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

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 B (NF B)-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 NF B activation. Three-dimensional alignment studies indicate that myotrophin/V-1 resembles a truncated I B␣ without the signal response domain (SRD) and PEST domains. Co-immunoprecipitation studies reveal that myotrophin/V-1 interacts with NF B proteins in vitro; however, it remains physically associated only with p65 and c-Rel proteins in vivo during NF B 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 Ad␤gal-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 NF B 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 B-luciferase reporter activity. Because myotrophin/V-1 is found at elevated levels during NF B 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 NF B dimers during this period.

An Unconventional Antigen Translated by a Novel Internal Ribosome Entry Site Elicits Antitumor Humoral Immune Reactions

“…1A). Myotrophin interacts with rel/NF-B (35,36) and converts NF-B p50-p65 heterodimers to homodimers (37). In contrast to DC6 encoded by the cDNA sense strand of tumor Ag CML66 that we previously reported (19), the coding sequence of the isolated clone was located in the mRNA-sense strand of myotrophin mRNA; however, the coding sequence was in the 3Ј-UTR (bp 3061-3237) of myotrophin mRNA (35) (Fig.…”

Transglutaminase-Catalyzed Site-Specific Conjugation of Small-Molecule Probes to Proteins in Vitro and on the Surface of Living Cells

“…After Ni-NTA purification to remove excess benzophenone, the protein was irradiated with UV light for 7 min at 4 °C, in the presence or absence of DNA (0.18 μM) or myotrophin (360 μM; 90 equivalents over p50). 26 The products were separated on 10 % SDS-PAGE and blotted with anti-p50 antibody. Formation of p50 dimer (MW 78.5 kD) is seen in lanes 8-10, but not in others where the alanine mutant was used, gpTGase was omitted, or the samples were not UVirradiated.…”

Nuclear Co-translocation of Myotrophin and p65 Stimulates Myocyte Growth

“…Myotrophin has been shown to interact with NF-B proteins (6,16), and myotrophin-induced myocyte growth appears to be mediated through activation of the NF-B pathway (10), although the details of these interactions are poorly characterized. Knuefermann et al (16) showed that myotrophin physically associates with p65 and prevents the reassociation of the p65-p50 heterodimer, resulting in activation of the NF-B pathway.…”

“…Knuefermann et al (16) showed that myotrophin physically associates with p65 and prevents the reassociation of the p65-p50 heterodimer, resulting in activation of the NF-B pathway. Separate studies have established the involvement of NF-B signaling in cardiac hypertrophy and dilated cardiomyopathy (17)(18)(19) and the importance of NF-B activation in the pathogenesis of cardiac remodeling and heart failure (20 -22).…”