Posttranslational Arginylation Research Articles

Identification of an intrinsically disordered region (IDR) in arginyltransferase 1 (ATE1).

Arginyltransferase 1 (ATE1) catalyzes arginylation, an important post-translational modification (PTM) in eukaryotes that plays a critical role in cellular homeostasis. The disruption of ATE1 function is implicated in mammalian neurodegenerative disorders and cardiovascular maldevelopment, while post-translational arginylation has also been linked to the activities of several important human viruses such as SARS-CoV-2 and HIV. Despite the known significance of ATE1 in mammalian cellular function, past biophysical studies of this enzyme have mainly focused on yeast ATE1, leaving the mechanism of arginylation in mammalian cells unclear. In this study, we sought to structurally and biophysically characterize mouse (Mus musculus) ATE1. Using size-exclusion chromatography (SEC), small angle X-ray scattering (SAXS), and hydrogen deuterium exchange mass spectrometry (HDX-MS), assisted by AlphaFold modeling, we found that mouse ATE1 is structurally more complex than yeast ATE1. Importantly, our data indicate the existence of an intrinsically disordered region (IDR) in all mouse ATE1 splice variants. However, comparative HDX-MS analyses show that yeast ATE1 does not have such an IDR, consistent with prior X-ray, cryo-EM, and SAXS analyses. Furthermore, bioinformatics approaches reveal that mammalian ATE1 sequences, as well as in a large majority of other eukaryotes, contain an IDR-like sequence positioned in proximity to the ATE1 GNAT active-site fold. Computational analysis suggests that the IDR likely facilitates the formation of the complex between ATE1 and tRNAArg, adding a new complexity to ATE1 structure and providing new insights for future studies of ATE1 functions.

Top-down Proteomics for the Characterization and Quantification of Calreticulin Arginylation.

Arginylation installed by arginyltransferase 1 (ATE1) features an addition of arginine (Arg) to the reactive amino acids (e.g., Glu and Asp) at the protein N-terminus or side chain. Systemic removal of arginylation after ATE1 knockout (KO) in mouse models resulted in heart defects leading to embryonic lethality. The biological importance of arginylation has motivated the discovery of arginylation sites on proteins using bottom-up approaches. While bottom-up proteomics is powerful in localizing peptide arginylation, it lacks the ability to quantify proteoforms at the protein level. Here we developed a top-down proteomics workflow for characterizing and quantifying calreticulin (CALR) arginylation. To generate fully arginylated CALR (R-CALR), we have inserted an R residue after the signaling peptide (AA1-17). Upon overexpression in ATE1 KO cells, CALR and R-CALR were purified by affinity purification and analyzed by LCMS in positive mode. Both proteoforms showed charge states ranging from 27-68 with charge 58 as the most intense charge state. Their MS2 spectra from electron-activated dissociation (EAD) showed preferential fragmentation at the protein N-terminals which yielded sufficient c ions facilitating precise localization of the arginylation sites. The calcium-binding domain (CBD) gave minimum characteristic ions possibly due to the abundant presence of >100 D and E residues. Ultraviolet photodissociation (UVPD) compared with EAD and ETD significantly improved the sequence coverage of CBD. This method can identify and quantify CALR arginylation at absence, endogenous (low), and high levels. To our knowledge, our work is the first application of top-down proteomics in characterizing post-translational arginylation in vitro and in vivo.

Iron-sulfur clusters are involved in post-translational arginylation

Eukaryotic arginylation is an essential post-translational modification that modulates protein stability and regulates protein half-life. Arginylation is catalyzed by a family of enzymes known as the arginyl-tRNA transferases (ATE1s), which are conserved across the eukaryotic domain. Despite their conservation and importance, little is known regarding the structure, mechanism, and regulation of ATE1s. In this work, we show that ATE1s bind a previously undiscovered [Fe-S] cluster that is conserved across evolution. We characterize the nature of this [Fe-S] cluster and find that the presence of the [Fe-S] cluster in ATE1 is linked to its arginylation activity, both in vitro and in vivo, and the initiation of the yeast stress response. Importantly, the ATE1 [Fe-S] cluster is oxygen-sensitive, which could be a molecular mechanism of the N-degron pathway to sense oxidative stress. Taken together, our data provide the framework of a cluster-based paradigm of ATE1 regulatory control.

