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• W2022267557 abstract "Myotrophin/V-1 is a cytosolic protein found at elevated levels in failing human hearts and in postnatal cerebellum. We have previously shown that it disrupts nuclear factor of κB (NFκB)-DNA complexes in vitro. In this study, we demonstrated that in HeLa cells native myotrophin/V-1 is predominantly present in the cytoplasm and translocates to the nucleus during sustained NFκB activation. Three-dimensional alignment studies indicate that myotrophin/V-1 resembles a truncated IκBα without the signal response domain (SRD) and PEST domains. Co-immunoprecipitation studies reveal that myotrophin/V-1 interacts with NFκB proteinsin vitro; however, it remains physically associated only with p65 and c-Rel proteins in vivo during NFκB activation. In vitro studies indicate that myotrophin/V-1 can promote the formation of p50-p50 homodimers from monomeric p50 proteins and can convert the preformed p50-p65 heterodimers into p50-p50 and p65-p65 homodimers. Furthermore, adenovirus-mediated overexpression of myotrophin/V-1 resulted in elevated levels of both p50-p50 and p65-p65 homodimers exceeding the levels of p50-p65 heterodimers compared with Adβgal-infected cells, where the levels of p50-p65 heterodimers exceeded the levels of p50-p50 and p65-p65 homodimers. Thus, overexpression of myotrophin/V-1 during NFκB activation resulted in a qualitative shift by quantitatively reducing the level of transactivating heterodimers while elevating the levels of repressive p50-p50 homodimers. Correspondingly, overexpression of myotrophin/V-1 resulted in significantly reduced κB-luciferase reporter activity. Because myotrophin/V-1 is found at elevated levels during NFκB activation in postnatal cerebellum and in failing human hearts, this study cumulatively suggests that myotrophin/V-1 is a regulatory protein for modulating the levels of activated NFκB dimers during this period. Myotrophin/V-1 is a cytosolic protein found at elevated levels in failing human hearts and in postnatal cerebellum. We have previously shown that it disrupts nuclear factor of κB (NFκB)-DNA complexes in vitro. In this study, we demonstrated that in HeLa cells native myotrophin/V-1 is predominantly present in the cytoplasm and translocates to the nucleus during sustained NFκB activation. Three-dimensional alignment studies indicate that myotrophin/V-1 resembles a truncated IκBα without the signal response domain (SRD) and PEST domains. Co-immunoprecipitation studies reveal that myotrophin/V-1 interacts with NFκB proteinsin vitro; however, it remains physically associated only with p65 and c-Rel proteins in vivo during NFκB activation. In vitro studies indicate that myotrophin/V-1 can promote the formation of p50-p50 homodimers from monomeric p50 proteins and can convert the preformed p50-p65 heterodimers into p50-p50 and p65-p65 homodimers. Furthermore, adenovirus-mediated overexpression of myotrophin/V-1 resulted in elevated levels of both p50-p50 and p65-p65 homodimers exceeding the levels of p50-p65 heterodimers compared with Adβgal-infected cells, where the levels of p50-p65 heterodimers exceeded the levels of p50-p50 and p65-p65 homodimers. Thus, overexpression of myotrophin/V-1 during NFκB activation resulted in a qualitative shift by quantitatively reducing the level of transactivating heterodimers while elevating the levels of repressive p50-p50 homodimers. Correspondingly, overexpression of myotrophin/V-1 resulted in significantly reduced κB-luciferase reporter activity. Because