Background-A number of distinct stress signaling pathways in myocardium cause cardiac hypertrophy and heart failure.Class II histone deacetylases (HDACs) antagonize several stress-induced pathways and hypertrophy. However, cardiac hypertrophy induced by transgenic overexpression of the homeodomain only protein, HOP, can be prevented by the nonspecific HDAC inhibitors trichostatin A and valproic acid, suggesting that alternate targets that oppose class II HDAC function might exist in myocardium. We tested the effects of several HDAC inhibitors, including a class I HDAC-selective inhibitor, SK-7041, on cardiac hypertrophy induced by angiotensin II (Ang II) treatment or aortic banding (AB). Methods and Results-Cardiac hypertrophy was induced by chronic infusion of Ang II or by AB in mice or rats and evaluated by determining the ratio of heart weight to body weight or to tibia length, cross-sectional area, or echocardiogram. Cardiac hypertrophy induced by Ang II or AB for 2 weeks was significantly reduced by simultaneous administration of trichostatin A, valproic acid, or SK-7041. Echocardiogram revealed that exaggerated left ventricular systolic dimensions were relieved by HDAC inhibitors. HDAC inhibitors partially reversed preestablished cardiac hypertrophy and improved survival of AB mice. The expressions of atrial natriuretic factor, ␣-tubulin, ␤-myosin heavy chain, and interstitial fibrosis were reduced by HDAC inhibition. Conclusions-These results suggest that the predominant effect of HDAC inhibition, mainly mediated by class I HDACs, is to prevent cardiac hypertrophy in response to a broad range of agonist and stretch stimuli.

Rationale: Histone deacetylases (HDACs) are closely involved in cardiac reprogramming. Although the functional roles of class I and class IIa HDACs are well established, the significance of interclass crosstalk in the development of cardiac hypertrophy remains unclear. Objective: Recently, we suggested that casein kinase 2α1–dependent phosphorylation of HDAC2 leads to enzymatic activation, which in turn induces cardiac hypertrophy. Here we report an alternative post-translational activation mechanism of HDAC2 that involves acetylation of HDAC2 mediated by p300/CBP-associated factor/HDAC5. Methods and Results: Hdac2 was acetylated in response to hypertrophic stresses in both cardiomyocytes and a mouse model. Acetylation was reduced by a histone acetyltransferase inhibitor but was increased by a nonspecific HDAC inhibitor. The enzymatic activity of Hdac2 was positively correlated with its acetylation status. p300/CBP-associated factor bound to Hdac2 and induced acetylation. The HDAC2 K75 residue was responsible for hypertrophic stress–induced acetylation. The acetylation-resistant Hdac2 K75R showed a significant decrease in phosphorylation on S394, which led to the loss of intrinsic activity. Hdac5, one of class IIa HDACs, directly deacetylated Hdac2. Acetylation of Hdac2 was increased in Hdac5-null mice. When an acetylation-mimicking mutant of Hdac2 was infected into cardiomyocytes, the antihypertrophic effect of either nuclear tethering of Hdac5 with leptomycin B or Hdac5 overexpression was reduced. Conclusions: Taken together, our results suggest a novel mechanism by which the balance of HDAC2 acetylation is regulated by p300/CBP-associated factor and HDAC5 in the development of cardiac hypertrophy.

The transcription of neuron-specific genes must be repressed in nonneuronal cells. REST͞NRSF is a transcription factor that restricts the expression of many neuronal genes through interaction with the neuron-restrictive silencer element at the promoter level. PAHX-AP1 is a neuronal gene that is developmentally up-regulated in the adult mouse brain but that has no functional NRSE motif in its 5 upstream sequence. Here, we report that the transcription factor AP4 and the corepressor geminin form a functional complex in which SMRT and histone deacetylase 3 are recruited. The functional complex represses PAHX-AP1 expression in nonneuronal cells and participates in regulating the developmental expression of PAHX-AP1 in the brain. This complex also serves as a transcriptional repressor of DYRK1A, a candidate gene for Down's syndrome. Furthermore, compared with that in normal fetal brain, the expression of AP4 and geminin is reduced in Down's syndrome fetal brain at 20 weeks of gestation age, at which time premature overexpression of dual-specificity tyrosine-phosphorylated and regulated kinase 1A (DYRK1A) is observed. Our findings indicate that AP4 and geminin act as a previously undescribed repressor complex distinct from REST͞NRSF to negatively regulate the expression of target genes in nonneuronal cells and suggest that the AP4 -geminin complex may contribute to suppressing the precocious expression of target genes in fetal brain.