Abstract 1553: Structural and Regulatory Elements of Post-Translational Arginylation

Abstract 1553: Structural and Regulatory Elements of Post-Translational Arginylation

The Structure of Saccharomyces cerevisiae Arginyltransferase 1 (ATE1)

Eukaryotic post-translational arginylation, mediated by the family of enzymes known as the arginyltransferases (ATE1s), is an important post-translational modification that can alter protein function and even dictate cellular protein half-life. Multiple major biological pathways are linked to the fidelity of this process, including neural and cardiovascular developments, cell division, and even the stress response. Despite this significance, the structural, mechanistic, and regulatory mechanisms that govern ATE1 function remain enigmatic. To that end, we have used X-ray crystallography to solve the crystal structure of ATE1 from the model organism Saccharomyces cerevisiae ATE1 (ScATE1) in the apo form. The three-dimensional structure of ScATE1 reveals a bilobed protein containing a GCN5-related N-acetyltransferase (GNAT) fold, and this crystalline behavior is faithfully recapitulated in solution based on size-exclusion chromatography-coupled small angle X-ray scattering (SEC-SAXS) analyses and cryo-EM 2D class averaging. Structural superpositions and electrostatic analyses point to this domain and its domain-domain interface as the location of catalytic activity and tRNA binding, and these comparisons strongly suggest a mechanism for post-translational arginylation. Additionally, our structure reveals that the N-terminal domain, which we have previously shown to bind a regulatory [Fe-S] cluster, is dynamic and disordered in the absence of metal bound in this location, hinting at the regulatory influence of this region. When taken together, these insights bring us closer to answering pressing questions regarding the molecular-level mechanism of eukaryotic post-translational arginylation.

The evolutionarily conserved arginyltransferase 1 mediates a pVHL-independent oxygen-sensing pathway in mammalian cells

The response to oxygen availability is a fundamental process concerning metabolism and survival/death in all mitochondria-containing eukaryotes. However, the known oxygen-sensing mechanism in mammalian cells depends on pVHL, which is only found among metazoans but not in other species. Here, we present an alternative oxygen-sensing pathway regulated by ATE1, an enzyme ubiquitously conserved in eukaryotes that influences protein degradation by posttranslational arginylation. We report that ATE1 centrally controls the hypoxic response and glycolysis in mammalian cells by preferentially arginylating HIF1α that is hydroxylated by PHD in the presence of oxygen. Furthermore, the degradation of arginylated HIF1α is independent of pVHL E3 ubiquitin ligase but dependent on the UBR family proteins. Bioinformatic analysis of human tumor data reveals that the ATE1/UBR and pVHL pathways jointly regulate oxygen sensing in a transcription-independent manner with different tissue specificities. Phylogenetic analysis suggests that eukaryotic ATE1 likely evolved during mitochondrial domestication, much earlier than pVHL.

Arginyltransferase1 mediates a pVHL‐independent oxygen sensing pathway in mammalian cells

The cellular response to oxygen availability is a fundamental process in all eukaryotes that regulates energy metabolism and death/survival decisions.The mechanisms for oxygen sensing likely co-evolved with the domestication of mitochondria as an adaptation to the oxygenation of the earth atmosphere. However, in mammalian cells, the only known oxygen sensing mechanism depends on the Von Hippel–Lindau tumor suppressor protein (pVHL), which is only found in metazoans but not in other species. This discrepancy points to a potential missing link in oxygen sensing pathways. In this study, we found that oxygen sensing in mammalian cells is also regulated by Arginyltransferase 1 (ATE1), which is ubiquitously conserved in eukaryotes and influences the degradation of many proteins by posttranslational arginylation via the principle of N-end rule. We report that ATE1 centrally controls the hypoxic response and glycolysis in mammalian cells by arginylating Hypoxia-inducible factor 1a (HIF1a), and subsequently regulating HIF1a signaling pathways in a transcription-independent manner. Moreover, we find that ATE1-mediated N-arginylation of HIF1a is sensitive to its oxygen-dependent hydroxylation mediated by the oxygen sensor prolyl hydroxylase (PHD). However, ATE1 promotes HIF1a degradation independently of the conventional pVHL E3 ubiquitin ligase pathway, but depending on typical N-end degradation machineries, the UBR E3 Ubiquitin ligase family. Bioinformatic analysis of human tumor data reveal that the ATE1/UBR and the pVHL pathway jointly regulate oxygen sensing in vivo with tissue specificities. Finally, a phylogenetic analysis suggests that ATE1 was derived from mitochondrial gene transfer and involved in oxygen-sensing in multiple lineages of eukaryotes. Our findings present an unexplored role of ATE1-mediated arginylation as a novel pathway for cellular oxygen sensing and adaptation in mammalian cells.