myotrophin/V-1 is found at elevated levels during NFκB activation in postnatal cerebellum and in failing human hearts, this study cumulatively suggests that myotrophin/V-1 is a regulatory protein for modulating the levels of activated NFκB dimers during this period. myotrophin or V-1 tumor necrosis factor-α cycloheximide gel-shift assay nuclear factor of κB inhibitor of κB-α N-tris(hydroxymethyl)methylglycine amino acid(s) fluorescein isothiocyanate 4,6-diamidino-2-phenylindole signal response domain oligonucleotide nuclear localization signal NFκB-inducing kinase IκBα kinase Myotrophin/V-1 (Myo/V1)1protein was initially characterized in the mammalian heart, where it was called myotrophin (1Sen S. Kundu G. Mekhail N. Castel J. Misono K. Healy B. J. Biol. Chem. 1990; 265: 16635-16643Abstract Full Text PDF PubMed Google Scholar), and in the rat cerebellum, where it was called V-1 (2Taoka M. Yamakuni T. Song S.Y. Yamakawa Y. Seta K. Okuyama T. Isobe T. Eur. J. Biochem. 1992; 207: 615-620Crossref PubMed Scopus (37) Google Scholar). It was later found to be ubiquitously expressed in all mammalian tissues (3Sivasubramanian N. Adhikary G. Sil P.C. Sen S. J. Biol. Chem. 1996; 271: 2812-2816Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 4Anderson K.M. Berrebi-Bertrand I. Kirkpatrick R.B. McQueney M.S. Underwood D.C. Rouanet S. Chabot-Fletcher M. J. Mol. Cell Cardiol. 1999; 31: 705-719Abstract Full Text PDF PubMed Scopus (23) Google Scholar). Myo/V1 is a 12-kDa ankyrin repeat-containing intracellular protein that has been found at elevated levels in failing human hearts (5Sil P. Misono K. Sen S. Circ. Res. 1993; 73: 98-108Crossref PubMed Scopus (37) Google Scholar) as well as in the hearts of spontaneously hypertensive rats (6Sil P. Mukherjee D. Sen S. Circ. Res. 1995; 76: 1020-1027Crossref PubMed Scopus (18) Google Scholar). Although Myo/V1 was originally described as a trophic protein (myotrophin) exhibiting growth properties exogenously on rat neonatal myocytes (1Sen S. Kundu G. Mekhail N. Castel J. Misono K. Healy B. J. Biol. Chem. 1990; 265: 16635-16643Abstract Full Text PDF PubMed Google Scholar), other studies showed that this protein was only present in intracellular space (2Taoka M. Yamakuni T. Song S.Y. Yamakawa Y. Seta K. Okuyama T. Isobe T. Eur. J. Biochem. 1992; 207: 615-620Crossref PubMed Scopus (37) Google Scholar, 7Taoka M. Isobe T. Okuyama T. Watanabe M. Kondo H. Yamakawa Y. Ozawa F. Hishinuma F. Kubota M. Minegishi A. J. Biol. Chem. 1994; 269: 9946-9951Abstract Full Text PDF PubMed Google Scholar, 8Song S.Y. Asakai R. Kenmotsu N. Taoka M. Isobe T. Yamakuni T. Endocrinology. 1996; 137: 1423-1428Crossref PubMed Scopus (13) Google Scholar, 9Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) and its trophic growth properties on neonatal myocytes were not confirmed (10Pennica D. Shaw K.J. Luoh S.M. Wood W.I. Gene (Amst.). 1995; 158: 305-306Crossref PubMed Scopus (9) Google Scholar). Moreover, this protein was originally identified and isolated only from an intracellular location (1Sen S. Kundu G. Mekhail N. Castel J. Misono K. Healy B. J. Biol. Chem. 1990; 265: 16635-16643Abstract Full Text PDF PubMed Google Scholar), and a transcriptional regulatory function has been proposed (3Sivasubramanian N. Adhikary G. Sil P.C. Sen S. J. Biol. Chem. 1996; 271: 2812-2816Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar,9Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Since its discovery, investigators have proposed various functions for Myo/V1 protein (1Sen S. Kundu G. Mekhail N. Castel J. Misono K. Healy B. J. Biol. Chem. 1990; 265: 16635-16643Abstract Full Text PDF PubMed Google Scholar, 2Taoka M. Yamakuni T. Song S.Y. Yamakawa Y. Seta K. Okuyama T. Isobe T. Eur. J. Biochem. 