Nuclear magnetic resonance assignment and secondary structure of an ankyrin‐like repeat‐bearing protein: Myotrophin

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Myotrophin, a 12-kDa ankyrin repeat protein, stimulates protein synthesis and cardiomyocyte growth to initiate cardiac hypertrophy by activating the NF-B signaling cascade. We found that, after internalization into myocytes, myotrophin cotranslocates into the nucleus with p65 to stimulate myocyte growth. We used structure-based mutations on the hairpin loops of myotrophin to determine the effect of the loops on myotrophin and p65 localization, induction of protein synthesis, and cardiac hypertrophy. Loop mutants, most prominently glutamic acid 33 3 alanine (E33A), stimulated protein synthesis much less than wild type. Myotrophin-E33A internalized into myocytes but did not translocate into the nucleus and failed to promote nuclear translocation of p65. In addition, two cardiac hypertrophy marker genes, atrial natriuretic factor and ␤-myosin heavy chain, were not up-regulated in E33A-treated cells. Myotrophin-induced myocyte growth and initiation of hypertrophy thus require nuclear co-translocation of myotrophin and p65, in a manner that depends crucially on the myotrophin hairpin loops.Myotrophin, a 12-kDa protein, is the smallest known ankyrin (ANK) 2 repeat protein (1) and contains four ANK repeats (2, 3). Myotrophin, also known as V-1 protein, was identified in spontaneously hypertensive rat hearts, cardiomyopathic human hearts (4), and neonatal rat cerebella (5). Myotrophin expression is ubiquitous in mammalian tissues (6). It is known to participate in the catecholamine synthesis signaling pathway (7), in regulation of actin polymerization (8), and in regulation of capping and uncapping by actin capping protein at the barbed ends of actin filaments (9). Myotrophin plays an important role in stimulation of cardiac myocyte growth and cardiac hypertrophy (4, 10). Sil et al. (11) reported an increase in myotrophin levels in human cardiomyopathic heart compared with normal heart. Overexpression of myotrophin in transgenic mice initiates cardiac hypertrophy that progresses toward heart failure (12, 13). Significantly, an increase in myotrophin level in blood plasma is observed after acute myocardial infarction (14) and in patients with heart failure (15). These data have established myotrophin as an initiator of cardiac hypertrophy and eventual heart failure.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. Separate studies have established the involvement of NF-B signaling in cardiac hypertrophy and dilated cardiomyopathy (17-19) and the importance of NF-B activation in the pathogenesis of cardiac remodeling and heart failure (20 -22). Recently, Gupta et al. (13) showed that progression of cardiac hypertrophy to heart failure is associated with...

Section: Expression and Purification Of Recombinant Myotrophin Proteimentioning

confidence: 99%

“…To dissect the structural basis of myotrophin function, we previously determined the solution structure of myotrophin (2,23), which consists of four ANK repeats. ANK repeat motifs are often involved in protein-protein and protein-nucleic acid interactions (24,25).…”

mentioning

confidence: 99%

Nuclear Co-translocation of Myotrophin and p65 Stimulates Myocyte Growth

Das

Gupta

Vasanji

et al. 2008

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“…7A) (11,12) and the three-dimensional structure of I B␣ (Fig. 7B) (13, 14), where NF B-interacting domains were well characterized.…”

Section: Three-dimensional Structural Alignment Of Myo/v1 With Rel-inmentioning

confidence: 99%

“…We recently reported that Myo/V1 protein exhibits significant homology to I B␣ protein and that Myo/V1 can disrupt the NF B-DNA complexes in vitro (3). Utilizing our recombinant Myo/V1 protein, the NMR structure of Myo/V1 was determined (11,12), and the ankyrin repeats of Myo/V1 exhibited structural features similar to those of I B␣ (13,14) at the three-dimensional level.…”

mentioning

confidence: 94%

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

Knuefermann¹,

Chen²,

Misra

et al. 2002

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.

“…A solution structure of V-1 shows two full ankyrin repeat motifs in tandem, with additional incomplete repeats at the amino and carboxyl termini (11,12). An ankyrin repeat consists of a ␤ hairpin loop followed by a pair of anti-parallel ␣ helices connected by a short turn sequence (13).…”

mentioning

confidence: 99%

Binding of Myotrophin/V-1 to Actin-capping Protein

Bhattacharya

Ghosh

Sept

et al. 2006

The heterodimeric actin-capping protein (CP) regulates actin assembly and cell motility by binding tightly to the barbed end of the actin filament. Here we demonstrate that myotrophin/V-1 binds directly to CP in a 1:1 molar ratio with a K d of 10 -50 nM. V-1 binding inhibited the ability of CP to cap the barbed ends of actin filaments. The actin-binding COOH-terminal region, the "tentacle," of the CP ␤ subunit was important for binding V-1, with lesser contributions from the ␣ subunit COOH-terminal region and the body of the protein. V-1 appears to be unable to bind to CP that is on the barbed end, based on the observations that V-1 had no activity in an uncapping assay and that the V-1⅐CP complex had no capping activity. Two loops of V-1, which extend out from the ␣-helical backbone of this ankyrin repeat protein, were necessary for V-1 to bind CP. Parallel computational studies determined a bound conformation of the ␤ tentacle with V-1 that is consistent with these findings, and they offered insight into experimentally observed differences between the ␣1 and ␣2 isoforms as well as the mutant lacking the ␣ tentacle. These results support and extend our "wobble" model for CP binding to the actin filament, in which the two COOH-terminal regions of CP bind independently to the actin filament, and bound CP is able to wobble when attached only via its mobile ␤-subunit tentacle. This model is also supported by molecular dynamics simulations of CP reported here. The existence of the wobble state may be important for actin dynamics in cells.Myotrophin is a 12-kDa protein identified in hypertrophied rat hearts (1) and dilated cardiomyopathic human hearts (2). Myotrophin was named for its ability to stimulate hypertrophy in cardiac myocytes when added to cells in culture (1). Transgenic mice overexpressing myotrophin in cardiac myocytes develop cardiac hypertrophy and heart failure (3). Myotrophin was found to be identical to the protein V-1 (4), which had been identified based on increased expression in granule cell neurons during development of the rat cerebellum (5). V-1 expression correlates with morphogenetic changes of cerebellar differentiation. V-1 is expressed in many vertebrate cells and tissues (6).V-1 homologues are present and highly conserved across vertebrates, and similar protein sequences are predicted by the genomes of other animal species, but not by those of fungi or plants. V-1 is important for expression of catecholamine-synthesizing enzymes (7), differentiation and regeneration of skeletal muscle (8), folliculogenesis and corpus luteum formation in the ovary (9), and regulation of insulin secretion (10). We will refer to the myotrophin/V-1 protein as V-1 for simplicity.Structurally, V-1 is a small ankyrin repeat protein. A solution structure of V-1 shows two full ankyrin repeat motifs in tandem, with additional incomplete repeats at the amino and carboxyl termini (11,12). An ankyrin repeat consists of a ␤ hairpin loop followed by a pair of anti-parallel ␣ helices connected by a short turn sequence ...