No abstract

Autocatalytic processing mediated by the carboxyterminal domain of the hedgehog (hh) protein precursor (Hh) generates an amino-terminal product that accounts for all known signaling activity. The role of autoprocessing biogenesis of the hh signal has been unclear, since a truncated unprocessed protein lacking all carboxy-terminal domain sequences retains signaling activity. Here, we present evidence that the autoprocessing reaction proceeds via an internal thioester intermediate and results in a covalent modification that increases the hydrophobic character of the signaling domain and influences its spatial and subcellular distribution. We demonstrate that truncated unprocessed amino-terminal protein causes embryonic mispatterning, even when expression is localized to cells that normally express Hh, thus suggesting a role for autoprocessing in spatial regulation of hh signaling. This type of processing also appears to operate in the biogenesis of other novel secreted proteins.

SR Ca2+ ATPase 2a (SERCA2a) is a critical ATPase responsible for Ca2+ re-uptake during excitation-contraction coupling. Impaired SR Ca2+ uptake resulting from decreased expression and reduced activity of SERCA2a is a hallmark of heart failure (HF)1. Accordingly, restoration of SERCA2a expression by gene transfer has proven to be effective in improving cardiac function in HF patients2 as well as in animal models3. The small ubiquitin-related modifier (SUMO) can be conjugated to lysine residues of target proteins4, which is involved in most cellular process5. Here, we show that SERCA2a is SUMOylated at lysine 480 and 585 and that this SUMOylation is essential for preserving SERCA2a ATPase activity and stability. The levels of SUMO1 and SUMOylation of SERCA2a itself were greatly reduced in failing hearts. SUMO1 restitution by adeno-associated virus-mediated gene delivery maintained protein abundance of SERCA2a and significantly improved cardiac function in HF mice. This effect was comparable to SERCA2a gene delivery. Moreover, SUMO1 overexpression in isolated cardiomyocytes augmented contractility and accelerated Ca2+ decay. Transgene-mediated SUMO1 overexpression rescued pressure overload-induced cardiac dysfunction concomitantly with increased SERCA2a function. By contrast, down-regulation of SUMO1 using shRNA accelerated pressure overload-induced deterioration of cardiac function and was accompanied by decreased SERCA2a function. However, knockdown of SERCA2a resulted in severe contractile dysfunction both in vitro and in vivo, which was not rescued by overexpression of SUMO1. Taken together, our data show that SUMOylation is a critical post-translational modification that regulates SERCA2a function and provides a platform for the design of novel therapeutic strategies for HF.

Heart failure is characterized by a debilitating decline in cardiac function1, and recent clinical trial results indicate that improving the contractility of heart muscle cells by boosting intracellular calcium handling might be an effective therapy2,3. microRNAs (miRs) are dysregulated with heart failure4,5 but whether they control contractility or constitute therapeutic targets remain speculative. Using high throughput, functional screening of the human microRNAome, we identified miRs that suppress intracellular calcium handling in heart muscle by interacting with mRNA encoding the sarcoplasmic reticulum calcium uptake pump SERCA2a. Of 875 miRs tested, miR-25 potently delayed calcium uptake kinetics in cardiomyocytes in vitro and was upregulated in heart failure, both in mice and humans. Whereas AAV9-mediated overexpression of miR-25 in vivo resulted in a significant loss of contractile function, injection of an antisense oligonucleotide (antagomiR) against miR-25 dramatically halted established heart failure in a mouse model, improving cardiac function and survival relative to a control antagomiR. These data reveal that increased expression of endogenous miR-25 contributes to declining cardiac function during heart failure and suggests that it might be targeted therapeutically to restore function.

Mitsugumin 53 (MG53) negatively regulates skeletal myogenesis by targeting insulin receptor substrate 1 (IRS-1). Here, we show that MG53 is a ubiquitin E3 ligase that induces IRS-1 ubiquitination with the help of an E2-conjugating enzyme UBE2H. Molecular manipulations that disrupt the E3 ligase function of MG53 abolishes IRS-1 ubiquitination and enhances skeletal myogenesis. Skeletal muscles derived from the MG53−/− mice show an elevated IRS-1 level with enhanced insulin signaling, which protects the MG53−/− mice from developing insulin resistance when challenged with a high fat/high sucrose diet. Muscle samples derived from human diabetic patients and mice with insulin resistance show normal expression of MG53, indicating that altered MG53 expression does not serve as a causative factor for the development of metabolic disorders. Thus, therapeutic interventions that target the interaction between MG53 and IRS-1 may be a novel approach for the treatment of metabolic diseases that are associated with insulin resistance.

Transition-metal-catalyzed activation of C-H and C-C bonds is a challenging area in synthetic organic chemistry. Among various methods to accomplish these processes, the approach using metal-organic cooperative catalytic systems is one of the most promising. In this protocol, organic molecules as well as transition metals act as catalysts to bring about reactions, which proceed with high efficiencies and selectivities. Various metal-organic cooperative catalytic systems developed for C-H and C-C bond activation reactions are discussed in this review. Also discussed are how each metal-organic cooperative catalyst affects the reaction mechanism and what kinds of substrates can be applied in each of the catalytic processes.

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BACKGROUND Cardiac fibrosis (CF) is associated with increased ventricular stiffness and diastolic dysfunction and is an independent predictor of long-term clinical outcomes of patients with heart failure (HF). We previously showed that the matricellular CCN5 protein is cardioprotective via its ability to inhibit CF and preserve cardiac contractility. OBJECTIVES This study examined the role of CCN5 in human heart failure and tested whether CCN5 can reverse established CF in an experimental model of HF induced by pressure overload. METHODS Human hearts were obtained from patients with end-stage heart failure. Extensive CF was induced by applying transverse aortic constriction for 8 weeks, which was followed by adeno-associated virus-mediated transfer of CCN5 to the heart. Eight weeks following gene transfer, cellular and molecular effects were examined. RESULTS Expression of CCN5 was significantly decreased in failing hearts from patients with end-stage heart failure compared to nonfailing hearts. Trichrome staining and myofibroblast content measurements revealed that the established CF had been reversed by CCN5 gene transfer. Anti-CF effects of CCN5 were associated with inhibition of the transforming growth factor beta signaling pathway. CCN5 significantly inhibited endothelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation, which are 2 critical processes for CF progression, both in vivo and in vitro. In addition, CCN5 induced apoptosis in myofibroblasts, but not in cardiomyocytes or fibroblasts, both in vivo and in vitro. CCN5 provoked the intrinsic apoptotic pathway specifically in myofibroblasts, which may have been due the ability of CCN5 to inhibit the activity of NFκB, an antiapoptotic molecule. CONCLUSIONS CCN5 can reverse established CF by inhibiting the generation of and enhancing apoptosis of myofibroblasts in the myocardium. CCN5 may provide a novel platform for the development of targeted anti-CF therapies.