) condition. Cells were harvested, and whole cell extracts were prepared essentially according to methods described (24Baler R. Gerhard D. Voellmy R. Mol. Cell. Biol. 1993; 13: 2486-2496Crossref PubMed Scopus (393) Google Scholar, 25Ausubel J Brent F. M Kingston R Moore R. D Smith D. D Seidman J. A Struhl J. G Current Protocols in Molecular Biology. John Wiley and Sons, New York1990: 10.2.1-10.2.8Google Scholar). Briefly, the freshly prepared cell pellet was placed in a liquid-N2 bath for 10 min. It was then resuspended in 2.5× pellet volume of a modified whole cell lysis Buffer C (20 mm HEPES (pH 7.9), 20% glycerol, 0.42m NaCl, 1.5 mm MgCl2, 0.5 mm iodoacetamide, 0.2 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride (PMSF), 1 μg/ml each of leupeptin and pepstatin, and 0.01 units/ml aprotinin) and thawed at 25 °C for 10 min with mild vortexing every 2 min. The mixture was then placed on ice for 10 min with periodic pulse vortexing and then centrifuged at 4 °C for 10 min at 12,000 × g. The supernatant containing whole cell extract was aliquoted and stored at −70 °C until use. Nitrosothiols (RSNO; orthionitrites) are unstable esters of thionitrous acid of the general structure R–S–N=O, and the biological activity of RSNO was likely due to the heterolytic decomposition and release of the nitrosonium cation (NO+) as oppose to the homolytic decomposition and release of nitric oxide (NO⋅) (26Arnelle D.R. Stamler J.S. Arch. Biochem. Biophys. 1995; 318: 279-285Crossref PubMed Scopus (539) Google Scholar). The RSNO reagents that we have used in this study include S-nitrosoglutathione (GSNO) andS-nitrosoacetylpenicillamine (SNAP). The other NO carrier that we used was sodium nitroprusside from Sigma. GSNO and SNAP were prepared according to procedures described (27Mathews W.R. Kerr S.W. J. Pharmacol. Exp. Ther. 1993; 267: 1523-1537Google Scholar). Briefly, sodium nitrate (NaNO2) was mixed with an equimolar concentration of GSH (dissolved in water) or N-acetylpenicillamine (dissolved in 25% methanol). The solutions were adjusted to pH 2.0 and incubated at 37 °C for 10 min at which time a characteristic color developed as follows: GSNO gave a deep orange hue, whereas SNAP formation was indicated by a light green color. The samples were then neutralized with NaOH to pH 7.4, aliquoted, frozen, and stored as stock at −70 °C to be used over the next 2 months. For controls, GSH and NAP without sodium nitrate were similarly processed. HeLa cells treated with these control reagents were used to validate the specificity of effects of the S-nitrosothiols. The in vitro activation of hHSF1 by heat was critically dependent on protein concentration (28Larson J.S. Schuetz T.J. Kingston R.E. Nature. 1988; 335: 372-375Crossref PubMed Scopus (189) Google Scholar, 29Mosser D.D. Kotzbauer P.T. Sarge K.D. Morimoto R.I. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3748-3752Crossref PubMed Scopus (236) Google Scholar). We routinely prepared and used S100 cell extracts with a protein concentration >4.5 μg/μl in order to get a robust and reliable activation of the HSF1 DNA binding activity. Aliquots of cell extracts were incubated at 42 °C for 30–60 min to activate hHSF1. Extracts incubated at 25 °C served as controls. In order that we may resolve and probe redox conformers of HSF1 by gel electrophoresis and immuno-Western blot, it was necessary for us to exclude SH-reducing reagents, such as β-mercaptoethanol or dithiothreitol, from the sample buffer used for gel electrophoresis. This is an important variation from the standard practice in protein gel electophoresis. Our 2× sample buffer for gel electrophoresis contained 125 mm Tris-HCl (pH 6.8), 20% glycerol, 25 μg/ml bromphenol blue, and SDS in concentrations indicated below. For SDS-PAGE, the sample, separation gel, and electrophoresis buffer contained 2, 0.1, and 0.1% SDS, respectively. For native gel electrophoresis, there was no SDS in the separation gel and electrophoresis buffer, although it was necessary to include 0.02% of SDS in the sample buffer. The omission of this 0.02% SDS (0.01 of that present in the normal SDS sample buffer) caused the hHSF1 signal to appear as a smear in immuno-Western blot analysis of proteins resolved by native gel electrophoresis. Perhaps the presence of a trace amount of SDS in the sample buffer had a salutary effect