Global Analysis of Post-Translational Side-Chain Arginylation Using Pan-Arginylation Antibodies.

Arginylation is a post-translational modification mediated by the arginyltransferase 1 (ATE1), which transfers the amino acid arginine to a protein or peptide substrate from a tRNA molecule. Initially, arginylation was thought to occur only on N-terminally exposed acidic residues, and its function was thought to be limited to targeting proteins for degradation. However, more recent data have shown that ATE1 can arginylate side chains of internal acidic residues in a protein without necessarily affecting metabolic stability. This greatly expands the potential targets and functions of arginylation, but tools for studying this process have remained limited. Here, we report the first global screen specifically for side-chain arginylation. We generate and validate "pan-arginylation" antibodies, which are designed to detect side-chain arginylation in any amino acid sequence context. We use these antibodies for immunoaffinity enrichment of side-chain arginylated proteins from wildtype and Ate1 knockout cell lysates. In this way, we identify a limited set of proteins that likely undergo ATE1-dependent side-chain arginylation and that are enriched in specific cellular roles, including translation, splicing, and the cytoskeleton.

Assaying for Arginyltransferase Activity and Specificity by Peptide Arrays.

Here, we describe arginylation assays performed on peptide arrays immobilized on cellulose membranes via chemical synthesis. In this assay, it is possible to simultaneously compare arginylation activity on hundreds of peptide substrates to analyze the specificity of arginyltransferase ATE1 toward its target site(s) and the amino acid sequence context. This assay was successfully employed in prior studies to dissect the arginylation consensus site and enable predictions of arginylated proteins encoded in eukaryotic genomes.

Analysis of Arginylated Peptides by Subtractive Edman Degradation.

During the early studies of N-terminal arginylation, Edman degradation was widely used to identify N-terminally added Arg on protein substrates. This old method is reliable, but highly depends on the purity and abundance of samples and can become misleading unless a highly purified highly arginylated protein can be obtained. Here, we report a mass spectrometry-based method that utilizes Edman degradation chemistry to identify arginylation in more complex and less abundant protein samples. This method can also apply to the analysis of other posttranslational modifications.

Identification of Arginylated Proteins by Mass Spectrometry.

Here, we describe the method for the identification of arginylated proteins by mass spectrometry. This method has been originally applied to the identification of N-terminally added Arg on proteins and peptides and then expanded to the side chain modification which has been recently described by our groups. The key steps in this method include the use of the mass spectrometry instruments that can identify peptides with very high pass accuracy (Orbitrap) and apply stringent mass cutoffs during automated data analysis, followed by manual validation of the identified spectra. These methods can be used with both complex and purified protein samples and, to date, constitute the only reliable way to confirm arginylation at a particular site on a protein or peptide.

Arginylation Regulates Cytoskeleton Organization and Cell Division and Affects Mitochondria in Fission Yeast.

Protein arginylation mediated by arginyltransferase Ate1 is a posttranslational modification of emerging importance implicated in the regulation of mammalian embryogenesis, the cardiovascular system, tissue morphogenesis, cell migration, neurodegeneration, cancer, and aging. Ate1 deletion results in embryonic lethality in mice but does not affect yeast viability, making yeast an ideal system to study the molecular pathways regulated by arginylation. Here, we conducted a global analysis of cytoskeleton-related arginylation-dependent phenotypes in Schizosaccharomyces pombe, a fission yeast species that shares many fundamental features of higher eukaryotic cells. Our studies revealed roles of Ate1 in cell division, cell polarization, organelle transport, and interphase cytoskeleton organization and dynamics. We also found a role of Ate1 in mitochondria morphology and maintenance. Furthermore, targeted mass spectrometry analysis of the total Sc. pombe arginylome identified a number of arginylated proteins, including those that play direct roles in these processes; lack of their arginylation may be responsible for ate1-knockout phenotypes. Our work outlines global biological processes potentially regulated by arginylation and paves the way to unraveling the functions of protein arginylation that are conserved at multiple levels of evolution and potentially constitute the primary role of this modification in vivo.

A new mechanism of fibronectin fibril assembly revealed by live imaging and super-resolution microscopy.