Regulation of Mitochondrial Respiratory Chain Complex Levels, Organization, and Function by Arginyltransferase 1

Arginyltransferase 1 (ATE1) is an evolutionary-conserved eukaryotic protein that localizes to the cytosol and nucleus. It is the only known enzyme in metazoans and fungi that catalyzes posttranslational arginylation. Lack of arginylation has been linked to an array of human disorders, including cancer, by altering the response to stress and the regulation of metabolism and apoptosis. Although mitochondria play relevant roles in these processes in health and disease, a causal relationship between ATE1 activity and mitochondrial biology has yet to be established. Here, we report a phylogenetic analysis that traces the roots of ATE1 to alpha-proteobacteria, the mitochondrion microbial ancestor. We then demonstrate that a small fraction of ATE1 localizes within mitochondria. Furthermore, the absence of ATE1 influences the levels, organization, and function of respiratory chain complexes in mouse cells. Specifically, ATE1-KO mouse embryonic fibroblasts have increased levels of respiratory supercomplexes I+III2+IVn. However, they have decreased mitochondrial respiration owing to severely lowered complex II levels, which leads to accumulation of succinate and downstream metabolic effects. Taken together, our findings establish a novel pathway for mitochondrial function regulation that might explain ATE1-dependent effects in various disease conditions, including cancer and aging, in which metabolic shifts are part of the pathogenic or deleterious underlying mechanism.

ATE1-Mediated Post-Translational Arginylation Is an Essential Regulator of Eukaryotic Cellular Homeostasis.

Arginylation is a protein post-translational modification catalyzed by arginyl-tRNA transferases (ATE1s), which are critical enzymes conserved across all eukaryotes. Arginylation is a key step in the Arg N-degron pathway, a hierarchical cellular signaling pathway that links the ubiquitin-dependent degradation of a protein to the identity of its N-terminal amino acid side chain. The fidelity of ATE1-catalyzed arginylation is imperative, as this post-translational modification regulates several essential biological processes such as cardiovascular maturation, chromosomal segregation, and even the stress response. While the process of ATE1-catalyzed arginylation has been studied in detail at the cellular level, much remains unknown about the structure of this important enzyme, its mechanism of action, and its regulation. In this work, we detail the current state of knowledge on ATE1-catalyzed arginylation, and we discuss both ongoing and future directions that will reveal the structural and mechanistic details of this essential eukaryotic cellular regulator.

TRNAArg-Derived Fragments Can Serve as Arginine Donors for Protein Arginylation

Arginyltransferase ATE1 mediates posttranslational arginylation and plays key roles in multiple physiological processes. ATE1 utilizes arginyl (Arg)-tRNAArg as the donor of Arg, putting this reaction into a direct competition with the protein synthesis machinery. Here, we address the question of ATE1- Arg-tRNAArg specificity as a potential mechanism enabling this competition invivo. Using invitro arginylation assays and Ate1 knockout models, we find that, in addition to full-length tRNA, ATE1 is also able to utilize short tRNAArg fragments that bear structural resemblance to tRNA-derived fragments (tRF), a recently discovered class of small regulatory non-coding RNAs with global emerging biological role. Ate1 knockout cells show a decrease in tRFArg generation and a significant increase in the ratio of tRNAArg:tRFArg compared with wild type, suggesting a functional link between tRFArg and arginylation. We propose that generation of physiologically important tRFs can serve as a switch between translation and protein arginylation.

Posttranslational Arginylation Enzyme Arginyltransferase1 Shows Genetic Interactions With Specific Cellular Pathways in vivo.