in preventing protein aggregation. Occasionally, when it was necessary for us to determine the total amount of HSF1, we included in the sample buffer 10 mm DTT, and the samples were then processed for gel electrophoresis and immuno-Western blot probing of HSF1. Aliquots of protein samples with 20 μg of protein were mixed with an equal volume of the specified 2× sample buffer and then loaded onto a mini-gel apparatus. Gel electrophoresis was done according to the method of Laemmli as described previously (25Ausubel J Brent F. M Kingston R Moore R. D Smith D. D Seidman J. A Struhl J. G Current Protocols in Molecular Biology. John Wiley and Sons, New York1990: 10.2.1-10.2.8Google Scholar) using a 4% spacer gel and, unless indicated otherwise, a 5.5% separation gel. Samples were electrophoresed at 100 V for 70 min or until the tracking dye, bromphenol blue, reached the bottom of the gel. For assessment of the stoichiometry of hHSF1, samples were incubated with glutaraldehyde (2 mm, 30 min at 25 °C) to cross-link protein subunits, followed by quenching of the cross-linking reaction with the addition of 100 mm lysine. These sample was then mixed with an equal volume of a reducing (10 mmDTT) SDS sample buffer and heated at 100 °C for 10 min to ensure the complete denaturation of proteins and reduction of disulfide-bonded cystines. Electrophoretic separation of the HSF1 monomer, dimer, and trimer was done using a SDS (4–10% gradient)-acrylamide gel. Immuno-Western blot detection of hHSF1 was done under either a standard or reducing condition. For the standard procedure, the gel after electrophoresis was equilibrated for 10 min in 50 ml of 20 mm Tris (pH 8.0), 150 mm glycine, and 20% methanol, and proteins were then transferred electrophoretically (50 mA, 60 min at 25 °C) onto a nitrocellulose membrane in the same buffer. For the reducing Western transfer procedure, the equilibration and transfer buffers contained 5 and 1 mm DTT, respectively. Nonspecific protein-binding site on the nitrocellulose membrane was blocked by incubation of the membrane with 10% nonfat milk in a Tris-buffered saline/Triton X-100 buffer (10 mmTris (pH 8.0), 150 mm NaCl, 0.1% Triton X-100). Immunodetection of the hHSF1 protein was done using a 1:2500 dilution of the anti-HSF1 antibody and 1:5000 dilution of the horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. Antigen-antibody complex was detected using the ECL Western Detection Kit from Amersham Pharmacia Biotech. In this as well as our previous studies (22Rabindran S.K. Haroun R.I. Clos J. Wisnieski J. Wu C. Science. 1993; 259: 230-234Crossref PubMed Scopus (386) Google Scholar, 30Park J. Liu A.Y.-C. J. Cell. Physiol. 2000; 185: 348-357Crossref PubMed Scopus (17) Google Scholar) of immuno-Western blot detection of hHSF1, the specific hHSF1 signal appeared as a cluster of bands with apparent molecular masses of ∼85–90 kDa in SDS-PAGE. Electrophoretic gel mobility shift assay of HSF DNA binding activity was performed essentially as described previously (31Choi H.S. Lin A. Li B. Liu A.Y.-C. J. Biol. Chem. 1990; 265: 18005-18011Abstract Full Text PDF PubMed Google Scholar, 32Liu A.Y.-C. Choi H.S. Lee Y.K. Chen K.Y. J. Cell. Physiol. 1991; 149: 560-566Crossref PubMed Scopus (38) Google Scholar). Aliquots of cell extracts containing 20–40 μg of protein were used to assay for binding to 32P-HSE. 500 ng of poly(dI/dC) was used to quench nonspecific protein-DNA binding. The reaction was initiated by adding 0.25–1.0 ng of the HSE-oligonucleotide probe (25,000–30,000 cpm) to the samples and incubation for 20–30 min at 25 °C. Protein-DNA complexes were resolved from the free 32P-HSE (DNA) probe by electrophoresis in a low ionic strength 4% nondenaturing polyacrylamide gel. A glycerol-bromphenol blue/xylene cyanol dye solution was added to flanking empty wells to monitor the migration of free probes. Samples were electrophoresed at 200 V for about 45 min. In the initial phase of this study, we noticed that detection of the hHSF1 monomer in HeLa S100 cell extracts using a standard immuno-Western blot detection protocol was markedly affected by cysteine SH-directed reagents. Fig. 1illustrates this. Thus, treatment of the latent hHSF1 with diamide significantly reduced the immunodetection of hHSF1 in a standard Western blot procedure (lanes 2–7). This effect of diamide was readily and completely reversed by DTT (5 mm) added to the sample prior to gel electrophoresis (lane 8), whereas DTT by itself had no effect (lane 9). This result suggests that the loss of immunoreactivity of the diamide-treated hHSF1 was not because of irreversible degradation of the protein. This cycle of treatment with diamide and then DTT could be repeated at least two times with little or no change in the signal intensity of hHSF1. Diamide is an oxidizing reagent known to promote protein disulfide cross-link (33Kosower N.S. Kosower E.M. Methods Enzymol. 