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Sulfoquinovosyl-diacylglycerol (SQDG) is one of the four lipids present in the thylakoid membranes. Depletion of SQDG causes different degrees of effects on photosynthetic growth and activities in different organisms. Four SQDG molecules bind to each monomer of photosystem II (PSII), but their role in PSII function has not been characterized in detail, and no PSII structure without SQDG has been reported. We analyzed the activities of PSII from an SQDG-deficient mutant of the cyanobacterium Thermosynechococcus elongatus by various spectroscopic methods, which showed that depletion of SQDG partially impaired the PSII activity by impairing secondary quinone (Q B ) exchange at the acceptor site. We further solved the crystal structure of the PSII dimer from the SQDG deletion mutant at 2.1 Å resolution and found that all of the four SQDG-binding sites were occupied by other lipids, most likely PG molecules. Replacement of SQDG at a site near the head of Q B provides a possible explanation for the Q B impairment. The replacement of two SQDGs located at the monomer-monomer interface by other lipids decreased the stability of the PSII dimer, resulting in an increase in the amount of PSII monomer in the mutant. The present results thus suggest that although SQDG binding in all of the PSII-binding sites is necessary to fully maintain the activity and stability of PSII, replacement of SQDG by other lipids can partially compensate for their functions., (© 2018 Nakajima et al.)

Interferon-α (IFN-α) is used clinically to treat hepatocellular carcinoma (HCC), although the detailed therapeutic mechanisms remain elusive. In particular, IFN-α has long been implicated in control of the cell cycle, but its actual point of action has not been clarified. Here, using time lapse imaging analyses of the human HCC cell line HuH7 carrying a fluorescence ubiquitination-based cell cycle indicator (Fucci), we found that IFN-α induced cell cycle arrest in the G0/G1 phases, leading to apoptosis through an IFN-α type-2 receptor (IFNAR2)-dependent signaling pathway. Detailed analyses by time lapse imaging and biochemical assays demonstrated that the IFN-α/IFNAR2 axis sensitizes cells to apoptosis in the S/G2/M phases in preparation for cell death in the G0/G1 phases. In summary, this study is the first to demonstrate the detailed mechanism of IFN-α as an anticancer drug, using Fucci-based time lapse imaging, which will be informative for treating HCC with IFN-α in clinical practice., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

A zinc finger protein GATA4 is one of the hypertrophy-responsive transcription factors and forms a complex with an intrinsic histone acetyltransferase, p300. Disruption of this complex results in the inhibition of cardiomyocyte hypertrophy and heart failure in vivo. By tandem affinity purification and mass spectrometric analyses, we identified cyclin-dependent kinase-9 (Cdk9) as a novel GATA4-binding partner. Cdk9 also formed a complex with p300 as well as GATA4 and cyclin T1. We showed that p300 was required for the interaction of GATA4 with Cdk9 and for the kinase activity of Cdk9. Conversely, Cdk9 kinase activity was required for the p300-induced transcriptional activities, DNA binding, and acetylation of GATA4. Furthermore, the kinase activity of Cdk9 was required for the phosphorylation of p300 as well as for cardiomyocyte hypertrophy. These findings demonstrate that Cdk9 forms a functional complex with the p300/GATA4 and is required for p300/GATA4- transcriptional pathway during cardiomyocyte hypertrophy.

Differentiation of embryonic stem (ES) cells into cardiac myocytes requires activation of a cardiac-specific gene program. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) govern gene expression patterns by being recruited to target genes through association with specific transcription factors. One of the HATs, p300, serves as a coactivator of cardiac-specific transcription factors such as GATA-4. The HAT activity of p300 is required for acetylation and DNA binding of GATA-4 and its full transcriptional activity as well as for promotion of a transcriptionally active chromatin configuration. However, the roles of HATs and HDACs in post-translational modification of GATA-4 during the differentiation of ES cells into cardiac myocytes remain unknown. In an ES cell model of developing embryoid bodies, an acetylated form of GATA-4 and its DNA binding increased concomitantly with the expression of p300 during the differentiation of ES cells into cardiac myocytes. Treatment of ES cells with trichostatin A (TSA), a specific HDAC inhibitor, induced acetylation of histone-3/4 near GATA sites within the atrial natriuretic factor promoter. In addition, TSA augmented the increase in an acetylated form of GATA-4 and its DNA binding during the ES cell differentiation. Finally, TSA facilitated the expression of green fluorescence protein under the control of the cardiac-specific Nkx-2.5 promoter and of endogenous cardiac beta-myosin heavy chain during the differentiation. These findings demonstrate that acetylation of GATA-4 as well as of histones is involved in the differentiation of ES cells into cardiac myocytes.