1992; 207: 615-620Crossref PubMed Scopus (37) Google Scholar, 3Sivasubramanian N. Adhikary G. Sil P.C. Sen S. J. Biol. Chem. 1996; 271: 2812-2816Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In the postnatal rat cerebellum, the cellular level of soluble Myo/V1 was found to be transiently up-regulated immediately after birth and later declined displaying a unique pattern of expression among 120 soluble proteins, implicating its role during postnatal cerebellum development (2Taoka M. Yamakuni T. Song S.Y. Yamakawa Y. Seta K. Okuyama T. Isobe T. Eur. J. Biochem. 1992; 207: 615-620Crossref PubMed Scopus (37) Google Scholar). Because of its aberrant expression in genetically defective cerebellar granular cells, this protein was proposed to play a role in granular cell differentiation process (Unigene Mm.4123) (7Taoka M. Isobe T. Okuyama T. Watanabe M. Kondo H. Yamakawa Y. Ozawa F. Hishinuma F. Kubota M. Minegishi A. J. Biol. Chem. 1994; 269: 9946-9951Abstract Full Text PDF PubMed Google Scholar). To date, however, the molecular function of Myo/V1 protein is still lacking. We recently reported that Myo/V1 protein exhibits significant homology to IκBα protein and that Myo/V1 can disrupt the NFκB-DNA complexes in vitro (3Sivasubramanian N. Adhikary G. Sil P.C. Sen S. J. Biol. Chem. 1996; 271: 2812-2816Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Utilizing our recombinant Myo/V1 protein, the NMR structure of Myo/V1 was determined (11Yang Y. Rao N.S. Walker E. Sen S. Qin J. Protein Sci. 1997; 6: 1347-1351Crossref PubMed Scopus (15) Google Scholar, 12Yang Y. Nanduri S. Sen S. Qin J. Structure. 1998; 6: 619-626Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), and the ankyrin repeats of Myo/V1 exhibited structural features similar to those of IκBα (13Huxford T. Huang D.B. Malek S. Ghosh G. Cell. 1998; 95: 759-770Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar, 14Jacobs M.D. Harrison S.C. Cell. 1998; 95: 749-758Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar) at the three-dimensional level. In response to a variety of pathophysiological and developmental signals, the NFκB/Rel family of transcription factors are activated and form different types of hetero- and homodimers among themselves to regulate the expression of target genes containing κB-specific binding sites (15Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2931) Google Scholar, 16Pahl H.L. Oncogene. 1999; 18: 6853-6866Crossref PubMed Scopus (3448) Google Scholar). Among the activated NFκB dimers, the p50-p65 heterodimers are known to be involved in enhancing the transcription of target genes and the p50-p50 homodimers in transcriptional repression (17Plaksin D. Baeuerle P.A. Eisenbach L. J. Exp. Med. 1993; 177: 1651-1662Crossref PubMed Scopus (113) Google Scholar, 18Brigelius-Flohe R. Bilgin B. Eickemeier S. Hipskind R. Singh M. Szamel M. Resch K. Biofactors. 1995; 5: 169-174PubMed Google Scholar, 19Goldring C.E. Narayanan R. Lagadec P. Jeannin J.F. Biochem. Biophys. Res. Commun. 1995; 209: 73-79Crossref PubMed Scopus (73) Google Scholar, 20Supakar P.C. Jung M.H. Song C.S. Chatterjee B. Roy A.K. J. Biol. Chem. 1995; 270: 837-842Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 21Kastenbauer S. Ziegler-Heitbrock H.W. Infect. Immun. 