Aminoacyl tRNA synthetase complex interacting multifunctional protein 1 simultaneously binds Glutamyl-Prolyl-tRNA synthetase and scaffold protein aminoacyl tRNA synthetase complex interacting multifunctional protein 3 of the multi-tRNA synthetase complex

Higher eukaryotes have developed extensive compartmentalization of amino acid (aa) - tRNA coupling through the formation of a multi-synthetase complex (MSC) that is composed of eight aa-tRNA synthetases (ARS) and three scaffold proteins: aminoacyl tRNA synthetase complex interacting multifunctional proteins (AIMP1, 2 and 3). Lower eukaryotes have a much smaller complex while yeast MSC consists of only two ARS (MetRS and GluRS) and one ARS cofactor 1 protein, Arc1p (Simos et al., 1996), the homolog of the mammalian AIMP1. Arc1p is reported to form a tripartite complex with GluRS and MetRS through association of the N-terminus GST-like domains (GST-L) of the three proteins (Koehler et al., 2013). Mammalian AIMP1 has no GST-L domain corresponding to Arc1p N-terminus. Instead, AIMP3, another scaffold protein of 18 kDa composed entirely of a GST-L domain, interacts with Methionyl-tRNA synthetase (MARS) (Quevillon et al., 1999) and Glutamyl-Prolyl-tRNA Synthetase (EPRS) (Cho et al., 2015). Here we report two new interactions between MSC members: AIMP1 binds to EPRS and AIMP1 binds to AIMP3. Interestingly, the interaction between AIMP1 and AIMP3 complex makes it the functional equivalent of a single Arc1p polypeptide in yeast. This interaction is not mapped to AIMP1 N-terminal coiled-coil domain, but rather requires an intact tertiary structure of the entire protein. Since AIMP1 also interacts with AIMP2, all three proteins appear to compose a core docking structure for the eight ARS in the MSC complex

Mutations in the mitochondrial cysteinyl-tRNA synthase gene, CARS2, lead to a severe epileptic encephalopathy and complex movement disorder

Background: Mitochondrial disease is often suspected in cases of severe epileptic encephalopathy especially when a complex movement disorder, liver involvement and progressive developmental regression are present. Although mutations in either mitochondrial DNA or POLG are often present, other nuclear defects in mitochondrial DNA replication and protein translation have been associated with a severe epileptic encephalopathy. Methods: and results We identified a proband with an epileptic encephalopathy, complex movement disorder and a combined mitochondrial respiratory chain enzyme deficiency. The child presented with neurological regression, complex movement disorder and intractable seizures. A combined deficiency of mitochondrial complexes I, III and IV was noted in liver tissue, along with increased mitochondrial DNA content in skeletal muscle. Incomplete assembly of complex V, using blue native polyacrylamide gel electrophoretic analysis and complex I, using western blotting, suggested a disorder of mitochondrial transcription or translation. Exome sequencing identified compound heterozygous mutations in CARS2, a mitochondrial aminoacyl-tRNA synthetase. Both mutations affect highly conserved amino acids located within the functional ligase domain of the cysteinyl-tRNA synthase. A specific decrease in the amount of charged mt-tRNACys was detected in patient fibroblasts compared with controls. Retroviral transfection of the wild-type CARS2 into patient skin fibroblasts led to the correction of the incomplete assembly of complex V, providing functional evidence for the role of CARS2 mutations in disease aetiology. Conclusions: Our findings indicate that mutations in CARS2 result in a mitochondrial translational defect as seen in individuals with mitochondrial epileptic encephalopathy

Three phases in the evolution of the standard genetic code: how translation could get started

A primordial genetic code is proposed, having only four codons assigned, GGC meaning glycine, GAC meaning aspartate/glutamate, GCC meaning alanine-like and GUC meaning valine-like. Pathways of ambiguity reduction enlarged the codon repertoire with CUC meaning leucine, AUC meaning isoleucine, ACC meaning threonine-like and GAG meaning glutamate. Introduction of UNN anticodons, in a next episode of code evolution in which nonsense elimination was the leading theme, introduced a family box structure superposed on the original mirror structure. Finally, growth rate was the leading theme during the remaining repertoire expansion, explaining the ordered phylogenetic pattern of aminoacyl-tRNA synthetases. The special role of natural aptamers in the process is high-lighted, and the error robustness characteristics of the code are shown to have evolved by way of a stepwise, restricted enlargement of the tRNA repertoire, instead of by an exhaustive selection process testing myriads of codes