Arginyltransferase1 (ATE1) is a conserved enzyme in eukaryotes mediating posttranslational arginylation, the addition of an extra arginine to an existing protein. In mammals, the dysregulations of the ATE1 gene (ate1) is shown to be involved in cardiovascular abnormalities, cancer, and aging-related diseases. Although biochemical evidence suggested that arginylation may be involved in stress response and/or protein degradation, the physiological role of ATE1 in vivo has never been systematically determined. This gap of knowledge leads to difficulties for interpreting the involvements of ATE1 in diseases pathogenesis. Since ate1 is highly conserved between human and the unicellular organism Schizosaccharomyces pombe (S. pombe), we take advantage of the gene-knockout library of S. pombe, to investigate the genetic interactions between ate1 and other genes in a systematic and unbiased manner. By this approach, we found that ate1 has a surprisingly small and focused impact size. Among the 3659 tested genes, which covers nearly 75% of the genome of S. pombe, less than 5% of them displayed significant genetic interactions with ate1. Furthermore, these ate1-interacting partners can be grouped into a few discrete clustered categories based on their functions or their physical interactions. These categories include translation/transcription regulation, biosynthesis/metabolism of biomolecules (including histidine), cell morphology and cellular dynamics, response to oxidative or metabolic stress, ribosomal structure and function, and mitochondrial function. Unexpectedly, inconsistent to popular belief, very few genes in the global ubiquitination or degradation pathways showed interactions with ate1. Our results suggested that ATE1 specifically regulates a handful of cellular processes in vivo, which will provide critical mechanistic leads for studying the involvements of ATE1 in normal physiologies as well as in diseased conditions.

Post-translational N-terminal Arginylation of Protein Fragments: A Pivotal Portal to Proteolysis.

The majority of tRNA studies has focused on tRNA molecules as pivotal player in the fundamental process of protein synthesis. Mounting studies have unveiled further functions for tRNA beyond protein synthesis, including non-ribosomal amino acid transfer, and regulation of targeted proteolysis. Post-translational N-terminal arginylation of protein fragments, a non-ribosomal amino acid transfer, is one of the crucial ways by which tRNA participates in various protein degradation trajectories and influences global cellular functions. Previous studies demonstrated a role of arginylation by arginyltransferases (ATEs) in protein degradation, autophagy, and cell death in mammalian cells. Notably, recent investigations in plants have revealed some of the crucial aspects regarding the biochemical nature of Nterminal arginylation and some of its physiological roles. Herein, we review some of the key data on Nterminal arginylation of protein fragments with respect to targeted proteolysis in mammalian cells. Future mechanistic studies using state of the art approaches and physiologically-relevant cellular models are warranted to enhance our molecular understanding of this important yet enigmatic protein modification.

Abstract A039: Arginyltransferase Ate1 reduction drives prostate cancer metastasis

Abstract This study demonstrates that diminished Arginyltransferase (Ate1) expression in prostate cancer is sufficient to drive progression and distal metastasis, as shown via in vitro and in vivo models, and in correlation analysis with human patient samples. While prostate cancer affects a large population of men, it is usually a dormant disease and is only lethal in the small subpopulation in which distal metastasis occurs. Unfortunately, distal metastasis is currently difficult to predict and treat. Understanding what provokes metastasis in prostate cancer is therefore clinically important. Ate1 is the enzyme solely responsible for mediating post-translational protein arginylation in most eukaryotic cells, including mammalian cells. Previous studies show that a complete loss of Ate1 in yeast and mammalian cells increases resistance to stress-induced cell death and promotes genomic instability. Ate1 loss further promotes cancer-relevant phenotypes in mouse fibroblasts, such as persisting cellular replication despite stress or cell-cell contacts. Furthermore, a lower level of Ate1 appears to correlate with shorter survival in patients with several types of cancer, including prostate cancer. However, the role of Ate1 in progression and metastasis of prostate cancer (or any cancer) remained unknown. Here we demonstrate in prostate-relevant models that a reduction of Ate1 drives prostate cancer progression and metastasis. We first examined Ate1’s impact on stress-induced cell death on prostate cancer cells in vitro in models relevant to the tumor microenvironment and cancer treatments. PC-3 and LNCaP prostate cancer cells with shRNA targeting Ate1 (or nontargeting control) were stressed with cellular oxidant hydrogen peroxide, gamma radiation, and apoptotic inducer staurosporine. Consistent with our hypothesis, the knockdown of Ate1 resulted in less cell death in these cells. Interestingly, while a reduction of Ate1 in these cell lines does not affect proliferation, it does cause an increase in anchorage-independent growth in LNCaP cells via the soft agar assay, and an increase in PC-3 cell invasion through Matrigel. Additional analysis of prostate cancer cell lines PC-3, LNCaP, metastatic PC-3-derivative PC3-ML, metastatic LNCaP-derivative C4-2B, and metastatic line DU145 showed a distinct and significant relationship between increased malignancy and decreased Ate1 expression. Next, to study the effect of Ate1 on metastasis in vivo, PC-3 cells expressing luciferase and with shRNA targeting Ate1 (or control) were injected into prostates of immune-compromised (nu/nu) mouse (orthotopic xenografts). PC3-ML, a derivative of PC-3 known to be metastasis-competent, was used as a positive metastatic control. We used luciferin luminescence to measure tumor growth and the presence of local invasions to adjacent tissues such as seminal vesicles and testicles, as well as metastasis to distal sites including lymph nodes, spleen, and lung. Sixty days after tumor inoculation, all three cell types consistently formed similar primary tumors but varied greatly in their metastatic behaviors. The PC-3 cells with nontargeting control shRNA largely failed to metastasize to distal sites and only occasionally exhibited local invasion, as expected from previous reports. However, PC-3 cells with Ate1 downregulated by shRNA consistently formed distal metastases in lungs, spleen, and lymph nodes with similar frequency to the positive control, PC3-ML. As further support for Ate1’s role in prostate cancer progression, results from data mining and immune-histologic examination of human tissue samples revealed a consistent downregulation of Ate1 protein and mRNA during prostate cancer progression and metastasis, offering promising value of predicting metastatic events before they occur. In conclusion, Ate1 reduction may be a potent indicator of prostate cancer progression, and provide insight into the molecular mechanism of prostate cancer metastasis. Citation Format: Michael D. Birnbaum, Ning Zhou, Kerry L. Burnstein, Fangliang Zhang. Arginyltransferase Ate1 reduction drives prostate cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A039.

