Arginylation regulates myofibrils to maintain heart function and prevent dilated cardiomyopathy

Abstract

Protein arginylation mediated by arginyltransferase (ATE1) is essential for heart formation during embryogenesis, however its cell-autonomous role in cardiomyocytes and the differentiated heart muscle has never been investigated. To address this question, we generated cardiac muscle-specific Ate1 knockout mice, in which Ate1 deletion was driven by α-myosin heavy chain promoter (αMHC-Ate1 mouse). These mice were initially viable, but developed severe cardiac contractility defects, dilated cardiomyopathy, and thrombosis over time, resulting in high rates of lethality after 6 months of age. These symptoms were accompanied by severe ultrastructural defects in cardiac myofibrils, seen in the newborns and far preceding the onset of cardiomyopathy, suggesting that these defects were primary and likely underlay the development of the future heart defects. Several major sarcomeric proteins were arginylated in vivo. Moreover, Ate1 deletion in the hearts resulted in a significant reduction of active and passive myofibril forces, suggesting that arginylation is critical for both myofibril structural integrity and contractility. Thus, arginylation is essential for maintaining the heart function by regulation of the major myofibril proteins and myofibril forces, and its absence in the heart muscle leads to progressive heart failure through cardiomyocyte-specific defects.

Analyzing N-terminal Arginylation through the Use of Peptide Arrays and Degradation Assays

“…If correct, this hypothesis may at least partly reconcile the direct and extensive discrepancy between the cited earlier reports (87)(88)(89)(90)(91)(92) and our arginylation data with peptide arrays and purified Ate1 (Figs. 2-5 and 8).…”

“…Abstract and Introduction, several publications, over the last decade, have suggested, largely on the basis of MS-based analyses of isolated cellular proteins (digested by trypsin or other proteases), that the arginylation of specific proteins or their natural fragments can involve, to a significant extent, non-canonical (non-Asp, non-Glu, non-Cys) N-terminal residues, including N-terminal Met, Leu, Phe, Val, Ala, Thr, Gly, Asn, Lys, and Pro (87)(88)(89)(90)(91)(92). In addition, a 2005 study (96) described a modified 13-residue neurotensin hormone that appeared to be arginylated, in vivo, at its internal Glu-4 residue.…”

“…Use of CelluSpots assays to address reports about arginylation of non-canonical N-terminal residues and specific internal residues of cellular proteins. A, list of 11-residue CelluSpots peptides whose sequences were identical to the N-terminal sequences of either a fulllength protein (item 2) or presumed natural fragments of other proteins (items 3-11) that have been reported to be Nt-arginylated by Ate1 at their non-canonical (non-Asp, non-Glu, non-Cys) N-terminal residues (87)(88)(89)(90)(91)(92). See "Results" for a detailed list of these peptides, which contains additional information and relevant references.…”

“…Several publications over the last decade, largely by the laboratory of A. Kashina, have reported that Ate1 R-transferase can also arginylate, to a significant extent, a variety of "noncanonical" (non-Asp, non-Glu, non-Cys) N-terminal residues in cellular proteins (87)(88)(89)(90)(91)(92). The same group described evidence that some isoforms of R-transferase were specific for substrates bearing N-terminal Cys (93).…”

Arginyltransferase ATE1 Catalyzes Midchain Arginylation of Proteins at Side Chain Carboxylates In Vivo

“…To test the possibility that addition of Arg to proteins can happen at internal sites and not on their N-termini, we performed mass spectrometry analysis of cell extracts and subcellular structures described in our prior studies (affinity enriched cell and embryo extracts (Wang et al, 2011), nuclear extracts (Saha et al, 2011), platelets, and myofibril preparations (Kurosaka et al, 2012)), to look for addition of Arg to any Asp, Glu, and Lys (the only three residues that can theoretically accept Arg directly on their side chain), using the search algorithms commonly applied to posttranslational modifications. While Lys searches did not yield any hits, we found a number of previously unidentified sites where Arg was added to mid-chain Asp and Glu (Fig.…”