Role of the Bifunctional Aminoacyl-tRNA Synthetase EPRS in Human Disease

Aminoacyl-tRNA synthetases (AARS) are a class of enzymes that catalyze the charging of tRNAs with cognate amino acids, a critical step that contributes to the fidelity of protein synthesis. Many AARSs also possess noncanonical functions such as regulation of apoptosis, mRNA translation, and RNA splicing. Some AARSs have evolved new domains with no apparent connection to their charging functions. For example, WHEP domains were originally identified in tryptophanyl-tRNA synthetase (WRS), histidyl-tRNA synthetase (HRS), and glutamyl-prolyl-tRNA synthetase (EPRS). EPRS is a unique bifunctional AARS, found only in higher eukaryotes, and consists of glutamyl-tRNA synthetase (ERS) and prolyl-tRNA synthetase (PRS) joined by a non-catalytic linker containing three WHEP domains in humans. Two compound heterozygous point mutations within human ERS (P14R and E205G) have been identified in the genomes of two patients with type 1 diabetes and bone disease. However, the mechanism by which these mutations contribute to disease is unknown. Our goal is to determine whether the point mutations affect the canonical catalytic activity of EPRS responsible for tRNA charging or noncanonical functions. Both P14 and E205 are highly conserved residues located in the GST and catalytic domain, respectively. An ERS variant appended to 2.5 WHEP domains (ERS 2.5W) has been purified and shown to display robust tRNA binding and aminoacylation activity in vitro. The P14R and E205G single mutants display the same binding affinity for tRNAGlu as WT ERS 2.5W, suggesting that the observed defect is at the catalytic step. Whereas the ERS 2.5W P14R mutant has near wild-type (WT) aminoacylation activity, the ERS 2.5W E205G variant has a severe aminoacylation defect. Both mutations, however, lead to reduced amino acid activation. Together with a collaborator, we are currently characterizing the effect of these two mutations on cell proliferation and the integrated stress response. Taken together, this work has important implications for the understanding of AARS-related human disease mechanisms and development of new therapeutics.College of Arts & SciencesOffice of Undergraduate Research & Creative InquiryNo embargoAcademic Major: Biochemistr

Modification of Aminoacyl tRNA Synthetase in Order to Incorporate an Unnatural Amino Acid

Proteins allow daily processes in the cell to occur. A protein consists of amino acids. There are twenty natural amino acids coded for in the DNA of organisms. The natural amino acids can be modified to form unnatural amino acids (UAAs). UAAs have useful characteristics when inserted into a protein of a cell, like the ability of fluoresce, which makes their incorporation important in research. For an UAA to be incorporated into a protein, it must be bound to a transport RNA molecule by an enzyme called aminoacyl tRNA synthetase (aaRS). An existing aaRS was modified in E. Coli bacterial cells to incorporate 3-(2-pyridyl)-L-Alanine since it has metal-binding capabilities. Once incorporated, the UAA acts as a sensor for a metal, making it useful to environmental fields. The aaRS was mutated using saturation mutagenesis at sites L32, V65, W108, G158, A159. The cells were run through a positive screen to determine if the mutated aaRS incorporated the UAA into a green fluorescent protein, which glowed if the UAA was inserted. The results of the positive screen showed mutated aaRSs 2, 4, 7, and 8 incorporated 3-(2-pyridyl)-L-Alanine, while mutated aaRSs 2, 5, 6, 7, 8, and 9 incorporated p-cyanophenylalanine. A negative screen to test if the mutated aaRS only incorporate an UAA, not natural amino acids still present in the cell, will be run on the mutated aaRSs passing the positive screen

Analysis of the Role of Aminoacyl tRNA Synthetase Genes in Global Protein Synthesis and mRNA Specific Regulation of Translation in Cancer Cells