Posttranslational arginylation enzyme Ate1 regulates cell death through a mitochondrial‐dependent pathway

Arginyltransferase1 (Ate1) is an evolutionarily conserved enzyme that mediates posttranslational addition of one extra arginine to a protein substrate in a process called arginylation. Recent evidence suggests that Ate1 is essential for normal stress response. The level of Ate1 and total arginylation activity appear to increase follow acute stress and leads to cell death. Vice versa, the removal or reduction of this enzyme minimized cellular sensitivity to high doses of common cellular stressors such as oxidants, radiations, heavy metal, and high temperature. All these evidence suggest that Ate1 is a master regulator of cell death/growth. However, how does Ate1 accomplish this role remain unknown. In this study, we found that Ate1 regulates cell death in a mitochondrial‐dependent manner. By using S.cerevisiae, which contains a conserved Ate1 gene, we found that during acute stress, Ate1 is highly recruited to mitochondria. By using recombinant Ate1 that is bound to cell membrane, we found that the ability of locating to mitochondria is essential for Ate1‐mediated cell death. By screening of dozens of other players with known roles in cell death, we discovered that Ate1‐mediated cell death appears to be independent of the pan‐caspase in yeast, the mitochondrial membrane potential, or the ubiquitin‐proteasome system in general. Instead, the Ate1‐mediated cell death is dependent on the integrity of the mitochondrial pore complex and the existence of apoptosis‐inducing factor (AIF), a mitochondrial protein that is often released into cytosol during mitochondrial stress. Consistently, the Ate1‐mediated cell death can be at least partially blocked by the over‐expression of mammalian pro‐survival protein bcl2, which is known to block the formation of mitochondrial permeability transition pore. Finally, in mammalian cells, where the intrinsic and extrinsic cell‐death pathways are well‐separated, we found that the absence of Ate1 only has impacts on intrinsic pathway that is mainly mediated by mitochondria, but has little effects on cell‐death triggered by death receptors such as TNF‐R.In summary, our study has discovered a novel cell death mechanism that is regulated by a posttranslational modification enzyme.Support or Funding InformationNIGNMS R01 GM107333DoD (CDMRP), Idea Award, PC140622This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Posttranslational arginylation enzyme Ate1 is a mitochondrial‐derived master regulator that coordinates glycolysis and respiration in the Warburg effect

