Explanation Mono-ADP-ribosylation is a post-translational protein modification, in which ADP-ribose is transferred from NAD to an acceptor amino acid. It was first identified as a mechanism of disease pathogenesis in the bacterial diseases such as cholera, diphtheria and pertussis, where toxins ADP-ribosylate critical regulatory and biosynthetic proteins. Mammalian tissues have enzymatic activities that mimic those of the bacterial toxins. Mammalian arginine-specific mono-ADP-ribosylation however, is a reversible modification of protein. Arginine-specific mono-ADP-ribosyltrans- ferases (ARTs) (e.g., ART1, ART5), transfer ADP-ribose from NAD to arginine residues of target proteins and ADP-ribosylarginine hydrolase 1 (ARH1) reverses the reaction by cleaving the ADP-ribose-(arginine)-protein bond. Data are consistent with ART and ARH1 serving as opposing arms of an arginine ADP-ribosylation cycle. Significant advances have led to the identification of mono-ADP-ribosylated proteins. Reanalysis of phosphoproteomic data identified 79 mono-ADP-ribosylated proteins including a muscle-specific membrane-repair protein tripartite motif 72 (TRIM72), also known as Mitsugumin 53 (MG53). Another method involved enrichment of ADP-ribose-acceptor proteins using the ADP-ribose-binding macro domain protein Af1521. Similar to other post-translational modifications, e.g., phosphorylation, mono-ADP-ribosylation can alter protein function. TRIM72 is abundant in heart and skeletal muscle and in lung and kidney epithelial cells where it is involved in membrane repair. Effective membrane repair protects cardiomyocytes from ischemic damage. Oligomerization of TRIM72 was required acutely for membrane repair, bringing TRIM72 to the site of injury. Nitrosylation of TRIM72 protected it from degradation under H2O2-induced oxidative stress. TRIM72 also countered cell damage due to ischemia-reperfusion injury. TRIM72 in complex with caveolin-3 (Cav-3) activated phosphatidylinositol-3-kinases (PI3K)-dependent reperfusion injury salvage kinase (RISK), thereby enhancing cell survival. Overexpression of TRIM72 protected cells from plasma membrane damage in muscular dystrophy, as well as lung, kidney and heart injury, suggesting that TRIM72 could be a therapeutic target in diseases involving cell membrane injury. ARH1-deficient mice developed cardiomyopathy with myocardial fibrosis, decreased myocardial function under dobutamine stress, and increased susceptibility to ischemia-reperfusion injury. We hypothesized that a mono-ADP-ribosylated protein serving as an ART1 and ARH1 substrate was involved in preserving cardiac function. Indeed, membrane-repair protein, TRIM72, was identified as a substrate for ART1 and ARH. Further, we observed in ARH1-knockout mouse heart in vivo and in the Langendorff perfused-heart model ex vivo, that the amount of mono-ADP-ribosylated TRIM72 was increased in ischemia-reperfusion injury. To understand better the role of TRIM72 and ADP-ribosylation, we used C2C12 myocytes. ARH1 knockdown in C2C12 myocytes increased ADP-ribosylation of TRIM72, and delayed wound healing in a scratch assay. Indeed, interrupting the TRIM72 mono-ADP-ribosylation cycle by knockdown of ART1, ARH1, or TRIM72 delayed membrane repair and wound healing. Effects of overexpressing mutant TRIM72 that lacks two primary ADP-ribosylation sites (R207K, R260K) and is not ADP-ribosylated, had a similar effect. We used a laser-injury model to test the role of ADP-ribosylated TRIM72 on acute membrane repair. After laser injury of C2C12 myocyte membranes, live-cell imaging revealed rapid accumulation of WT TRIM72-GFP at the site, which was markedly lower in cells depleted of ARH1 or ART1. Moreover, accumulation of double mutant TRIM72 (R207K, R260K)-GFP at the injury site was significantly less than that of WT TRIM72-GFP. The regulatory enzymes ART1 and ARH1 and their substrate TRIM72 were found in complexes, which were co-immunoprecipitated from mouse heart lysates. The complexes found in association with TRIM72 appeared to be relatively heterogeneous and could be resolved by FPLC and HPLC. Some of the complexes included Cav-3. It has been reported that TRIM72 oligomerization serves as a key mechanism for regulation of acute membrane repair. Oligomerization of TRIM72, observed in WT mice, was delayed in ARH1-deficient, heart lysates, where a dimer of TRIM72 rather than a trimer was seen. In addition, at a cellular level, oligomerization of TRIM72 at the sites of injury required the presence of the mono-ADP-ribosylation cycle, that is, ART1 and ARH1. The mono-ADP-ribosylation inhibitors, vitamin K1 and novobiocin, inhibited oligomerization of TRIM72, the mechanism by which TRIM72 is recruited to the site of injury. We propose that a mono-ADP-ribosylation cycle involving recruitment of TRIM72 and other regulatory factors to sites of membrane damage is critical for membrane repair and wound healing following myocardial injury.

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