Protein Arginylation Research Articles

Arginylation of Myosin Heavy Chain Regulates Skeletal Muscle Strength

Protein arginylation is a posttranslational modification with an emerging global role in the regulation of actin cytoskeleton. To test the role of arginylation in the skeletal muscle, we generated a mouse model with Ate1 deletion driven by the skeletal muscle-specific creatine kinase (Ckmm) promoter. Ckmm-Ate1 mice were viable and outwardly normal; however, their skeletal muscle strength was significantly reduced in comparison to controls. Mass spectrometry of isolated skeletal myofibrils showed a limited set of proteins, including myosin heavy chain, arginylated on specific sites. Atomic force microscopy measurements of contractile strength in individual myofibrils and isolated myosin filaments from these mice showed a significant reduction of contractile forces, which, in the case of myosin filaments, could be fully rescued by rearginylation with purified Ate1. Our results demonstrate that arginylation regulates force production in muscle and exerts a direct effect on muscle strength through arginylation of myosin.

Loss of ATE1-mediated arginylation leads to impaired platelet myosin phosphorylation, clot retraction, and in vivo thrombosis formation

Protein arginylation by arginyl-transfer RNA protein transferase (ATE1) is emerging as a regulator protein function that is reminiscent of phosphorylation. For example, arginylation of β-actin has been found to regulate lamellipodial formation at the leading edge in fibroblasts. This finding suggests that similar functions of β-actin in other cell types may also require arginylation. Here, we have tested the hypothesis that ATE1 regulates the cytoskeletal dynamics essential for in vivo platelet adhesion and thrombus formation. To test this hypothesis, we generated conditional knockout mice specifically lacking ATE1 in their platelets and in their megakaryocytes and analyzed the role of arginylation during platelet activation. Surprisingly, rather than finding an impairment of the actin cytoskeleton structure and its rearrangement during platelet activation, we observed that the platelet-specific ATE1 knockout led to enhanced clot retraction and in vivo thrombus formation. This effect might be regulated by myosin II contractility since it was accompanied by enhanced phosphorylation of the myosin regulatory light chain on Ser19, which is an event that activates myosin in vivo. Furthermore, ATE1 and myosin co-immunoprecipitate from platelet lysates. This finding suggests that these proteins directly interact within platelets. These results provide the first evidence that arginylation is involved in phosphorylation-dependent protein regulation, and that arginylation affects myosin function in platelets during clot retraction.

Calreticulin-dimerization induced by post-translational arginylation is critical for stress granules scaffolding

Protein arginylation mediated by arginyl-tRNA protein transferase is a post-translational modification that occurs widely in biology, it has been shown to regulate protein and properties and functions. Post-translational arginylation is critical for embryogenesis, cardiovascular development and angiogenesis but the molecular effects of proteins arginylated in vivo are largely unknown. In the present study, we demonstrate that arginylation reduces CRT (calreticulin) thermostability and induces a greater degree of dimerization and oligomerization. R-CRT (arginylated calreticulin) forms disulfide-bridged dimers that are increased in low Ca2+ conditions at physiological temperatures, a similar condition to the cellular environment that it required for arginylation of CRT. Moreover, R-CRT self-oligomerizes through non-covalent interactions that are enhanced at temperatures above 40°C, condition that mimics the heat shock treatment where R-CRT is the only isoespecies of CRT that associates in cells to SGs (stress granules). We show that in cells lacking CRT the scaffolding of larger SGs is impaired; the transfection with CRT (hence R-CRT expression) restores SGs assembly whereas the transfection with CRT mutated in Cys146 does not. Thus, R-CRT disulfide-bridged dimers (through Cys146) are essential for the scaffolding of larger SGs under heat shock, although these dimers are not required for R-CRT association to SGs. The alteration in SGs assembly is critical for the normal cellular recover of cells after heat induced stress.We conclude that R-CRT is emerging as a novel protein that has an impact on the regulation of SGs scaffolding and cell survival.

Arginylation regulates myofibrils to maintain heart function and prevent dilated cardiomyopathy

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 6months 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.