In the Warburg effect observed in vast majority of cancer cells, glycolysis is preferred over mitochondrial respiration. However, the role of mitochondria and its relationship with glycolysis in cancer metabolism is poorly understood. Arginyltransferase 1(Ate1) is an evolutionarily conserved enzyme that exist in most eukaryotes and a large subset of bacteria. In eukaryotes, this enzyme mediates arginylation, the posttranslational addition of one extra arginine to a protein. Recently, Ate1 was identified to be down‐regulated in many types of cancer but the significance of such observation remained unclear. Here, by examining classic glycolytic markers such glucose intake rate, sensitivity to glucose starvation, lactate generation, and the effects of glycolysis inhibitors including 2‐DG and 2‐FDG, we found that a deletion of Ate1 gene is sufficient to causes the Warburg effect on the cell level. This effect was reproduced by shRNA‐mediated knockdown of Ate1 and can be reversed by the expression of a recombinant Ate1 in the KO cells. In a screening of typical players involved in glycolysis, we found that the deletion of Ate1 increases of the protein level of hypoxia‐inducible factor 1a (HIF1a), a protein with known role in glycolysis. We found that the knockdown of HIF1a in Ate1‐KO cells was sufficient to reverse the glycolytic phenoytype in these cells, suggesting Ate1 regulates glycolysis through HIF1a. Furthermore, we found that Ate1 regulates the protein level of HIF1a through both conventional and unconventional pathways. In particularly, HIF1a appears to be a previous unknown substrate of Ate1‐mediated arginylation, which promotes the degradation of HIF1a through the N‐end rule. In addition, the deletion of Ate1 generate prominent effects on the morphology and functions of mitochondria, with significant defect on the assembly of mitochondrial complex II and a consequential accumulation of succinate, which is expected to stabilize the HIF1a protein by an inhibition of the PHD proteins. Finally, we found that eukaryotic Ate1 has significant homology to its counterpart in alpha‐proteobacteria, the ancestor of mitochondria. Also, although the majority of Ate1 is located in the cytosol, a detectable portion of Ate1 is located inside mitochondria. These evidence suggest that the Ate1 enzyme was derived from a gene transfer during the domestication of mitochondria, which further explains the previous unknown role of Ate1 as a master regulator of the balance between glycolysis and mitochondrial function.In summary, our study discovered a novel regulator of cell metabolism with a evolution root concomitant with mitochondria and our data suggest this regulator coordinates the balance between glycolysis and mitochondrial respiration in the cancer Warburg effect. Our data also reveal the existence of a non‐canonical degradation pathway of HIF1a.Support or Funding InformationNIGMS R01 GM107333This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Posttranslational arginylation enzyme Ate1 affects DNA mutagenesis by regulating stress response

Arginylation is a protein posttranslational modification (PTM) which leads to addition of an extra arginine at the N‐terminus of the target proteins and peptides. This process is evolutionarily conserved, and is mediated by arginyltransferase1 (Ate1). While earlier studies found that the overall arginylation activity often increases during stressing conditions including heat shock, oxidative stress, and injury, little is known about the role of arginylation or Ate1 in stress response. Here we report that arginylation promotes growth arrest and cell death, depending on the nature and intensity of the stressing factor. Specifically, in yeast, mouse and human cells, deletion or downregulation of the ATE1 gene disrupts typical stress responses by bypassing growth arrest and suppressing cell death events under stressing conditions such as oxidative stress, heat shock, and osmotic stresses, as well as the exposure to heavy metals or radiation. Conversely, in wild‐type cells with normal stress response, there is an increase of cellular Ate1 protein level and arginylation activity. Furthermore, the increase of Ate1 protein directly promotes cell death in a manner dependent on its arginylation activity. Finally, we found that Ate1 is required to suppress mutation frequency in yeast and mammalian cells during DNA‐damaging conditions such as ultraviolet irradiation. Our data thereby explain the basis for the involvement of arginylation in physiological and diseased conditions by its regulation of cell death and/or mutagenesis. We also uncover a novel mechanism by which a posttranslational modification exerts a global effect on mutagenesis.

Posttranslational arginylation enzyme Ate1 affects DNA mutagenesis by regulating stress response.