Previously, the authors cloned and characterized murine brain-specific angiogenesis inhibitor 1 (mBAI1). In this study, the authors cloned mBAI2 and analyzed its functional characteristics. Northern and Western blot analyses demonstrated a unique developmental expression pattern of mBAI2 in the brain. The expression level of mBAI2 appeared to increase as the development of the brain progressed. Reverse transcription-polymerase chain reaction (RT-PCR) analyses demonstrated the existence of alternative splice variants of mBAI2, which were defective in parts of type I repeat of thrombospondin or the third cytoplasmic loop of the seven-span transmembrane domain that were considered essential to the functions of mBAI2. The expressions of spliced variants in the brain were differently regulated compared with wild-type mBAI2 during development and ischemic conditions. In situ hybridization analyses of the brain showed the same localization of BAI2 as BAI1, such as in most neurons of cerebral cortex. In the in vivo focal cerebral ischemia model and the in vitro hypoxic cell culture model with cobalt, BAI2 expression decreased after hypoxia and preceded the increased expression of vascular endothelial growth factor (VEGF). RT-PCR analysis of antisense BAI2 cDNA-transfected SHSY5Y cells showed an increased VEGF expression as well as a decreased BAI2 expression. Immunohistochemical study of focal ischemic cortex showed that the regional localization of decreased BAI2 was related to the formation of new vessels. These results suggest that the brain-specific developmental expression pattern of angiostatic BAI2 is correlated with the decreased neovascularization in the adult brain, and that angiostatic BAI2 participates in the ischemia-induced brain angiogenesis in concert with angiogenic VEGF.

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Murine brain-specific angiogenesis inhibitor 1 and 2 (mBAI1, mBAI2) are involved in angiogenesis after cerebral ischemia. In this study, mBAI3 was cloned and characterized. Northern and Western blot analyses demonstrated a unique developmental expression pattern in the brain. The level of mBAI3 in brain peaked 1 day after birth, unlike mBAI1 and mBAI2, which peaked 10 days after birth. In situ hybridization analyses of the brain showed the same localization of BAI3 as BAI1 and BAI2, which includes most neurons of cerebral cortex and hippocampus. In the in vivo focal cerebral ischemia model, BAI3 expression decreased from 0.5 h after hypoxia until 8 h, but returned to control level after 24 h. The expression of vascular endothelial growth factor following ischemia showed an inverse pattern. The decreased expressions of BAIs in high-grade gliomas were observed, but BAI3 expression was generally lower in malignant gliomas than in normal brain. Our results indicate that the expression and distribution of BAI3 in normal brain, but not its developmental expression, are very similar to those of BAI1 and BAI2, and that BAI3 may participate in the early phases of ischemia-induced brain angiogenesis and in brain tumor progression.