Contractile Properties of Myocardium Myofibrils Isolated from Mice with Heart-Specific Deletion of Arginyl-tRNA-Protein Transferase

Protein arginylation is a cellular process catalyzed by arginyl-tRNA-protein transferase (Ate1). Mice with the Ate1 knockout present high embryonic lethality and defects in cardiovascular development including myofibril disorganization, which may lead to heart disease. In this study we compared passive forces at sarcomere lengths (SL) between 1.8 and 2.6μm, and contractile properties of myofibrils isolated from the myocardium muscle conditional knockout mice with heart-specific deletion of Ate1 with those from wild-type (WT) mice. The maximal isometric forces were lower in Ate1 KO myofibrils (102.2 ± 11.0 nN/μm2) than in WT myofibrils (151.3 ± 11.7 nN/μm2), which was accompanied by a downwards shift in the passive force-SL curve, suggesting that Ate1 KO myofibrils are weaker and less stiff. The rate of force development (Kact) was similar between groups (Ate1 KO: 3.1 ± 0.4 sec−1, WT: 3.3 ± 0.5 sec−1). The rate of force redevelopment (Ktr) following a shortening-stretch protocol was also similar between groups (Ate1 KO: 4.7 ± 2.2 sec−1, WT: 6.6 ± 0.8 sec−1), which suggests that changes in force are not associated with cross-bridge kinetics and thin filament activation. The rates of relaxation upon muscle deactivation (Krel) was similar among groups at most SL investigated, although there was a tendency for Ate1 KO myofibrils to become slower when measurements were made at short SL (∼1.8μm). Overall, these results imply that arginylation is an important mechanism associated with force regulation in cardiac myofibrils, and it likely affects contractile proteins and passive structures within sarcomeres.

Arginylation and Methylation Double Up to Regulate Nuclear Proteins and Nuclear Architecture In Vivo

Protein arginylation and arginine methylation are two posttranslational modifications of emerging importance that involve Arg residues and their modifications. To test a hypothesis that posttranslationally added arginines can be methylated, we used high-precision mass spectrometry and metabolic labeling to find whether posttranslationally added arginines can serve as methylation sites. We identifieda number of proteins invivo, on which posttranslationally added Arg have undergone mono- and dimethylation. This double modification predominantly affects the chromatin-containing nuclear fraction and likely plays an important regulatory role in chromatin-associated proteins. Moreover, inhibition of arginylation and Arg methylation results ina significant reduction of the nucleus size in cultured cells, suggesting changes in chromatin compaction and nuclear architecture. Our findings suggesta functional link between protein regulation by arginylation and methylation that affects nuclear structure invivo.

Differential arginylation of actin isoforms is regulated by coding sequence-dependent degradation.

The mammalian cytoskeletal proteins β- and γ-actin are highly homologous, but only β-actin is amino-terminally arginylated in vivo, which regulates its function. We examined the metabolic fate of exogenously expressed arginylated and nonarginylated actin isoforms. Arginylated γ-actin, unlike β-, was highly unstable and was selectively ubiquitinated and degraded in vivo. This instability was regulated by the differences in the nucleotide coding sequence between the two actin isoforms, which conferred different translation rates. γ-actin was translated more slowly than β-actin, and this slower processing resulted in the exposure of a normally hidden lysine residue for ubiquitination, leading to the preferential degradation of γ-actin upon arginylation. This degradation mechanism, coupled to nucleotide coding sequence, may regulate protein arginylation in vivo.

Arginylation-Dependent Neural Crest Cell Migration Is Essential for Mouse Development

Coordinated cell migration during development is crucial for morphogenesis and largely relies on cells of the neural crest lineage that migrate over long distances to give rise to organs and tissues throughout the body. Recent studies of protein arginylation implicated this poorly understood posttranslational modification in the functioning of actin cytoskeleton and in cell migration in culture. Knockout of arginyltransferase (Ate1) in mice leads to embryonic lethality and severe heart defects that are reminiscent of cell migration–dependent phenotypes seen in other mouse models. To test the hypothesis that arginylation regulates cell migration during morphogenesis, we produced Wnt1-Cre Ate1 conditional knockout mice (Wnt1-Ate1), with Ate1 deletion in the neural crest cells driven by Wnt1 promoter. Wnt1-Ate1 mice die at birth and in the first 2–3 weeks after birth with severe breathing problems and with growth and behavioral retardation. Wnt1-Ate1 pups have prominent defects, including short palate and altered opening to the nasopharynx, and cranial defects that likely contribute to the abnormal breathing and early death. Analysis of neural crest cell movement patterns in situ and cell motility in culture shows an overall delay in the migration of Ate1 knockout cells that is likely regulated by intracellular mechanisms rather than extracellular signaling events. Taken together, our data suggest that arginylation plays a general role in the migration of the neural crest cells in development by regulating the molecular machinery that underlies cell migration through tissues and organs during morphogenesis.