1987; 143: 264-270Crossref PubMed Scopus (81) Google Scholar). This being given, the result in Fig. 1 suggested to us that perhaps disulfide bond formation selectively stabilized a form of hHSF1 with a shielded antigenic core that was difficult if not impossible to detect using the anti-hHSF1 polyclonal antibody. Accordingly, we reasoned that breaking the disulfide bond(s) after the completion of gel electrophoresis may allow for the immunorecognition and detection of the oxidized hHSF1 protein. We devised a “reducing” Western blot procedure, in which the gel after electrophoresis was equilibrated in a buffer containing 5 mm DTT, and proteins were transferred from the gel to nitrocellulose membrane in buffer containing 1 mm DTT. A diagrammatic illustration of this experimental strategy is shown in Fig. 2, and the result comparing immuno-Western blot detection of hHSF1 using the standardversus the reducing Western transfer procedure is shown in Fig. 3. We showed that whereas diamide treatment caused the hHSF1 protein to evade immunodetection in a standard Western blot procedure (Fig. 3 A), the equilibration and then transfer of proteins after electrophoresis in a buffer containing DTT allowed for the detection of a set of faster moving, and presumably more compact forms of ox-hHSF in both SDS- and native gel electrophoresis (Fig. 3, B and C, respectively). The fact that the ox-hHSF1 could be resolved from and in fact had a greater electrophoretic mobility than hHSF1, particularly in native gel electrophoresis (Fig. 3 C), has two important implications. First, it suggests that the reduced HSF1 protein was metastable and unable to maintain its compact conformation during electrophoresis. Second, the result argues for intramolecular rather than intermolecular disulfide cross-link. Together, the results in Figs. 1 and 3 showed that diamide promoted the formation of ox-hHSF1, an intramolecularly disulfide cross-linked hHSF1 conformer. This effect of diamide could be assessed either by the apparent loss of immunoreactivity of hHSF1 in a standard Western blot procedure or by the appearance of a more compact form (faster mobility) of the protein in both SDS- and native polyacrylamide gel electrophoresis detectable with a “reducing” Western transfer procedure.Figure 3Diamide promoted formation of a compact disulfide cross-linked hHSF1 , and the immuno-Western blot detection of this oxidized hHSF1 required reduction of the disulfide bond after the gel electrophoresis procedure. Conditions of sample treatment are as follows: lane 1, untreated S100 control; lane 2, sample treated with 1 mm diamide (DM) for 30 min at 25 °C; lane 3, sample treated first with 1 mm diamide for 30 min at 25 °C followed by 5 mm dithiothreitol and incubation for another 30 min. The treated samples were mixed with an equal volume of the 2× non-reducing sample buffer and then subjected to analysis by gel electrophoresis and immuno-Western blot probing for hHSF1 as specified below. A,standard Western blot detection of hHSF1 in SDS-polyacrylamide gel. Samples were mixed with an equal volume of SDS sample buffer and subjected to analysis by SDS-PAGE. All procedures and buffers used (gel electrophoresis, equilibration, and transfer of proteins from the gel to nitrocellulose membrane) were done without dithiothreitol.B, reducing Western blot detection of hHSF1 in SDS-polyacrylamide gel. Upon completion of the SDS-PAGE procedure, the gel was equilibrated in a buffer containing 5 mm DTT, and proteins were transferred in buffer containing 1 mm DTT. The membrane was then probed for hHSF1 according to methods described in the text. C, reducing Western blot detection of hHSF1 in native polyacrylamide gel. Samples were mixed with an equal volume of the 2× sample buffer for native gel electrophoresis and then loaded onto a native 5.5% polyacrylamide gel. After the electrophoresis procedure, the gel was equilibrated in buffer containing 5 mm DTT; proteins were transferred in a buffer containing 1 mm DTT, and the presence of hHSF1 was probed as inB. The position of the native (and presumably reduced) hHSF1 protein is indicated by a brace. The position of the oxidized hHSF1 is indicated by *. We note that in both SDS- and native PAGE the mobility of the oxidized hHSF1 conformer is faster than the reduced hHSF1, suggesting intramolecular rather then intermolecular disulfide cross-link.