Sougata Saha

Differential Arginylation of Actin Isoforms Is Regulated by Coding Sequence–Dependent Degradation

Making Modifications Arginylation of β-actin regulates cell motility and the actin cytoskeleton, but how differential arginylation of the two highly similar actin isoforms—β and γ—is achieved in vivo is unclear. Zhang et al. (p. 1534; see the Perspective by Weygand-Durasevic and Ibba) describe a cotranslational mechanism that selectively regulates the arginylation of proteins through degradation and is dependent on the nucleotide coding sequence coupled to the translation speed. The work provides an explanation for the different N-terminal arginylation states of β- and γ-actin in vivo and suggests translation rate affected by nucleotide coding sequence confers different posttranslational states to proteins and selectively regulates protein degradation. The translational speed of proteins influences whether they are cotranslationally degraded after arginylation. 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.

Posttranslational arginylation as a global biological regulator.

Posttranslational modifications constitute a major field of emerging biological significance as mounting evidence demonstrates their key role in multiple physiological processes. Following in the footsteps of protein phosphorylation studies, new modifications are being shown to regulate protein properties and functions in vivo. Among such modifications, an important role belongs to protein arginylation - posttranslational tRNA-mediated addition of arginine, to proteins by arginyltransferase, ATE1. Recent studies show that arginylation is essential for embryogenesis in many organisms and that it regulates such important processes as heart development, angiogenesis, and tissue morphogenesis in mammals. This review summarizes the key data in the protein arginylation field since its original discovery to date.

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.

Arginylation regulates intracellular actin polymer level by modulating actin properties and binding of capping and severing proteins.

Actin arginylation regulates lamella formation in motile fibroblasts, but the underlying molecular mechanisms are unknown. Here, we found that actin regulation by arginylation affects its biochemical properties and binding of actin-associated proteins, modulating the overall structural organization of actin filaments in the cell.

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.

Arginylation-dependent regulation of a proteolytic product of talin is essential for cell–cell adhesion

A proteolytic fragment of talin is regulated by arginylation and promotes cadherin-dependent cell–cell adhesion.

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.

Excellent N-fixing and P-solubilizing traits in earthworm gut-isolated bacteria: A vermicompost based assessment with vegetable market waste and rice straw feed mixtures

Vermicomposting is a dependable waste recycling technology which greatly augments N and P levels mainly through microbial action. This paper aims to identify efficient N-fixing (NFB) and P-solubilizing (PSB) bacteria from earthworm intestines. Various combinations of vegetable market waste, rice straw, and cowdung were fed to two earthworm species (Eisenia fetida and Perionyx excavatus). Total organic C decreased, pH shifted towards neutrality, and NPK availability, and microbial (NFB, PSB, and total bacteria) population increased remarkably during vermicomposting with E. fetida. Therefore, 45 NFB and 34 PSB strains isolated from Eisenia gut were initially screened, their inter-dominance assessed, and 8 prolific strains were identified through 16SrRNA sequencing. Interestingly, two novel N-fixing strains of Kluyvera ascorbata emerged as an efficient biofertilizer candidate. Moreover, both N-fixing and P-solubilizing strains of Serratia and Bacillus were isolated from earthworm gut. All the isolated strains significantly improved soil health and facilitated crop growth as compared to commercial biofertilizers.

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.

Arginylation regulates purine nucleotide biosynthesis by enhancing the activity of phosphoribosyl pyrophosphate synthase

Protein arginylation is an emerging post-translational modification that targets a number of metabolic enzymes, however the mechanisms and downstream effects of this modification are unknown. Here we show that lack of arginylation renders cells vulnerable to purine nucleotide synthesis inhibitors and affects the related glycine and serine biosynthesis pathways. We show that the purine nucleotide biosynthesis enzyme PRPS2 is selectively arginylated, unlike its close homologue PRPS1, and that arginylation of PRPS2 directly facilitates its biological activity. Moreover, selective arginylation of PRPS2 but not PRPS1 is regulated through a coding sequence-dependent mechanism that combines elements of mRNA secondary structure with lysine residues encoded near the N-terminus of PRPS1. This mechanism promotes arginylation-specific degradation of PRPS1 and selective retention of arginylated PRPS2 in vivo. We therefore demonstrate that arginylation affects both the activity and stability of a major metabolic enzyme.