Conditional Tek Promoter-Driven Deletion of Arginyltransferase in the Germ Line Causes Defects in Gametogenesis and Early Embryonic Lethality in Mice

Posttranslational protein arginylation mediated by Ate1 is essential for cardiovascular development, actin cytoskeleton functioning, and cell migration. Ate1 plays a role in the regulation of cytoskeleton and is essential for cardiovascular development and angiogenesis—capillary remodeling driven by in-tissue migration of endothelial cells. To address the role of Ate1 in cytoskeleton-dependent processes and endothelial cell function during development, we produced a conditional mouse knockout with Ate1 deletion driven by Tek endothelial receptor tyrosine kinase promoter expressed in the endothelium and in the germ line. Contrary to expectations, Tek-Ate1 mice were viable and had no visible angiogenesis-related phenotypes; however, these mice showed reproductive defects, with high rates of embryonic lethality in the second generation, at stages much earlier than the complete Ate1 knockout strain. While some of the early lethality originated from the subpopulation of embryos with homozygous Tek-Cre transgene—a problem that has not previously been reported for this commercial mouse strain—a distinct subpopulation of embryos had lethality at early post-implantation stages that could be explained only by a previously unknown defect in gametogenesis originating from Tek-driven Ate1 deletion in premeiotic germs cells. These results demonstrate a novel role of Ate1 in germ cell development.

Global Analysis of Posttranslational Protein Arginylation

Posttranslational arginylation is critical for embryogenesis, cardiovascular development, and angiogenesis, but its molecular effects and the identity of proteins arginylated in vivo are largely unknown. Here we report a global analysis of this modification on the protein level and identification of 43 proteins arginylated in vivo on highly specific sites. Our data demonstrate that unlike previously believed, arginylation can occur on any N-terminally exposed residue likely defined by a structural recognition motif on the protein surface, and that it preferentially affects a number of physiological systems, including cytoskeleton and primary metabolic pathways. The results of our study suggest that protein arginylation is a general mechanism for regulation of protein structure and function and outline the potential role of protein arginylation in cell metabolism and embryonic development.

Microglial expression of peptidylarginine deiminase 2 in the prenatal rat brain

Peptidylarginine deiminases (PADs) are Ca2t+-dependant post-translational modification enzymes that catalyze the citrullination of protein arginyl residues. PAD type 2 (PAD2) is thought to be involved in some processes of neurodegeneration and myelination in the central nervous system. In this study, we found PAD2-positive cells in rat cerebra in 19-to 21-day old embryos, i.e. at a developmental stage well before myelination begins. Most of the cells were microglial marker-positive cells found mainly in the prospective medulla, and others were microglial marker-negative cells found mainly in the prospective dentate gyrus of the hippocampus. The former seemed to be in an activated state as judged by morphological criteria. The specificity of the enzyme activity, immunoblotting and reverse transcriptase-polymerase chain reaction analyses revealed that these cells expressed PAD2 and not PAD1, PAD3 or PAD4. Our data is indicative of microglial expression of PAD2 in the prenatal developing cerebrum.

Molecular dissection of arginyltransferases guided by similarity to bacterial peptidoglycan synthases

Post-translational protein arginylation is essential for cardiovascular development and angiogenesis in mice and is mediated by arginyl-transfer RNA-protein transferases Ate1-a functionally conserved but poorly understood class of enzymes. Here, we used sequence analysis to detect the evolutionary relationship between the Ate1 family and bacterial FemABX family of aminoacyl-tRNA-peptide transferases, and to predict the functionally important residues in arginyltransferases, which were then used to construct a panel of mutants for further molecular dissection of mouse Ate1. Point mutations of the residues in the predicted regions of functional importance resulted in changes in enzymatic activity, including complete inactivation of mouse Ate1; other mutations altered its substrate specificity. Our results provide the first insights into the mechanisms of Ate1-mediated arginyl transfer reaction and substrate recognition, and define a new protein superfamily called Dupli-GNAT to reflect its origin by the duplication of the GNAT acetyltransferase domain.

Identification of mammalian arginyltransferases that modify a specific subset of protein substrates

Posttranslational N-terminal protein arginylation, mediated by Arg-tRNA-protein transferase 1 (ATE1), is essential for cardiovascular development and angiogenesis in mammals but is nonessential in yeast. Evidence suggests that many proteins are arginylated in vivo in both mammals and yeast; however, in yeast, N-terminal arginylation can occur only on proteins bearing an N-terminal Asp or Glu, whereas in mammals, N-terminal Cys residues are also arginylation targets, suggesting that Cys arginylation contributes to the essential role of ATE1 in mammals. To date, all of the characterized forms of ATE1 in yeast and mammals have been shown to arginylate only Asp and Glu, leaving open to speculation whether Cys arginylation is possible only through other components of mammalian arginylation machinery and whether Cys-specific forms of Arg-transferase exist in mammals. Here, we report the identification of two forms of Arg-transferase in mice that are specific for N-terminal Cys. We also show that the two previously identified mammalian forms of ATE1 can arginylate Cys-containing substrates in addition to Asp- and Glu-containing substrates. This finding provides insights into the significance of Cys-specific protein arginylation in mammals and suggests possibilities of the determinants of substrate specificity within the ATE1 molecule.