Arginyltransferase 1 (Ate1) mediates protein arginylation, a poorly understood protein posttranslational modification (PTM) in eukaryotic cells. Previous evidence suggest a potential involvement of arginylation in stress response and this PTM was traditionally considered anti-apoptotic based on the studies of individual substrates. However, here we found that arginylation promotes cell death and/or growth arrest, depending on the nature and intensity of the stressing factor. Specifically, in yeast, mouse and human cells, deletion or downregulation of the ATE1 gene disrupts typical stress responses by bypassing growth arrest and suppressing cell death events in the presence of disease-related stressing factors, including oxidative, heat, and osmotic stresses, as well as the exposure to heavy metals or radiation. Conversely, in wild-type cells responding to stress, there is an increase of cellular Ate1 protein level and arginylation activity. Furthermore, the increase of Ate1 protein directly promotes cell death in a manner dependent on its arginylation activity. Finally, we found Ate1 to be required to suppress mutation frequency in yeast and mammalian cells during DNA-damaging conditions such as ultraviolet irradiation. Our study clarifies the role of Ate1/arginylation in stress response and provides a new mechanism to explain the link between Ate1 and a variety of diseases including cancer. This is also the first example that the modulation of the global level of a PTM is capable of affecting DNA mutagenesis.

Post-translational protein arginylation in the normal nervous system and in neurodegeneration.

Post-translational arginylation of proteins is an important regulator of many physiological pathways in cells. This modification was originally noted in protein degradation during neurodegenerative processes, with an apparently different physiological relevance between central and peripheral nervous system. Subsequent studies have identified a steadily increasing number of proteins and proteolysis-derived polypeptides as arginyltransferase (ATE1) substrates, including β-amyloid, α-synuclein, and TDP43 proteolytic fragments. Arginylation is involved in signaling processes of proteins and polypeptides that are further ubiquitinated and degraded by the proteasome. In addition, it is also implicated in autophagy/lysosomal degradation pathway. Recent studies using mutant mouse strains deficient in ATE1 indicate additional roles of this modification in neuronal physiology. As ATE1 is capable of modifying proteins either at the N-terminus or middle-chain acidic residues, determining which proteins function are modulated by arginylation represents a big challenge. Here, we review studies addressing various roles of ATE1 activity in nervous system function, and suggest future research directions that will clarify the role of post-translational protein arginylation in brain development and various neurological disorders. Arginyltransferase (ATE1), the enzyme responsible for post-translational arginylation, modulates the functions of a wide variety of proteins and polypeptides, and is also involved in the main degradation pathways of intracellular proteins. Regulatory roles of ATE1 have been well defined for certain organs. However, its roles in nervous system development and neurodegenerative processes remain largely unknown, and present exciting opportunities for future research, as discussed in this review.

Posttranslational Arginylation Regulates Striated Muscle Function.

In this article, we propose the hypothesis that the posttranslational arginylation of proteins, a process catalyzed by the enzyme arginyl-tRNA-transferase, regulates active and passive force generation in striated muscles. Specifically, we propose that proteins essential for muscle contraction and force production are regulated by arginylation, including myosin heavy chain, troponin, actin, and titin filaments.

Ate1‐mediated posttranslational arginylation is essential for stress response regulation and mutagenesis suppression

Arginylation is a posttranslational modification which leads to addition of an extra arginine to an existing peptide chain, thus changing the de facto primary sequence and the surface change. This process is evolutionarily conserved and mainly mediated by arginyltransferase1 (Ate1). While earlier studies found that the overall arginylation activity often increases during stressing conditions including heat shock, oxidative stress, and injury, little is known about the role of arginylation or Ate1 in these conditions. In our study by using multiple types of eukaryotic cells as test models, we found that Ate1/arginylation is essential for stress response, apoptosis and mutagenesis suppression. Knockout of Ate1 in cells leads to a retardation of stress response and the diminish of apoptosis and cell death during exposure of high doses of stressing factors including heat shock, oxidative and osmotic stress, heavy metal and radiation. We demonstrated that Ate1 and its arginylation activity are up‐regulated during stress and activate the intrinsic apoptotic pathway with dependence on the mitochondria‐related apoptosis‐inducing factor (Aif1), but not directly on the ubiquitin‐proteasome system or Caspase (Yca1). Moreover, over‐expression of Ate1 also leads to cell death which depends on its enzymatic activity. Furthermore, we found that Ate1/arginylation is required to suppress mutagenesis in the population during DNA damaging stress. Our data thereby explain the basis for the involvement of arginylation in physiological and diseased conditions by its regulation of cell death and/or mutagenesis. We also uncover a novel mechanism by which a posttranslational modification exerts a global effect on mutagenesis.Support or Funding InformationNIH R01GM107333DoD/CDMRP PC140622 and PC141013ACS IRG‐98‐277‐13Sylvester Comprehensive Cancer Center Developmental Grant