1999; 67: 1553-1559Crossref PubMed Google Scholar, 22Udalova I.A. Richardson A. Denys A. Smith C. Ackerman H. Foxwell B. Kwiatkowski D. Mol. Cell. Biol. 2000; 20: 9113-9119Crossref PubMed Scopus (208) Google Scholar). However, the p65-p65 homodimers are known for both transcriptional activation and repressive activity against target genes (23Sheppard K.A. Rose D.W. Haque Z.K. Kurokawa R. McInerney E. Westin S. Thanos D. Rosenfeld M.G. Glass C.K. Collins T. Mol. Cell. Biol. 1999; 19: 6367-6378Crossref PubMed Google Scholar, 24Ashburner B.P. Westerheide S.D. Baldwin A.S., Jr. Mol. Cell. Biol. 2001; 21: 7065-7077Crossref PubMed Scopus (627) Google Scholar, 25Nozaki S. Sledge G.W., Jr. Nakshatri H. Oncogene. 2001; 20: 2178-2185Crossref PubMed Scopus (101) Google Scholar, 26Waltner-Law M. Daniels M.C. Sutherland C. Granner D.K. J. Biol. Chem. 2000; 275: 31847-31856Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 27Kalkhoven E. Wissink S. van der Saag P.T. van der Burg B. J. Biol. Chem. 1996; 271: 6217-6224Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 28Sohur U.S. Dixit M.N. Chen C.L. Byrom M.W. Kerr L.A. Gene Exp. 1999; 8: 219-229PubMed Google Scholar, 29Farmer P.K., He, X. Schmitz M.L. Rubin J. Nanes M.S. Am. J. Physiol. 2000; 279: E213-E220Crossref PubMed Google Scholar, 30Nagarajan R.P. Chen F., Li, W. Vig E. Harrington M.A. Nakshatri H. Chen Y. Biochem. J. 2000; 348: 591-596Crossref PubMed Scopus (110) Google Scholar, 31Guttridge D.C. Mayo M.W. Madrid L.V. Wang C.Y. Baldwin A.S., Jr. Science. 2000; 289: 2363-2366Crossref PubMed Scopus (763) Google Scholar). NFκB activation is regulated at multiple levels. The dynamic shuttling of the inactive NFκB dimers between the cytoplasm and nucleus by IκB proteins (32Johnson C. Van Antwerp D. Hope T.J. EMBO J. 1999; 18: 6682-6693Crossref PubMed Google Scholar, 33Carlotti F. Dower S.K. Qwarnstrom E.E. J. Biol. Chem. 2000; 275: 41028-41034Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 34Malek S. Chen Y. Huxford T. Ghosh G. J. Biol. 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Immunol. 1998; 28: 2960-2970Crossref PubMed Scopus (39) Google Scholar, 41Liou H.C. Sha W.C. Scott M.L. Baltimore D. Mol. Cell. Biol. 1994; 14: 5349-5359Crossref PubMed Google Scholar, 42Miyamoto S. Schmitt M.J. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5056-5060Crossref PubMed Scopus (104) Google Scholar) have all been identified as key regulatory steps in NFκB-mediated transcription process. κB DNA binding sites with varied affinities to different NFκB dimers (43Kunsch C. Ruben S.M. Rosen C.A. Mol. Cell. Biol. 1992; 12: 4412-4421Crossref PubMed Google Scholar) have been discovered in the promoters of several eukaryotic genes (16Pahl H.L. Oncogene. 1999; 18: 6853-6866Crossref PubMed Scopus (3448) Google Scholar, 20Supakar P.C. Jung M.H. Song C.S. Chatterjee B. Roy A.K. J. Biol. Chem. 1995; 270: 837-842Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 22Udalova I.A. Richardson A. Denys A. Smith C. Ackerman H. Foxwell B. Kwiatkowski D. Mol. Cell. Biol. 2000; 20: 9113-9119Crossref PubMed Scopus (208) Google Scholar, 44Pan J. McEver R.P. J. Biol. Chem. 1995; 270: 23077-23083Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 45Marks-Konczalik J. Chu S.C. Moss J. J. Biol. Chem. 1998; 273: 22201-22208Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar), and the balance between activated NFκB homo- and heterodimers ultimately determines the nature and level of gene expression within the cell (18Brigelius-Flohe R. Bilgin B. Eickemeier S. Hipskind R. Singh M. Szamel M. Resch K. Biofactors. 