Post-translational arginylation of proteins in cultured cells.

The aim of this study was to analyze the N-terminal post-translational incorporation of arginine into cytosolic proteins from cultured cells and the in vitro incorporation of arginine into soluble proteins of PC12 cells after serum deprivation. Arginine incorporation was measured in the presence of protein synthesis inhibitors. None of the inhibitors used affected significantly the arginylation reaction while the novo synthesis of protein was reduced by 98%. Under these conditions, we found that of the total [14C]arginine incorporated into the proteins, around 20% to 40% was incorporated into the N-terminal position of soluble proteins by a post-translational mechanism. These results suggest that this post-translational aminoacylation may be a widespread reaction in neuronal and non-neuronal cells. We also found that in PC12 cells, the in vitro post-translational arginylation was 60% higher in apoptotic cells with respect to control cells. These findings suggest that the post-translational arginylation of proteins may be involved in programmed cell death.

What is the signal for the posttranslational arginylation of proteins?

The N-terminal, posttranslational arginylation of proteins is ubiquitous in eukaryotic cells. Previous experiments, using purified components of the reaction incubated in the presence of exogenous substrates, have shown that only those proteins containing acidic residues at their N-terminals are arginylation substrates. However, data from experiments that used crude extracts of brain and nerve as the source of the arginylating molecules, suggest that the in vivo targets for arginylation are more complex than those demonstrated using purified components. One of the proposed functions for arginylation is as a signal for protein degradation and proteins that have undergone oxidative damage have been shown to be rapidly degraded. In the present experiments we have tested the hypothesis that the presence of an oxidatively damaged residue in a protein is a signal for its arginylation. These experiments have been performed by adding synthetic oxidized peptides to crude extracts of rat brain, incubating them with [3H]Arg and ATP and assaying for arginylated peptides using RP-HPLC. Results showed that while the oxidized A-chain of insulin was arginylated in this system, confirming previous experiments, other peptides containing oxidized residues were not. When a peptide containing Glu in the N-terminus was incubated under the same conditions it too was not a substrate for arginylation. These findings show that neither the presence of an N-terminal acidic residue nor an oxidized residue alone are sufficient to signal arginylation. Thus, another feature of the oxidized A-chain of insulin is required for arginylation. That feature remains to be identified.

Posttranslational arginylation of soluble rat brain proteins after whole body hyperthermia

We have previously reported the posttranslational addition of [14C]-arginine in the N-terminus of several soluble rat brain proteins. One of these proteins was identified as the microtubule-associated protein, the stable tubule only polypeptide (STOP). However, despite the fact that the biological significance of arginylation is not completely understood, some evidence associates it with proteolysis via the ubiquitin pathway. Since this degradative via is exacerbated as a response to stress, we studied in vitro the posttranslational [14C]-arginylation of cytosolic brain proteins of rats subjected to hyperthermia in vivo. Immediately after subjecting the animals to hyperthermia, a minor reduction (16%) in the acceptor capacity of [14C]-arginine into proteins was observed in comparison with animals maintained at 28°C. However, in the animals allowed to recover for 3 h, an increase (46%) in the arginylation was observed concomitantly with a significant accumulation of the heat shock protein (70 kDa; hsp 70) when compared to the control animals. These findings suggest that the posttranslational arginylation of proteins participate in the heat shock response. The STOP protein of the soluble brain fraction of control animals, which in Western blot appears as a doublet band (125 and 130 kDa, respectively), is seen, after the hyperthermic treatment, as a single band of 125 kDa. The amount of 125 kDa protein, as well as the in vitro incorporation of [14C]-arginine, increases after hyperthermia in comparison with control animals. Following hyperthermic treatment, we observed a decrease in the amount of in vivo [35S]-methionine-labeled brain proteins. We speculate that, as observed for STOP protein, the increase in the degradation of protein that occurs in hyperthermia, would produce an increase in the amount of arginine acceptor proteins. J. Neurosci. Res. 56:85–92, 1999. © 1999 Wiley-Liss, Inc.

