Role of a TRIM72 ADP-ribosylation cycle in myocardial injury and membrane repair
Hiroko Ishiwata-Endo, Jiro Kato, Akihiko Tonouchi, Youn Wook Chung, Junhui Sun, Linda A. Stevens, Jianfeng Zhu, Angel M. Aponte, Danielle A. Springer, Hong San, Kazuyo Takeda, Zu-Xi Yu, Victoria Hoffmann, Elizabeth Murphy, Joel Moss
Hiroko Ishiwata-Endo, Jiro Kato, Akihiko Tonouchi, Youn Wook Chung, Junhui Sun, Linda A. Stevens, Jianfeng Zhu, Angel M. Aponte, Danielle A. Springer, Hong San, Kazuyo Takeda, Zu-Xi Yu, Victoria Hoffmann, Elizabeth Murphy, Joel Moss
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Research Article Cardiology Muscle biology

Role of a TRIM72 ADP-ribosylation cycle in myocardial injury and membrane repair

Abstract

Mono-ADP-ribosylation of an (arginine) protein catalyzed by ADP-ribosyltransferase 1 (ART1) — i.e., transfer of ADP-ribose from NAD to arginine — is reversed by ADP-ribosylarginine hydrolase 1 (ARH1) cleavage of the ADP-ribose–arginine bond. ARH1-deficient mice developed cardiomyopathy with myocardial fibrosis, decreased myocardial function under dobutamine stress, and increased susceptibility to ischemia/reperfusion injury. The membrane repair protein TRIM72 was identified as a substrate for ART1 and ARH1; ADP-ribosylated TRIM72 levels were greater in ARH1-deficient mice following 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. Mutant TRIM72 (R207K, R260K) that is not ADP-ribosylated interfered with assembly of TRIM72 repair complexes at a site of laser-induced injury. The regulatory enzymes ART1 and ARH1 and their substrate TRIM72 were found in multiple complexes, which were coimmunoprecipitated from mouse heart lysates. In addition, 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.

Authors

Hiroko Ishiwata-Endo, Jiro Kato, Akihiko Tonouchi, Youn Wook Chung, Junhui Sun, Linda A. Stevens, Jianfeng Zhu, Angel M. Aponte, Danielle A. Springer, Hong San, Kazuyo Takeda, Zu-Xi Yu, Victoria Hoffmann, Elizabeth Murphy, Joel Moss

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Figure 2

Identification of ADP-ribosylated TRIM72 as an ARH1 substrate in mouse heart.

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Identification of ADP-ribosylated TRIM72 as an ARH1 substrate in mouse h...
(A) ADP-ribosylarginine content of ARH1-deficient (KO), heterozygous (HET), and WT mouse hearts. (B) Cardiac ADP-ribosyltransferase activity of ARH1-KO, HET and WT mice. (C) Identification of ADP-ribosylated TRIM72 in ARH1-KO mouse hearts. Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) image. Heart lysates from ARH1-KO mice were incubated with recombinant mouse ARH1 (Cy5, red) or PBS (Cy3, green). Two-dimensional gel separation, followed by Western blot (WB) images show TRIM72 status after treatment with urea, PBS, ARH1, NaCl, and NH2OH. (D) Active Af1521 macrodomain–GST (MacD), but not inactive Af1521 macrodomain–GST (IMD), bound to ADP-ribose (ADPr) on TRIM72. Af1521 macrodomain–GST (0.5 μmol/100 μl) or inactive Af1521 macrodomain–GST (0.5 μmol/100 μl) were preincubated for 1 hour at 4°C with ADP-ribose (1 μmol/100 μl) or PBS, then incubated with TCA-treated (Supplemental Figure 2) mouse heart lysates (500 μg/reaction) overnight at 4°C in a pull-down assay to concentrate ADP-ribosylated TRIM72 (ADPr-TRIM72), which then was identified by immunoreactivity using Western blots with anti-TRIM72 antibody. Box-and-whisker plots show median, first and third quartiles (boxes), and minimum and maximum values (whiskers). n = 3 in each group; *P < 0.05, **P < 0.01 vs. WT of same sex by 1-way ANOVA and Bonferroni’s post hoc test. Representative images of 3 or 4 independent experiments are shown.