1995; 5: 169-174PubMed Google Scholar,22Udalova I.A. Richardson A. Denys A. Smith C. Ackerman H. Foxwell B. Kwiatkowski D. Mol. Cell. Biol. 2000; 20: 9113-9119Crossref PubMed Scopus (208) Google Scholar). However, thus far the underlying molecular mechanism for the generation and dynamic reorganization of NFκB dimers during chronic activation is unknown. Here, for the first time, we show that Myo/V1 acts as a “zipper chaperone” protein to generate NFκB homodimers from monomeric p50 proteins and with its “unzipping” function converts the transcriptionally active p50-p65 heterodimers to transcriptionally repressive homodimers in HeLa cells, thus attenuating NFκB-mediated transcription. Recombinant Myo/V1 protein was expressed inEscherichia coli in two different forms (∼12-kDa full-length and histidine-tagged ∼14-kDa fusion protein) using pET expression vectors as described before (3Sivasubramanian N. Adhikary G. Sil P.C. Sen S. J. Biol. Chem. 1996; 271: 2812-2816Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The following mammalian expression plasmids were constructed by recombinant DNA methods. For pcDNA-AM1.1-Myo/V1, because of the poor translation initiation Kozak site in the Myo/V1 mRNA, we engineered a heterologous highly efficient Kozak site into 5′-untranslated region of Myo/V1 so that Myo/V1 is expressed at high levels in mammalian cells. In vitro transcription and translation with pcDNA3-AM1.1-Myo/V1 template DNA confirmed the synthesis of 12-kDa Myo/V1 protein at higher levels than the native Myo/V1 mRNA (data not shown). For pκB-tk-luc, the parent chloramphenicol acetyltransferase reporter plasmid containing a minimal thymidine kinase promoter and two κB enhancer sites was obtained and replaced with the coding region of luciferase enzyme. pRSV-RelA vector expressing p65 was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health. The expression plasmid is from Dr. Gary Nabel and Dr. Neil Perkins. The recombinant adenoviruses expressing Myo/V1 and β-galactosidase were constructed as follows. For AdMyo/V1/Adβgal, the respective expression plasmids pcDNA3-AM1.1-Myo/V1 and pcDNA3-βgal were incorporated into Ad5 adenovirus through allelic recombination. Recombinant adenoviruses were propagated, purified, and titered as previously reported (46Vallejo J.G. Knuefermann P. Mann D.L. Sivasubramanian N. J. Immunol. 2000; 165: 419-425Crossref PubMed Scopus (34) Google Scholar). HeLa cells (ATCC-CCL2) were maintained in minimal essential medium. Cell fixation and indirect immunofluorescence studies were performed as previously described (47Sivasubramanian N. Nayak D.P. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1-5Crossref PubMed Scopus (22) Google Scholar). Cytoplasmic and nuclear extracts were prepared using NE-PER™ nuclear and cytoplasmic extraction reagents (Pierce). Plasmid DNA transient transfection experiments were performed using FuGENE6 reagent (Roche). Luciferase assays were conducted with reagents from Promega Inc. The protein concentration was determined by BCA method using Pierce reagents. HeLa cells were treated for 2 h with TNF (50 ng/ml) to induce NFκB. For superinduction of NFκB, cells were further treated with cycloheximide (10 μg/ml). After 2 h of treatment, cells were harvested and subcellular fractionation for cytoplasmic and nuclear extracts were carried out as previously described (48Baeuerle P.A. Baltimore D. Cell. 1988; 53: 211-217Abstract Full Text PDF PubMed Scopus (798) Google Scholar). Briefly, HeLa cells were harvested at 150 × g and Dounce-homogenized in a hypotonic lysis buffer (buffer A; Ref. 49Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar), and nuclei were collected at 4300 × g. The nuclei were extracted with buffer C (49Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar) and were used for GSA and Western blot analysis. After the removal of the nuclei, the supernatant was further centrifuged at 20,000 × g for isolating mitochondrial fractions. The remaining cytosolic supernatant was further concentrated by acetone precipitation. Identical amounts of cytosolic (40 μg), nuclear extract (30 μg), and mitochondrial proteins (35 μg) were fractionated on a 10% Tris-Tricine SDS-PAGE and processed for Myo/V1 ECL immunoblotting. Pure recombinant p50 protein was obtained from Promega Inc., and pure truncated recombinant p65Δ (p65 RHRs) protein was obtained from Dr. Gaurishankar Ghosh (50Malek S. Huxford T. Ghosh G. J. Biol. Chem. 1998; 273: 25427-25435Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The p65Δ contains only the Rel DNA binding domain (∼31 kDa; aa 19–291) of p65, and the carboxyl terminus containing the transactivating domain of p65 has been removed (50Malek S. Huxford T. Ghosh G. J. Biol. Chem. 1998; 273: 25427-25435Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Monomeric p50 (2.5 ng in 50 μl; 2 nm) and p65Δ (0.5 μg in 50 μl; 7 μm) proteins were separately incubated with increasing concentrations of Myo/V1 (0–100 ng) and the resulting κB-DNA complexes from these reactions were analyzed by GSA (3Sivasubramanian N. Adhikary G. Sil P.C. Sen S. J. Biol. Chem. 1996; 271: 2812-2816Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 46Vallejo J.G. Knuefermann P. Mann D.L. Sivasubramanian N. J. Immunol. 2000; 165: 419-425Crossref PubMed Scopus (34) Google Scholar). Because p50 and p65 proteins exhibit high affinity to each other, equimolar concentrations of p50 (16 pmol; 0.8 μg) and p65Δ (16 pmol; 0.5 μg) proteins were mixed and incubated in a binding buffer at 4 °C to preform the p50-p65Δ heterodimers. Later, increasing concentrations of 12-kDa Myo/V1 protein (0–56 pmol; 0–800 ng) were added and incubated for an additional 30 min at 4 °C. Finally, the radiolabeled κB oligonucleotides (25,000 cpm) were added and incubated for another 10 min at 4 °C. The resulting κB-DNA complexes from these reactions were analyzed by GSA (3Sivasubramanian N. Adhikary G. Sil P.C. Sen S. J. Biol. Chem. 1996; 271: 2812-2816Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 46Vallejo J.G. Knuefermann P. Mann D.L. Sivasubramanian N. J. Immunol. 2000; 165: 419-425Crossref PubMed Scopus (34) Google Scholar). The mobility patterns of the p50-p65Δ, p50-p50, and p65Δ-p65Δ dimers on GSA exactly resemble the previously published patterns for these dimers (43Kunsch C. Ruben S.M. Rosen C.A. Mol. Cell. Biol. 1992; 12: 4412-4421Crossref PubMed Google Scholar, 51Chen F.E. Kempiak S. Huang D.B. Phelps C. Ghosh G. Protein Eng. 1999; 12: 423-428Crossref PubMed Scopus (45) Google Scholar, 52Ruben S.M. Narayanan R. Klement J.F. Chen C.H. Rosen C.A. Mol. Cell. Biol. 1992; 12: 444-454Crossref PubMed Google Scholar). To identify the nature of Myo/V1-generated NFκB dimers, antibodies to p50 (#sc114x; Santa Cruz Biotechnology, Santa Cruz, CA) and p65 (#ab243, Abcam, Cambridge, UK) were added to preformed heterodimers (equimolar mixture of p50 (80 ng) and p65Δ (50 ng) proteins) with increasing concentrations of Myo/V1 (0, 100, 400 ng), and its effects were studied on GSA. This experiment was also repeated with preformed p50-p65Δ heterodimers with 100-fold excess p65Δ protein (5 μg). We developed a more accurate method to quantify NFκB