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Studies with other cysteine-SH-directed reagents provided additional support that formation of the compact hHSF1 conformer is a result of disulfide cross-link. Fig. 4 Ashowed that the effect of diamide was blocked by prior treatment of the hHSF1 with N-ethylmaleimide, a cysteine-SH-alkylating agent (compare lanes 3 and 6). Similarly, in Fig.4 B, we showed that preincubation of the S100 extract with GSNO, a physiologically important carrier and donor of nitric oxide (34Stamler J.S. Jaraki O. Osborne J. Simon D.I. Kearney J. Vita J. Singel D. Valeri C.R. Loscalzo J. Proc. Natl. Acad. Sci., U. S. A. 1992; 89: 7674-7677Crossref PubMed Scopus (1129) Google Scholar, 35Clancy R.M. Levartovsky D. Leszczynska-Piziak J. Yegudin J. Abramson S.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3680-3684Crossref PubMed Scopus (305) Google Scholar), blocked the effect of diamide (compare lanes 3 and6). NEM (Fig. 4 A, lane 5) and GSNO (Fig.4 B, lane 5) were by themselves without effect. The effects of GSNO were complex and dependent on the temperature of incubation. In Fig. 5 we showed that whereas incubation of the S100 extract with GSNO at 25 °C by itself had little or no effect on the mobility of hHSF1 in native PAGE, incubation at 42 °C promoted the formation of ox-hHSF1 (comparelanes 4 and 7), and this effect of GSNO was readily and completely reversed by DTT (compare lanes 7 and8). As controls, we showed that incubation at 42 °C without (lane 9) and with glutathione (GSH,lane 10) or sodium nitrate (NO2,lane 11) had no effect. Our observation of these distinct effects of GSNO on hHSF1 regulation at 25 versus 42 °C as presented in Fig. 5 is consistent with the suggested importance of both additive (trans-nitrosylation) and redox (e.g. disulfide bond formation) chemistry in subserving the many and diverse biological effects of GSNO (36Lipton S.A. Neurochem. Int. 1996; 29: 111-114Crossref PubMed Scopus (18) Google Scholar, 37Wong P.S.-Y. Hyun J. Fukuto J.M. Shirota F.N. DeMaster E.G. Shoeman D.W. Nagasawa H.T. Biochemistry. 1998; 37: 5362-5371Crossref PubMed Scopus (327) Google Scholar). The hHSF1 protein can be induced to undergo monomer to trimer conversion in vitro by heat (42 °C) or by treatment with Nonidet P-40 or low pH buffer (28Larson J.S. Schuetz T.J. Kingston R.E. Nature. 1988; 335: 372-375Crossref PubMed Scopus (189) Google Scholar, 29Mosser D.D. Kotzbauer P.T. Sarge K.D. Morimoto R.I. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3748-3752Crossref PubMed Scopus (236) Google Scholar). As disulfide bridges are known to increase protein stability by reducing the number of unfolded conformations and decreasing the entropic costs of folding a protein into its single native state (3Brandon C. Tooze J. Introduction to Protein Structure. 2nd Ed. Garland Publishing, New York1998: 353-356Google Scholar, 4Jaenicke R. Chadwick D.J. Widdows K. Protein Conformati" @default.
• W2019425933 created "2016-06-24" @default.
• W2019425933 creator A5010011763 @default.
• W2019425933 creator A5065233587 @default.
• W2019425933 date "2001-06-01" @default.
• W2019425933 modified "2023-10-11" @default.
• W2019425933 title "Resolution, Detection, and Characterization of Redox Conformers of Human HSF1" @default.
• W2019425933 cites W1504187225 @default.
• W2019425933 cites W1581914543 @default.
• W2019425933 cites W1934753590 @default.
• W2019425933 cites W1967138582 @default.
• W2019425933 cites W1971819783 @default.
• W2019425933 cites W1973328612 @default.
• W2019425933 cites W1973475159 @default.
• W2019425933 cites W1974716551 @default.
• W2019425933 cites W1987938397 @default.
• W2019425933 cites W1993577562 @default.
• W2019425933 cites W2002192155 @default.