Analysis of the Role of Aminoacyl tRNA Synthetase Genes in Global Protein Synthesis and mRNA Specific Regulation of Translation in Cancer Cells Elyse Nguyen, Depts. of Biology and Chemistry, Dipak Poria, & Esta Sterneck, with Dr. Sarah Williams, Dept. of Forensic Science Coordinated control of transcription and translation of gene expression impels cellular fate decision under different microenvironmental stresses. Cancer cells often usurp these regulatory machineries to adapt under microenvironmental stress or under therapeutic intervention. The transcription factor CEBPδ is induced by various stressors and mediates cellular adaptation and survival. RNA-seq analysis of a CEBPD-silenced human melanoma cell line, MB-435s, showed decreased expression of 12 aminoacyl-tRNA synthetase (aaRS) genes. Our group recently found that deletion of CEBPD by CRISPR/Cas9 (CEBPD-KO) compromised aminoacyl tRNA synthetase (aaRS) expression and global protein synthesis. However, despite this decrease in global protein production, the synthesis of certain proteins, such as ATF4, which promotes survival and/or death under stress conditions, is increased. Aminoacyl tRNA synthetases are essential enzymes in the process of protein synthesis which catalyze the addition of appropriate amino acid to its corresponding tRNA, and therefore act as a rate limiting step in cellular protein synthesis. In the current project, we sought to investigate the effect of silencing of specific aaRS genes, glutamyl-prolyl-tRNA synthetase (EPRS) and valyl-tRNA synthetase (VARS) on global protein translation and ATF4 expression. To address this question, we silenced the EPRS and VARS gene expression using two independent short-hairpin-RNA (shRNA) targeting two different regions of EPRS and VARS mRNAs in MB-435s cells. Silencing of EPRS gene showed compensatory upregulation of VARS and vice versa. Interestingly, our preliminary data suggested an upregulation of global protein synthesis after EPRS and VARS silencing in MB435s cells measured by puromycin pulse labelling. Ongoing experiments to validate the preliminary data and ATF4 expression will be discussed.https://scholarscompass.vcu.edu/uresposters/1326/thumbnail.jp

Developing an Unnatural Amino Acid-Specific Aminoacyl tRNA Synthetase

Unnatural Amino Acids (UAAs), amino acids not present in the human genetic code, have been synthesized to have a broad range of useful properties, in this case, as metal-binders which could have drug delivery applications. In order for the cell to place a UAA into the protein, two components, a unique aminoacyl tRNA synthetase and a corresponding tRNA must be present. If an amino acid is successfully charged to the tRNA, a stop codon is suppressed and a functional protein is built with the UAA at the mutation site. Such a tRNA molecule has previously been developed, as well as many synthetases specific to UAAs. In this work, the range of UAAs which can be incorporated into proteins using the E. coli’s own machinery is expanded by the development of a novel aminoacyl tRNA synthetase. By making a library of synthetase-coding plasmid variants and performing positive and negative screenings, the binding pocket of the synthetase can be modified for specificity to a UAA while not allowing the tRNA to be charged with a natural amino acid. In this work, we are attempting to evolve new tRNA synthetases for the incorporation of metal-binding amino acids by developing the plasmid library and a screening system to find synthetase variants meeting these criteria

Developing an Unnatural Amino Acid-Specific Aminoacyl tRNA Synthetase

Unnatural Amino Acids (UAAs), amino acids not present in the human genetic code, have been synthesized to have a broad range of useful properties, in this case, as metal-binders which could have drug delivery applications. In order for the cell to place a UAA into the protein, two components, a unique aminoacyl tRNA synthetase and a corresponding tRNA must be present. If an amino acid is successfully charged to the tRNA, a stop codon is suppressed and a functional protein is built with the UAA at the mutation site. Such a tRNA molecule has previously been developed, as well as many synthetases specific to UAAs. In this work, the range of UAAs which can be incorporated into proteins using the E. coli’s own machinery is expanded by the development of a novel aminoacyl tRNA synthetase. By making a library of synthetase-coding plasmid variants and performing positive and negative screenings, the binding pocket of the synthetase can be modified for specificity to a UAA while not allowing the tRNA to be charged with a natural amino acid. In this work, we are attempting to evolve new tRNA synthetases for the incorporation of metal-binding amino acids by developing the plasmid library and a screening system to find synthetase variants meeting these criteria

Unexpectedly fast transfer of positron-emittable artificial substrate into N-terminus of peptide/protein mediated by wild-type L/F-tRNA-protein transferase

This article demonstrates the fastest enzymatic introduction of a positron emission tomography (PET) probe into acceptor peptides/proteins. It is site-specifically introduced at the basic N-terminus of the acceptors by using L/F-transferase in combination with aminoacyl-tRNA synthetase, namely the NEXT-A/PET reaction. Estimated from kinetic analysis, the transfer efficiency of O-(2-fluoromethyl)-L-tyrosine as an artificial amino acid PET probe mediated by the wild-type transferase is almost as good as that of the natural substrate, phenylalanine