dimers in mammalian cell nuclear extracts. We chose three κB sites from native genes, which have been previously well characterized to exhibit high affinity to individual NFκB dimers. The rationale behind this approach is with varied amounts of activated NFκB dimers in the mammalian cell, using a single κB oligo, which exhibits high affinity to one dimer, and not to others and using it to quantify the various NFκB dimers will yield erroneous results. Moreover, the ideal κB oligo, which exhibits equal affinity toward all NFκB dimers with varied mobilities on GSA to distinguish them, does not exist. Therefore, the conventional κB-Igκ/HIV oligonucleotide (5′-AGTTGAGGGGACTTTCCCAGGC-3′ from Santa Cruz Biotechnology, Inc.) was used to quantify only p50-p65 heterodimers because it exhibited high affinity only toward p50-p65 heterodimers. Because this κB site exhibited lower affinities toward p50-p50 and p65-p65 homodimers (5- and 15-fold, respectively) (53Phelps C.B. Sengchanthalangsy L.L. Malek S. Ghosh G. J. Biol. Chem. 2000; 275: 24392-24399Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), we chose two other κB oligonucleotides κB#SeqB (44Pan J. McEver R.P. J. Biol. Chem. 1995; 270: 23077-23083Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) and κB#u-iNOS (45Marks-Konczalik J. Chu S.C. Moss J. J. Biol. Chem. 1998; 273: 22201-22208Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar), which were previously shown to exhibit high affinity and exclusive specificity toward p50-p50 and p65-p65 homodimers (44Pan J. McEver R.P. J. Biol. Chem. 1995; 270: 23077-23083Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 45Marks-Konczalik J. Chu S.C. Moss J. J. Biol. Chem. 1998; 273: 22201-22208Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). The κB#SeqB (5′-GTAGGGGGCCTCCCCGGCTCGAGATCCTATG-3′) and κB#u-iNOS (5′-GTACCGGAAATTCCGGGCTCGAGATCCTATG-3′) oligonucleotides were custom synthesized (Synthetic Genetics, CA) and used for quantifying respective NFκB homodimers. To determine the relative levels of various NFκB dimers, nuclear extracts (∼20 μg) from AdMyo/V1- and Adβgal-infected cells were incubated with radiolabeled individual κB oligonucleotides (κB-Igκ, #SeqB; #u-iNOS; 25,000 cpm with similar specific activities), and the resulting DNA-protein complexes were fractionated on the same 4% PAGE. Radiographic images of the gels were captured using a STORM 860 imager (Molecular Dynamics, CA) and quantified using ImageQuant 4.2 software. NFκB antibody supershifts were done as described previously; however, p65 antibody from Geneka Biotechnology (Montreal, Canada) was used in these experiments. Additionally, heterologous chase experiments were done to further confirm the nature of the NFκB dimers. Indirect immunofluorescence studies were conducted to locate the native Myo/V1 protein in HeLa cells. Under basal conditions (Fig. 1 A), Myo/V1 was predominantly observed in a wide area of the cytoplasm surrounding the nucleus. Further analysis with confocal microscopy revealed that native Myo/V1 was also present in the nucleus to a lesser extent (spotted green fluorescence in Fig.1 C). Upon treatment with TNF for 1 h, Myo/V1 was found to cluster around the perinuclear region in the cytoplasm. Additionally, Myo/V1 was found to increase slightly within the nucleus (green FITC masking the blue DAPI nuclear staining, resulting in pale blue nucleus) suggesting migration of Myo/V1 to the nucleus (Fig.1 B). Because TNF is known to reorganize the cytoskeleton (54Koukouritaki S.B. Vardaki E.A. Papakonstanti E.A. Lianos