Glutamyl-tRNAGln Amidotransferase Research Articles

Characterization of the mitochondrial GlutamyltRNAGln amidotransferase (GatCAB) as a new model for mitochondrial translation disorders.

Mitochondrial OXPHOS diseases are provoked by dysfunction of the OXPHOS system, showing heterogeneity from a genetic, biochemical and clinical perspective. This complexity is partly due to the involvement of proteins encoded in two genomes located in different cellular compartments, nuclear and mitochondrial DNA (mtDNA). mtDNA encodes part of the mtRNAs translation machinery, including 22 tRNAs. All these are charged by their corresponding aminoacyl-tRNA synthetases (ARS2), except mt-Gln-tRNAGln whose synthesis is carried out through an indirect pathway. The mt-tRNAGln is charged with glutamic acid (Glu) by a non-discriminating mitochondrial glutamyl-tRNA synthetase (EARS2), then the Glu is converted to Gln by a Glutamyl-tRNAGln amidotransferase, using free glutamine as an amide donor, yielding Gln- tRNAGln. This mitochondrial amidotransferase activity lies in the GatCAB complex, which is formed by three subunits: GatA (QSRL1), GatB (GATB), and GatC (GATC). Patients with mutations in these genes presented with severe cardiomyopathy and lactic acidosis, which underscores the importance of the GatCAB complex as an essential component in the translational machinery of mitochondrial protein synthesis. We have generated Knockout (KO) lines for all subunits using the genomic editing system CRISPR/Cas9 in HEK293T cells. We measured the levels of the GatCAB subunits and mitochondrial proteins by western blot. We studied the oxygen consumption levels using a Clark electrode and detected the GatCAB complex by Blue Native-western blotting. In all cases, KO cells show reduced levels of mitochondrial proteins, decreased oxygen consumption and an absence of the GatCAB complex compared to wildtype cells. In the QRSL1 and GATC KO cells the levels of all subunits diminish, but in GATB KO cells the levels of subunits A and C remain stable. Possibly, QRSL1 and GATC could form a stable but nonfunctional dimer, to which GATB would join. GATB has two potential ATG codons at the start of its sequence separated by 12 nucleotides. From over 90 clones analyzed, we only obtained one KO, by generating an upstream ORF, and two cell lines with very low expression of GATB. The latter present a duplicated region at the beginning of GATB which changes the reading frame from the first ATG; suggesting the downstream ATG as an alternative translation start codon for those ribosome small subunits that do not assemble on the first one, yielding a GATB protein missing 4 amino acids. Characterization of the molecular mechanisms that lead to the synthesis of mt-Gln-tRNAGln and its role in the physiology of the cell could allow us to comprehend the pathological manifestation of the defects in these genes and propose possible therapeutic avenues.

Structural insights into Pseudomonas aeruginosaType six secretion system exported effector 8

Recent reports indicate that the Type six secretion system exported effector 8 (Tse8) is a cytoactive effector secreted by the Type VI secretion system (T6SS) of the human pathogen Pseudomonas aeruginosa. The T6SS is a nanomachine that assembles inside of the bacteria and injects effectors/toxins into target cells, providing a fitness advantage over competing bacteria and facilitating host colonisation. Here we present the first crystal structure of Tse8 revealing that it conserves the architecture of the catalytic triad Lys84-transSer162-Ser186 that characterises members of the Amidase Signature superfamily. Furthermore, using binding affinity experiments, we show that the interaction of phenylmethylsulfonyl fluoride (PMSF) to Tse8 is dependent on the putative catalytic residue Ser186, providing support for its nucleophilic reactivity. This work thus demonstrates that Tse8 belongs to the Amidase Signature (AS) superfamily. Furthermore, it highlights Tse8 similarity to two family members: the Stenotrophomonas maltophilia Peptide Amidase and the Glutamyl-tRNAGln amidotransferase subunit A from Staphylococcus aureus.

Glutamyl-tRNAGln amidotransferase is essential for mammalian mitochondrial translation in vivo

Translational accuracy depends on the correct formation of aminoacyl-tRNAs, which, in the majority of cases, are produced by specific aminoacyl-tRNA synthetases that ligate each amino acid to its cognate isoaceptor tRNA. Aminoacylation of tRNAGln, however, is performed by various mechanisms in different systems. Since no mitochondrial glutaminyl-tRNA synthetase has been identified to date in mammalian mitochondria, Gln-tRNAGln has to be formed by an indirect mechanism in the organelle. It has been demonstrated that human mitochondria contain a non-discriminating glutamyl-tRNA synthetase and the heterotrimeric enzyme GatCAB (where Gat is glutamyl-tRNAGln amidotransferase), which are able to catalyse the formation of Gln-tRNAGln invitro. In the present paper we demonstrate that mgatA (mouse GatA) interference in mouse cells produces a strong defect in mitochondrial translation without affecting the stability of the newly synthesized proteins. As a result, interfered cells present an impairment of the oxidative phosphorylation system and a significant increase in ROS (reactive oxygen species) levels. MS analysis of mitochondrial proteins revealed no glutamic acid found in the position of glutamines, strongly suggesting that misaminoacylated Glu-tRNAGln is rejected from the translational apparatus to maintain the fidelity of mitochondrial protein synthesis in mammals.

A dual-specific Glu-tRNA Gln and Asp-tRNA Asn amidotransferase is involved in decoding glutamine and asparagine codons in Acidithiobacillus ferrooxidans

The gatC, gatA and gatB genes encoding the three subunits of glutamyl-tRNA Gln amidotransferase from Acidithiobacillus ferrooxidans, an acidophilic bacterium used in bioleaching of minerals, have been cloned and expressed in Escherichia coli. As in Bacillus subtilis the three gat genes are organized in an operon-like structure in A. ferrooxidans. The heterologously overexpressed enzyme converts Glu-tRNA Gln to Gln-tRNA Gln and Asp-tRNA Asn to Asn-tRNA Asn. Biochemical analysis revealed that neither glutaminyl-tRNA synthetase nor asparaginyl-tRNA synthetase is present in A. ferrooxidans, but that glutamyl-tRNA synthetase and aspartyl-tRNA synthetase enzymes are present in the organism. These data suggest that the transamidation pathway is responsible for the formation of Gln-tRNA and Asn-tRNA in A. ferrooxidans.