A multizinc finger protein, FOG-2, associates with a cardiac transcription factor, GATA-4, and represses GATA-4-dependent transcription. GATA-4 is required not only for normal heart development but is also involved in hypertrophic responses in cardiac myocytes; however, the effects of FOG-2 on these responses are unknown. The interaction of GATA-4 with a transcriptional coactivator p300 is required for its full transcriptional activity and the activation of the embryonic program during myocardial cell hypertrophy. We show here that exogenous FOG-2 represses phenylephrine-induced hypertrophic responses such as myofibrillar organization, increases in cell size, and hypertrophy-associated gene transcription. Using immunoprecipitation Western blotting, we demonstrate that FOG-2 physically interacted with p300 and reduced the binding of GATA-4 to p300. In addition, in COS7 cells, in which the function of endogenous p300 is disrupted, FOG-2 is unable to repress the GATA-4-dependent transcriptional activities; however, FOG-2 markedly repressed the p300-mediated increase in the DNA-binding and transcriptional activities of GATA-4 in these cells. Similarly, FOG-2 inhibited a phenylephrine-induced increase in the p300/GATA-4 interaction, the GATA-4/DNA-binding, and transcriptional activities of GATA-4-dependent promoters in cardiac myocytes as well. These findings demonstrate that FOG-2 represses hypertrophic responses in cardiac myocytes and that p300 is involved in these repressive effects.

Although somatic stem cells have been reported to exist in various adult organs, there have been few reports concerning stem cells in the heart. We here demonstrate that Sca-1-positive (Sca-1+) cells in adult hearts have some of the features of stem cells. Sca-1+ cells were isolated from adult murine hearts by a magnetic cell sorting system and cultured on gelatin-coated dishes. A fraction of Sca-1+ cells stuck to the culture dish and proliferated slowly. When treated with oxytocin, Sca-1+ cells expressed genes of cardiac transcription factors and contractile proteins and showed sarcomeric structure and spontaneous beating. Isoproterenol treatment increased the beating rate, which was accompanied by the intracellular Ca(2+) transients. The cardiac Sca-1+ cells expressed oxytocin receptor mRNA, and the expression was up-regulated after oxytocin treatment. Some of the Sca-1+ cells expressed alkaline phosphatase after osteogenic induction and were stained with Oil-Red O after adipogenic induction. These results suggest that Sca-1+ cells in the adult murine heart have potential as stem cells and may contribute to the regeneration of injured hearts.

Substrate specificity of condensing enzymes is a predominant factor determining the nature of fatty acyl chains synthesized by type II fatty acid synthase (FAS) enzyme complexes composed of discrete enzymes. The gene (mtKAS) encoding the condensing enzyme, beta-ketoacyl-[acyl carrier protein] (ACP) synthase (KAS), constituent of the mitochondrial FAS was cloned from Arabidopsis thaliana, and its product was purified and characterized. The mtKAS cDNA complemented the KAS II defect in the E. coli CY244 strain mutated in both fabB and fabF encoding KAS I and KAS II, respectively, demonstrating its ability to catalyze the condensation reaction in fatty acid synthesis. In vitro assays using extracts of CY244 containing all E. coli FAS components, except that KAS I and II were replaced by mtKAS, gave C(4)-C(18) fatty acids exhibiting a bimodal distribution with peaks at C(8) and C(14)-C(16). Previously observed bimodal distributions obtained using mitochondrial extracts appear attributable to the mtKAS enzyme in the extracts. Although the mtKAS sequence is most similar to that of bacterial KAS IIs, sensitivity of mtKAS to the antibiotic cerulenin resembles that of E. coli KAS I. In the first or priming condensation reaction of de novo fatty acid synthesis, purified His-tagged mtKAS efficiently utilized malonyl-ACP, but not acetyl-CoA as primer substrate. Intracellular targeting using green fluorescent protein, Western blot, and deletion analyses identified an N-terminal signal conveying mtKAS into mitochondria. Thus, mtKAS with its broad chain length specificity accomplishes all condensation steps in mitochondrial fatty acid synthesis, whereas in plastids three KAS enzymes are required.