Fibronectin (Fn1) fibrils have long been viewed as continuous fibers composed of extended, periodically aligned Fn1 molecules. However, our live-imaging and single-molecule localization microscopy data are inconsistent with this traditional view and show that Fn1 fibrils are composed of roughly spherical nanodomains containing six to eleven Fn1 dimers. As they move toward the cell center, Fn1 nanodomains become organized into linear arrays, in which nanodomains are spaced with an average periodicity of 105±17 nm. Periodical Fn1 nanodomain arrays can be visualized between cells in culture and within tissues; they are resistant to deoxycholate treatment and retain nanodomain periodicity in the absence of cells. The nanodomain periodicity in fibrils remained constant when probed with antibodies recognizing distinct Fn1 epitopes or combinations of antibodies recognizing epitopes spanning the length of Fn1. Treatment with FUD, a peptide that binds the Fn1 N-terminus and disrupts Fn1 fibrillogenesis, blocked the organization of Fn1 nanodomains into periodical arrays. These studies establish a new paradigm of Fn1 fibrillogenesis. This article has an associated First Person interview with the first author of the paper.

Arginyl-tRNA-protein transferase 1 (ATE1) promotes melanoma cell growth and migration.

Arginyl-tRNA-protein transferase 1 (ATE1) catalyses N-terminal protein arginylation, a post-translational modification implicated in cell migration, invasion and the cellular stress response. Herein, we report that ATE1 is overexpressed in NRAS-mutant melanomas, while it is downregulated in BRAF-mutant melanomas. ATE1 expression was higher in metastatic tumours, compared with primary tumours. Consistent with these findings, ATE1 depletion reduced melanoma cell viability, migration and colony formation. Reduced ATE1 expression also affected cell responses to mTOR and MEK inhibitors and to serum deprivation. Among putative ATE1 substrates is the tumour suppressor AXIN1, pointing to the possibility that ATE1 may fine-tune AXIN1 function in melanoma. Our findings highlight an unexpected role for ATE1 in melanoma cell aggressiveness and suggest that ATE1 constitutes a potential new therapeutic target.

Cysteine-Based Mimic of Arginylation Reproduces Neuroprotective Effects of the Authentic Post-Translational Modification on α-Synuclein.

Arginylation is an understudied post-translational modification (PTM) involving the transfer of arginine to aspartate or glutamate sidechains in a protein. Among the targets of this PTM is α-synuclein (αS), a neuronal protein involved in regulating synaptic vesicles. The aggregation of αS is implicated in neurodegenerative diseases, particularly in Parkinson's disease, and arginylation has been found to protect against this pathological process. Arginylated αS has been studied through semisynthesis involving multipart native chemical ligation (NCL), but this can be very labor-intensive with low yields. Here, we present a facile way to introduce a mimic of the arginylation modification into a protein of interest, compatible with orthogonal installation of labels such as fluorophores. We synthesize bromoacetyl arginine and react it with recombinant, site-specific cysteine mutants of αS. We validate the mimic by testing the vesicle binding affinity of mimic-arginylated αS, as well as its aggregation kinetics and monomer incorporation into fibrils, and comparing these results to those of authentically arginylated αS produced through NCL. In cultured neurons, we compare the fibril seeding capabilities of preformed fibrils carrying a small percentage of arginylated αS. We find that, consistent with authentically arginylated αS, mimic-arginylated αS does not perturb the protein's native function but alters aggregation kinetics and monomer incorporation. Both mimic and authentically modified αS suppress aggregation in neuronal cells. Our results provide further insight into the neuroprotective effects of αS arginylation, and our alternative strategy to generate arginylated αS enables the study of this PTM in proteins not accessible through NCL.

Post-translational Modifications of the Protein Termini.

Post-translational modifications (PTM) involve enzyme-mediated covalent addition of functional groups to proteins during or after synthesis. These modifications greatly increase biological complexity and are responsible for orders of magnitude change between the variety of proteins encoded in the genome and the variety of their biological functions. Many of these modifications occur at the protein termini, which contain reactive amino- and carboxy-groups of the polypeptide chain and often are pre-primed through the actions of cellular machinery to expose highly reactive residues. Such modifications have been known for decades, but only a few of them have been functionally characterized. The vast majority of eukaryotic proteins are N- and C-terminally modified by acetylation, arginylation, tyrosination, lipidation, and many others. Post-translational modifications of the protein termini have been linked to different normal and disease-related processes and constitute a rapidly emerging area of biological regulation. Here we highlight recent progress in our understanding of post-translational modifications of the protein termini and outline the role that these modifications play in vivo.

Bio-Inspired Amphiphilic Block-Copolymers Based on Synthetic Glycopolymer and Poly(Amino Acid) as Potential Drug Delivery Systems.