Pseudomonas putida contains an amine dehydrogenase that is called a quinohemoprotein as it contains a quinone and two hemes c as redox active groups. Amino acid sequence analysis of the smallest (8.5 kDa), quinone-cofactor-bearing subunit of this heterotrimeric enzyme encountered difficulties in the interpretation of the results at several sites of the polypeptide chain. As this suggested posttranslational modifications of the subunit, the structural genes for this enzyme were determined and mass spectrometric de novo sequencing was applied to several peptides obtained by chemical or enzymatic cleavage. In agreement with the interpretation of the X-ray electronic densities in the diffraction data for the holoenzyme, our results show that the polypeptide of the small subunit contains four intrachain cross-linkages in which the sulfur atom of a cysteine residue is involved. Two of these cross-linkages occur with the ␤-carbon atom of an aspartic acid, one with the ␥-carbon atom of a glutamic acid and the fourth with a tryptophanquinone residue, this adduct constituting the enzyme's quinone cofactor, CTQ. The thioether type bond in all four of these adducts has never been found in other proteins. CTQ is a novel cofactor in the series of the recently discovered quinone cofactors.A diversity of enzymes appears to be involved in amine oxidation, as reflected by the number of different cofactors found in the types established so far (1). Based on the natural electron acceptor used, a further distinction can be made between amine oxidases and amine dehydrogenases. Both classes convert amines into their corresponding aldehydes, but oxidases produce toxic peroxides, while the reducing equivalents in the case of dehydrogenases are directly transferred to the respiratory chain (2).Depending on the identity of their cofactor(s), amine dehydrogenases are subdivided into quinoproteins, flavoproteins, quinohemoproteins, and flavohemoproteins (2). Pseudomonas putida strain ATCC 12633, as well as strain IFO 15633, contain a novel type of amine dehydrogenase; a quinohemoprotein (QH-AmDH) 1 as a quinone compound is present in the small subunit and two heme c groups in the large subunit (3). Although the quinone cofactor was not liberated on denaturing the enzyme, spectroscopic data (4) already indicated that it is different from tryptophan tryptophylquinone (5), topaquinone (6), or lysine tyrosylquinone (7), forming part of the protein chain of several amine dehydrogenases (EC 1.4.99.3/4), several amine oxidases (EC 1.4.3.6), and protein-lysine 6-oxidase (EC 1.4.3.13), respectively.To reveal the identity of the quinone cofactor and its position in the protein, the genes for QH-AmDH were cloned and sequenced, and the small subunit subjected to chemical analysis. The latter was carried out in a combination of automated Edman degradation, mass spectrometry, liquid chromatography, and fragmentation MS applied to the underivatized and the derivatized form (the quinone cofactor converted into a hydrazone with a hydrazine) of the small sub...

The crystal structure of a quinohemoprotein amine dehydrogenase from Pseudomonas putida has been determined at 1.9-Å resolution. The enzyme comprises three non-identical subunits: a four-domain ␣-subunit that harbors a di-heme cytochrome c, a seven-bladed ␤-propeller ␤-subunit that provides part of the active site, and a small ␥-subunit that contains a novel crosslinked, proteinous quinone cofactor, cysteine tryptophylquinone. More surprisingly, the catalytic ␥-subunit contains three additional chemical cross-links that encage the cysteine tryptophylquinone cofactor, involving a cysteine side chain bridged to either an Asp or Glu residue all in a hitherto unknown thioether bonding with a methylene carbon atom of acidic amino acid side chains. Thus, the structure of the 79-residue ␥-subunit is quite unusual, containing four internal cross-links in such a short polypeptide chain that would otherwise be difficult to fold into a globular structure.Recently, a number of modified amino acids have been identified in proteins that are generated by post-translational oxidation or non-oxidation processes (1, 2). Such a controlled modification of a specific amino acid residue forming part of the active site provides catalytic power to the protein. In the case of a certain class of amine-oxidizing enzymes, depending on the enzyme concerned, oxidation of a specific tyrosine or tryptophan residue leads to the generation of a redox-active quinone cofactor: 2,4,5-trihydroxyphenylalanine quinone (topaquinone) (3), lysine tyrosylquinone (4), or tryptophan tryptophylquinone (TTQ) 1 (5). Together with several enzymes containing the first identified, non-proteinous quinone cofactor, pyrroloquinoline quinone (PQQ), the enzymes containing those cofactors constitute a quinoprotein family of enzymes (6).Quinohemoprotein amine dehydrogenases (QH-AmDH) from Gram-negative bacteria represent a new type in the quinoprotein class of amine-oxidizing enzymes because they contain not only a quinone but also one or two hemes as a redox active group (7, 8) providing an opportunity for intramolecular electron transfer. Intermolecular electron transfer from QHAmDH has been demonstrated with the natural electron acceptors azurin for the enzyme from Pseudomonas putida (7) and cytochrome c-550 for the enzyme from Paracoccus denitrificans (9). The structure of the presumed quinone cofactor in QH-AmDH remain to be settled, although biochemical and spectroscopic analyses have suggested the presence of a quinone group similar to, but not identical, with TTQ (7, 8).To identify the quinone cofactor and its position in the protein, we (10) have recently determined the primary structure of the quinone-containing small subunit of QH-AmDH from P. putida using a combination of automated Edman degradation and mass spectrometry, and we have also cloned the genes coding for the three subunits of the heterotrimeric enzyme. Although we initially encountered difficulties in the interpretation of the chemical and mass data, the progress in elucidating the crystal struct...