• W2019425933 cites W2005205544 @default.
• W2019425933 cites W2006172842 @default.
• W2019425933 cites W2006609702 @default.
• W2019425933 cites W2014914786 @default.
• W2019425933 cites W2015238819 @default.
• W2019425933 cites W2017240909 @default.
• W2019425933 cites W2020754722 @default.
• W2019425933 cites W2026728447 @default.
• W2019425933 cites W2030566353 @default.
• W2019425933 cites W2043593153 @default.
• W2019425933 cites W2045117885 @default.
• W2019425933 cites W2045899523 @default.
• W2019425933 cites W2048438868 @default.
• W2019425933 cites W2060836836 @default.
• W2019425933 cites W2064638709 @default.
• W2019425933 cites W2067706269 @default.
• W2019425933 cites W2069594598 @default.
• W2019425933 cites W2070693959 @default.
• W2019425933 cites W2082739985 @default.
• W2019425933 cites W2091477850 @default.
• W2019425933 cites W2107919301 @default.
• W2019425933 cites W2110076050 @default.
• W2019425933 cites W2121667843 @default.
• W2019425933 cites W2145760315 @default.
• W2019425933 cites W2148130200 @default.
• W2019425933 cites W2154975058 @default.
• W2019425933 cites W2164779212 @default.
• W2019425933 cites W248833920 @default.
• W2019425933 cites W32366060 @default.
• W2019425933 cites W944568383 @default.
• W2019425933 doi "https://doi.org/10.1074/jbc.m011300200" @default.
• W2019425933 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11320084" @default.
• W2019425933 hasPublicationYear "2001" @default.
• W2019425933 type Work @default.
• W2019425933 sameAs 2019425933 @default.
• W2019425933 citedByCount "25" @default.
• W2019425933 countsByYear W20194259332012 @default.
• W2019425933 countsByYear W20194259332014 @default.
• W2019425933 countsByYear W20194259332016 @default.
• W2019425933 countsByYear W20194259332018 @default.
• W2019425933 crossrefType "journal-article" @default.
• W2019425933 hasAuthorship W2019425933A5010011763 @default.
• W2019425933 hasAuthorship W2019425933A5065233587 @default.
• W2019425933 hasBestOaLocation W20194259331 @default.
• W2019425933 hasConcept C12554922 @default.
• W2019425933 hasConcept C171250308 @default.
• W2019425933 hasConcept C178790620 @default.
• W2019425933 hasConcept C179104552 @default.
• W2019425933 hasConcept C185592680 @default.
• W2019425933 hasConcept C18705241 @default.
• W2019425933 hasConcept C192562407 @default.
• W2019425933 hasConcept C2780841128 @default.
• W2019425933 hasConcept C32909587 @default.
• W2019425933 hasConcept C55904794 @default.
• W2019425933 hasConcept C86803240 @default.
• W2019425933 hasConceptScore W2019425933C12554922 @default.
• W2019425933 hasConceptScore W2019425933C171250308 @default.
• W2019425933 hasConceptScore W2019425933C178790620 @default.
• W2019425933 hasConceptScore W2019425933C179104552 @default.
• W2019425933 hasConceptScore W2019425933C185592680 @default.
• W2019425933 hasConceptScore W2019425933C18705241 @default.
• W2019425933 hasConceptScore W2019425933C192562407 @default.
• W2019425933 hasConceptScore W2019425933C2780841128 @default.
• W2019425933 hasConceptScore W2019425933C32909587 @default.
• W2019425933 hasConceptScore W2019425933C55904794 @default.
• W2019425933 hasConceptScore W2019425933C86803240 @default.
• W2019425933 hasIssue "26" @default.
• W2019425933 hasLocation W20194259331 @default.
• W2019425933 hasOpenAccess W2019425933 @default.
• W2019425933 hasPrimaryLocation W20194259331 @default.
• W2019425933 hasRelatedWork W1531601525 @default.
• W2019425933 hasRelatedWork W2319480705 @default.
• W2019425933 hasRelatedWork W2384464875 @default.
• W2019425933 hasRelatedWork W2398689458 @default.
• W2019425933 hasRelatedWork W2606230654 @default.
• W2019425933 hasRelatedWork W2607424097 @default.
• W2019425933 hasRelatedWork W2748952813 @default.
• W2019425933 hasRelatedWork W2899084033 @default.
• W2019425933 hasRelatedWork W2948807893 @default.
• W2019425933 hasRelatedWork W2778153218 @default.

• next