Posttranslational arginylation of soluble rat brain proteins after whole body hyperthermia

We have previously reported the posttranslational addition of [14C]-arginine in the N-terminus of several soluble rat brain proteins. One of these proteins was identified as the microtubule-associated protein, the stable tubule only polypeptide (STOP). However, despite the fact that the biological significance of arginylation is not completely understood, some evidence associates it with proteolysis via the ubiquitin pathway. Since this degradative via is exacerbated as a response to stress, we studied in vitro the posttranslational [14C]-arginylation of cytosolic brain proteins of rats subjected to hyperthermia in vivo. Immediately after subjecting the animals to hyperthermia, a minor reduction (16%) in the acceptor capacity of [14C]-arginine into proteins was observed in comparison with animals maintained at 28 degrees C. However, in the animals allowed to recover for 3 h, an increase (46%) in the arginylation was observed concomitantly with a significant accumulation of the heat shock protein (70 kDa; hsp 70) when compared to the control animals. These findings suggest that the posttranslational arginylation of proteins participate in the heat shock response. The STOP protein of the soluble brain fraction of control animals, which in Western blot appears as a doublet band (125 and 130 kDa, respectively), is seen, after the hyperthermic treatment, as a single band of 125 kDa. The amount of 125 kDa protein, as well as the in vitro incorporation of [14C]-arginine, increases after hyperthermia in comparison with control animals. Following hyperthermic treatment, we observed a decrease in the amount of in vivo [35S]-methionine-labeled brain proteins. We speculate that, as observed for STOP protein, the increase in the degradation of protein that occurs in hyperthermia, would produce an increase in the amount of arginine acceptor proteins.

Evidence that oxidized proteins are substrates for N-terminal arginylation.

While the posttranslational N-terminal arginylation of proteins has been demonstrated in a variety of eukaryotic cells including neurons and their axons, the targets of the reaction are poorly understood. Several lines of evidence suggest that arginylation may be a cytoprotective mechanism used by cells to target oxidatively damaged (and thus potentially toxic) proteins for degradation. In the present experiments, we have begun to test this hypothesis by incubating oxidized test proteins in a rat brain extract capable of arginylating endogenous proteins. Bovine serum albumin, pancreatic ribonuclease-A and the A-chain of insulin were chosen as test proteins and either oxidized by metal catalyzed oxidation or purchased in their oxidized forms and incubated with the extract and [3H]Arg. SDS PAGE of the incubation product showed [3H]Arg migrating with the oxidized forms of BSA and RNase but not with the un-oxidized form of BSA. Following incubation with the oxidized A-chain of insulin, analysis of the [3H]product by SDS PAGE and HPLC showed co-migration of [3H]Arg with A-chain standards and amino acid sequencing showed [3H]Arg at the N-terminus of the A-chain of insulin. The data suggest that oxidative damage to a protein may be a signal for its N-terminal arginylation.

Posttranslational arginylation of brain proteins.

The knowledge of brain protein metabolism is important in understanding nervous system brain function. Protein synthesis rates are high in young brain, decline rapidly at adult stages, and thereafter continue falling slowly with age. The breakdown of protein appears to follow a similar rate (1). Protein synthesis and degradation however, are only the two extremes of a complex phenomena which includes a variety of other protein modifications. Proteolytic cleavage is the most common covalent modification of proteins; probably all proteins that have been isolated were modified by proteolysis, since only few are found with the starting amino acid (methionine) attached. This suggests that most proteins were subject to one or more co- and/or posttranslational modifications (2). One of these posttranslational modifications is the arginylation of proteins, described 30 years ago, which now is being recognized as a widespread modification of proteins. In this review, the current status of posttranslational arginylation of brain proteins is discussed.

N-terminal arginylation of sciatic nerve and brain proteins following injury.

N-terminal protein arginylation has been demonstrated in vitro and in situ and has been reported to increase following injury to sciatic nerves of rats. The present study attempts to demonstrate these reactions in vivo by applying [3H]Arg to the cut end of sciatic nerves in anesthetized rats and assaying for N-terminal arginylation using Edman chemistry and acid precipitation of labeled proteins in the proximal nerve segment. No evidence was found for arginylation in an aqueous soluble fraction. However, N-terminal arginylation was detected in a urea soluble fraction at 2 hours after nerve crush. The data show that arginylation of rat sciatic nerve proteins occurs in vivo and suggest that the arginylated proteins formed an aqueous insoluble/urea soluble aggregate after arginylation. In other experiments, rat brains were injured and assayed for arginylation in vitro to test the hypothesis that injury causes an up-regulation of these reactions. Results showed an activation of the reaction at 2 hours post crush and indicate that increases in N-terminal arginylation are likely to be a general response to injury in nervous tissue.