The low molecular weight GTPase Rho mediates a variety of cytoskeleton-dependent cell functions and stretch- and G(q) protein-induced hypertrophic responses in cardiac myocytes. Although ROCK, one of Rho's effectors, has been suggested to mediate hypertrophic signals, the relationship of Rho/ROCK with downstream signals is unknown. A zinc finger transcription factor, GATA-4, is activated by extracellular signal-regulated kinase 1/2 and is required for the up-regulation of the endothelin-1 gene during myocardial cell hypertrophy. However, it is unknown whether Rho/ROCK signals are linked to downstream GATA-4. By transient transfection assays using a dominant-negative mutant and an activated derivative of ROCK-I, we showed that ROCK-I participates in GATA-4-dependent endothelin-1 transcription. Inhibition of the Rho/ROCK pathway by Y-27632, a selective inhibitor of ROCK, suppressed phenylephrine-stimulated phosphorylation of extracellular signal-regulated kinase 1/2 and increased the DNA binding activity of cardiac GATA-4. Interestingly, latrunculin B, which inhibits actin polymerization, also prevents phenylephrine-induced responses. These findings demonstrate that the Rho/ROCK pathway is linked to downstream GATA-4 via the activation of extracellular signal-regulated kinases during myocardial cell hypertrophy. The results also suggest that changes in actin dynamics provide a convergence point for Rho/ROCK to the downstream signals during this process.

Increases in the expression of endothelin-1 (ET-1) in cardiac myocytes play a critical role in the development of heart failure in vivo. Whereas norepinephrine (NE) is a potent inducer of ET-1 expression in cardiac myocytes, the signaling pathways that link NE to inducible cardiac ET-1 expression are unknown. Adrenergic stimulation results in an increase in intracellular calcium levels, which in turn activates calcineurin. Here, we have shown that stimulation with NE markedly increased the expression of the ET-1 gene in primary cardiac myocytes from neonatal rats. This increase was severely attenuated by a beta-adrenergic antagonist, metoprolol, but not by an alpha-adrenergic antagonist, prazosin. Consistent with these data, the beta-adrenergic agonist isoproterenol (ISO) activated the rat ET-1 promoter activity to an extent that was similar to NE. The ISO-stimulated increase in promoter activity was significantly inhibited by a Ca(2+)-antagonist, nifedipine, and an immunosuppressant, cyclosporin A, which blocks calcineurin. Mutation analysis indicated that the GATA4 binding site is required for ISO-responsive ET-1 transcription. Stimulation with ISO enhanced the interaction between NFATc and GATA4 in cardiac myocytes. Consistent with this interaction, overexpression of GATA4 and NFATc synergistically activated the ET-1 promoter. These findings demonstrate that NE-stimulated ET-1 expression in cardiac myocytes is mediated predominantly via a beta-adrenergic pathway, and that calcium-activated calcineurin-GATA4 plays a role in this process.

The mechanisms that regulate smooth muscle development and differentiation are poorly understood. Although recent studies have suggested the possible role of a zinc finger transcription factor, GATA-6, in the differentiation of vascular smooth muscle cells (VSMCs), the downstream gene targeted by GATA-6 is unknown. The expression of smooth muscle-myosin heavy chain (Sm-MHC) provides a highly specific marker for the differentiated phenotype of VSMCs as well as the smooth muscle cell lineage. Here, we show that GATA-6 bound to a GATA-like motif (-810/-805) within the rat Sm-MHC promoter in a sequence-specific manner and activated this promoter through this site. In addition, we show that the transcriptional coactivator p300 associated with GATA-6 during the transcription of the Sm-MHC gene. A p300/GATA-6 complex in VSMCs was up-regulated by induction of the quiescent phenotype. A wild-type E1A, which interferes with endogenous p300, but not a mutant E1A defective for p300 binding, markedly down-regulated the expression of endogenous Sm-MHC in quiescent-phenotype VSMCs. These studies provide the first identification of a functionally important GATA-6 binding site within a smooth muscle-specific promoter and suggest a role for p300 in the maintenance of the differentiated phenotype in VSMCs as a coactivator of GATA-6.

We have previously shown that human cullin-2 (Cul-2) is covalently modified at Lys-689 by NEDD8 (Wada, H., Yeh, E. T. H., and Kamitani, T. (1999) Biochem. Biophys. Res. Commun. 257, 100-105). Cul-2 has also been reported to form a multiprotein complex, Cul-2.VBC, with the von Hippel-Lindau tumor suppressor gene product (pVHL) and elongins B and C. In this study, using an in vivo coexpression system in COS cells, we show that NEDD8 conjugation to Cul-2 is promoted by coexpression with wild-type pVHL and elongins B and C. Interestingly, tumorigenic mutants and deletion mutants of pVHL, which are unable to form a Cul-2.VBC complex, do not have the activity to promote NEDD8 conjugation to Cul-2. These results suggest that the complex formation is required for NEDD8 conjugation to Cul-2. Furthermore, we used a pVHL-deficient cell line, 786-0, to show that Cul-2 is poorly but clearly conjugated by NEDD8, indicating that pVHL is not the only molecule that promotes NEDD8 conjugation to Cul-2. Taken together, the VBC complex appears to have ligase activity in the conjugation of NEDD8 to Cul-2.