In this work, a method to prepare hybrid amphiphilic block copolymers consisting of biocompatible synthetic glycopolymer with non-degradable backbone and biodegradable poly(amino acid) (PAA) was developed. The glycopolymer, poly(2-deoxy-2-methacrylamido-D-glucose) (PMAG), was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Two methods for modifying the terminal dithiobenzoate-group of PMAG was investigated to obtain the macroinitiator bearing a primary aliphatic amino group, which is required for ring-opening polymerization of N-carboxyanhydrides of hydrophobic α-amino acids. The synthesized amphiphilic block copolymers were carefully analyzed using a set of different physico-chemical methods to establish their composition and molecular weight. The developed amphiphilic copolymers tended to self-assemble in nanoparticles of different morphology that depended on the nature of the hydrophobic amino acid present in the copolymer. The hydrodynamic diameter, morphology, and cytotoxicity of polymer particles based on PMAG-b-PAA were evaluated using dynamic light scattering (DLS) and transmission electron microscopy (TEM), as well as CellTiter-Blue (CTB) assay, respectively. The redox-responsive properties of nanoparticles were evaluated in the presence of glutathione taken at different concentrations. Moreover, the encapsulation of paclitaxel into PMAG-b-PAA particles and their cytotoxicity on human lung carcinoma cells (A549) and human breast adenocarcinoma cells (MCF-7) were studied.

Hijacking tRNAs From Translation: Regulatory Functions of tRNAs in Mammalian Cell Physiology.

Transfer tRNAs (tRNAs) are small non-coding RNAs that are highly conserved in all kingdoms of life. Originally discovered as the molecules that deliver amino acids to the growing polypeptide chain during protein synthesis, tRNAs have been believed for a long time to play exclusive role in translation. However, recent studies have identified key roles for tRNAs and tRNA-derived small RNAs in multiple other processes, including regulation of transcription and translation, posttranslational modifications, stress response, and disease. These emerging roles suggest that tRNAs may be central players in the complex machinery of biological regulatory pathways. Here we overview these non-canonical roles of tRNA in normal physiology and disease, focusing largely on eukaryotic and mammalian systems.

Protein arginylation of cytoskeletal proteins in the muscle: modifications modifying function.

The cytoskeleton drives many essential processes in normal physiology, and its impairments underlie many diseases, including skeletal myopathies, cancer, and heart failure, that broadly affect developed countries worldwide. Cytoskeleton regulation is a field of investigation of rapidly emerging global importance and a new venue for the development of potential therapies. This review overviews our present understanding of the posttranslational regulation of the muscle cytoskeleton through arginylation, a tRNA-dependent addition of arginine to proteins mediated by arginyltransferase 1. We focus largely on arginylation-dependent regulation of striated muscles, shown to play critical roles in facilitating muscle integrity, contractility, regulation, and strength.

Protein arginylation targets alpha synuclein, facilitates normal brain health, and prevents neurodegeneration.

Alpha synuclein (α-syn) is a central player in neurodegeneration, but the mechanisms triggering its pathology are not fully understood. Here we found that α-syn is a highly efficient substrate for arginyltransferase ATE1 and is arginylated in vivo by a novel mid-chain mechanism that targets the acidic side chains of E46 and E83. Lack of arginylation leads to increased α-syn aggregation and causes the formation of larger pathological aggregates in neurons, accompanied by impairments in its ability to be cleared via normal degradation pathways. In the mouse brain, lack of arginylation leads to an increase in α-syn's insoluble fraction, accompanied by behavioral changes characteristic for neurodegenerative pathology. Our data show that lack of arginylation in the brain leads to neurodegeneration, and suggests that α-syn arginylation can be a previously unknown factor that facilitates normal α-syn folding and function in vivo.

Mitochondrial dysfunction and mitochondrial dynamics-The cancer connection.

Mitochondrial dysfunction is a hallmark of many diseases. The retrograde signaling initiated by dysfunctional mitochondria can bring about global changes in gene expression that alters cell morphology and function. Typically, this is attributed to disruption of important mitochondrial functions, such as ATP production, integration of metabolism, calcium homeostasis and regulation of apoptosis. Recent studies showed that in addition to these factors, mitochondrial dynamics might play an important role in stress signaling. Normal mitochondria are highly dynamic organelles whose size, shape and network are controlled by cell physiology. Defective mitochondrial dynamics play important roles in human diseases. Mitochondrial DNA defects and defective mitochondrial function have been reported in many cancers. Recent studies show that increased mitochondrial fission is a pro-tumorigenic phenotype. In this paper, we have explored the current understanding of the role of mitochondrial dynamics in pathologies. We present new data on mitochondrial dynamics and dysfunction to illustrate a causal link between mitochondrial DNA defects, excessive fission, mitochondrial retrograde signaling and cancer progression. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux.