E. Stournaras C. Emmanouel D.S. Mol. Med. 1999; 5: 382-392Crossref PubMed Google Scholar, 55Yan S.R. Fumagalli L. Dusi S. Berton G. J. Leukoc. Biol. 1995; 58: 595-606Crossref PubMed Scopus (59) Google Scholar, 56De Vos K. Goossens V. Boone E. Vercammen D. Vancompernolle K. Vandenabeele P. Haegeman G. Fiers W. Grooten J. J. Biol. Chem. 1998; 273: 9673-9680Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), the present observation of increased perinuclear clustering and nuclear migration of Myo/V1 in TNF-treated HeLa cells (Fig.1 B) suggests that Myo/V1 might be associated with the cytoskeleton and its associated organelles and might participate in the signal transduction process from these locations. Cellular partitioning studies were performed to determine the location of Myo/V1 under the NFκB inducing conditions. Cells were treated with TNF or TNF plus CHX for 2 h. Cytoplasmic, mitochondrial, and nuclear extracts were prepared and immunoblotted for Myo/V1 protein (Fig.2). Under basal conditions the majority of Myo/V1 was present in the cytoplasmic fraction (lane 5 in Fig. 2 B) and only a small quantity in the nuclear fraction (lane 3 in Fig. 2 B). After TNF stimulation for 2 h, Myo/V1 levels in the cytoplasmic fraction did not change significantly (data not shown). However, with TNF plus CHX stimulation, the levels of Myo/V1 reduced significantly in the cytoplasmic fraction compared with control (lanes 5 and 6 in Fig.2 B). Additionally, a simultaneous increase in the levels of Myo/V1 was observed in the nucleus (lane 4 in Fig.2 B), indicating that the translocated Myo/V1 migrated from cytoplasm. We did not observe any Myo/V1 in mitochondrial fractions (lanes 1 and 2 in Fig. 2 B). These data suggest that, under acute stress conditions, a major relocalization of Myo/V1 first occurs within the cytoplasm (Fig. 1 B), following which Myo/V1 is translocated to the nucleus. To confirm that Myo/V1 physically interacts with NFκB proteins, co-immunoprecipitation studies were conducted both in vitro and in vivo. Fig.3 A shows that, in vitro, p50, p65, and c-Rel proteins strongly interacted with Myo/V1 protein (lanes 2–4). To further confirm these physical interactions in vivo, HeLa cells were treated with diluent (lanes 8–10 in Fig. 3 B), TNF plus CHX (lanes 5–7 in Fig. 3 B), or phorbol ester (lanes 11–13 in Fig. 3 C) for 2 h or infected with AdMyo/V1 for 12 h (lanes 14and 15 in Fig. 3 C), cellular extracts were prepared, and co-immunoprecipitation experiments were conducted. In control diluent-treated cells, Myo/V1 did not associate with any of the NFκB proteins (lanes 8–10 in Fig. 3 B). However, in TNF plus CHX- or AdMyo/V1-treated cells, Myo/V1 predominantly associated with p65 protein (lane 7 in Fig.3 B and lane 15 in Fig. 3 C) but not with p50 protein (lane 6 in Fig. 3 B andlane 14 in Fig. 3 C). In phorbol ester-treated HeLa cells (Fig. 3 C), Myo/V1 co-immunoprecipitated with c-Rel and p65 protein (lanes 11 and12 in Fig. 3 C) compared with p50 protein (lane 13 in Fig. 3 C). Because our earlier studies indicated that Myo/V1 disrupts and induces the formation of new NFκB-DNA complexes (3Sivasubramanian N. Adhikary G. Sil P.C. Sen S. J. Biol. Chem. 1996; 271: 2812-2816Abstract Full Text Full Text PDF Pub" @default.
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• W2022267557 title "Myotrophin/V-1, a Protein Up-regulated in the Failing Human Heart and in Postnatal Cerebellum, Converts NFκB p50-p65 Heterodimers to p50-p50 and p65-p65 Homodimers" @default.
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