Identification of Arginylated Proteins by Mass Spectrometry.

Here we describe the method for identification of arginylated proteins by mass spectrometry. This method has been originally applied to the identification of N-terminally added Arg on proteins and peptides, and then expanded to identification of side chain arginylation which has been recently described by our groups. The key steps in this method include the use of the mass spectrometry instruments that can identify peptides with very high pass accuracy (Orbitrap) and apply stringent mass cutoffs during automated data analysis, followed by manual validation of the identified spectra. These methods can be used with both complex and purified protein samples and, to date, constitute the only reliable way to confirm arginylation at a particular site on a protein or peptide.

Analysis of Arginylated Peptides by Subtractive Edman Degradation.

During the early studies of N-terminal arginylation, Edman degradation was widely used to identify N-terminally added Arg on protein substrates. This old method is reliable but highly depends on the purity and abundance of samples and can become misleading unless a highly purified, highly arginylated protein can be obtained. Here we report a mass spectrometry-based method that utilizes Edman degradation chemistry to identify arginylation in more complex and less abundant protein samples. This method can also apply to the analysis of other posttranslational modifications.

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 ß-actin has been found to regulate lamellipodial formation at the leading edge in fibroblasts. This finding suggests that similar functions of ß-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.

Sounds of silence: synonymous nucleotides as a key to biological regulation and complexity.

Messenger RNA is a key component of an intricate regulatory network of its own. It accommodates numerous nucleotide signals that overlap protein coding sequences and are responsible for multiple levels of regulation and generation of biological complexity. A wealth of structural and regulatory information, which mRNA carries in addition to the encoded amino acid sequence, raises the question of how these signals and overlapping codes are delineated along non-synonymous and synonymous positions in protein coding regions, especially in eukaryotes. Silent or synonymous codon positions, which do not determine amino acid sequences of the encoded proteins, define mRNA secondary structure and stability and affect the rate of translation, folding and post-translational modifications of nascent polypeptides. The RNA level selection is acting on synonymous sites in both prokaryotes and eukaryotes and is more common than previously thought. Selection pressure on the coding gene regions follows three-nucleotide periodic pattern of nucleotide base-pairing in mRNA, which is imposed by the genetic code. Synonymous positions of the coding regions have a higher level of hybridization potential relative to non-synonymous positions, and are multifunctional in their regulatory and structural roles. Recent experimental evidence and analysis of mRNA structure and interspecies conservation suggest that there is an evolutionary tradeoff between selective pressure acting at the RNA and protein levels. Here we provide a comprehensive overview of the studies that define the role of silent positions in regulating RNA structure and processing that exert downstream effects on proteins and their functions.

Small molecule inhibitors of arginyltransferase regulate arginylation-dependent protein degradation, cell motility, and angiogenesis.

Posttranslational arginylation mediated by arginyltransferase (ATE1) is an emerging major regulator of embryogenesis and cell physiology. Impairments of ATE1 are implicated in congenital heart defects, obesity, cancer, and neurodegeneration making this enzyme an important therapeutic target, whose potential has been virtually unexplored. Here we report the development of a biochemical assay for identification of small molecule inhibitors of ATE1 and application of this assay to screen a library of 3280 compounds. Our screen identified two compounds which specifically affect ATE1-regulated processes in vivo, including tannic acid, which has been previously shown to inhibit protein degradation and angiogenesis and to act as a therapeutic agent in heart disease and cancer. Our data suggest that these actions of tannic acid are mediated by its direct effect on ATE1, which regulates protein degradation and angiogenesis in vivo.

Arginyltransferase is an ATP-independent self-regulating enzyme that forms distinct functional complexes in vivo.

Posttranslational arginylation mediated by arginyl transferase (ATE1) plays an important role in cardiovascular development, cell motility, and regulation of cytoskeleton and metabolic enzymes. This protein modification was discovered decades ago, however, the arginylation reaction and the functioning of ATE1 remained poorly understood because of the lack of good biochemical models. Here, we report the development of an in vitro arginylation system, in which ATE1 function and molecular requirements can be tested using purified recombinant ATE1 isoforms supplemented with a controlled number of components. Our results show that arginylation reaction is a self-sufficient, ATP-independent process that can affect different sites in a polypeptide and that arginyl transferases form different molecular complexes in vivo, associate with components of the translation machinery, and have distinct, partially overlapping subsets of substrates, suggesting that these enzymes play different physiological functions.