Human histidyl-tRNA Synthetase Research Articles

Orphan designation: 505 amino acid protein, corresponding to amino acids 2-506 of the wild type human histidyl-tRNA synthetase, treatment of facioscapulohumeral muscular dystrophy

Orphan designation: 505 amino acid protein, corresponding to amino acids 2-506 of the wild type human histidyl-tRNA synthetase, treatment of facioscapulohumeral muscular dystrophy

Orphan designation: 505 amino acid protein, corresponding to amino acids 2-506 of the wild-type human histidyl-tRNA synthetase, Treatment of limb-girdle muscular dystrophy

Orphan designation: 505 amino acid protein, corresponding to amino acids 2-506 of the wild-type human histidyl-tRNA synthetase, Treatment of limb-girdle muscular dystrophy

Evolutionary gain of highly divergent tRNA specificities by two isoforms of human histidyl-tRNA synthetase.

The discriminator base N73 is a key identity element of tRNAHis. In eukaryotes, N73 is an "A" in cytoplasmic tRNAHis and a "C" in mitochondrial tRNAHis. We present evidence herein that yeast histidyl-tRNA synthetase (HisRS) recognizes both A73 and C73, but somewhat prefers A73 even within the context of mitochondrial tRNAHis. In contrast, humans possess two distinct yet closely related HisRS homologues, with one encoding the cytoplasmic form (with an extra N-terminal WHEP domain) and the other encoding its mitochondrial counterpart (with an extra N-terminal mitochondrial targeting signal). Despite these two isoforms sharing high sequence similarities (81% identity), they strongly preferred different discriminator bases (A73 or C73). Moreover, only the mitochondrial form recognized the anticodon as a strong identity element. Most intriguingly, swapping the discriminator base between the cytoplasmic and mitochondrial tRNAHis isoacceptors conveniently switched their enzyme preferences. Similarly, swapping seven residues in the active site between the two isoforms readily switched their N73 preferences. This study suggests that the human HisRS genes, while descending from a common ancestor with dual function for both types of tRNAHis, have acquired highly specialized tRNA recognition properties through evolution.

A Loss-of-Function Variant in the Human Histidyl-tRNA Synthetase (HARS) Gene is Neurotoxic In Vivo

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes responsible for ligating amino acids to cognate tRNA molecules. Mutations in four genes encoding an ARS have been implicated in inherited peripheral neuropathy with an axonal pathology, suggesting that all ARS genes are relevant candidates for disease in patients with related phenotypes. Here, we present results from a mutation screen of the histidyl-tRNA synthetase (HARS) gene in a large cohort of patients with peripheral neuropathy. These efforts revealed a rare missense variant (c.410G>A/p.Arg137Gln) that resides at a highly conserved amino acid, represents a loss-of-function allele when evaluated in yeast complementation assays, and is toxic to neurons when expressed in a worm model. In addition to the patient with peripheral neuropathy, p.Arg137Gln HARS was detected in three individuals by genome-wide exome sequencing. These findings suggest that HARS is the fifth ARS locus associated with axonal peripheral neuropathy. Implications for identifying ARS alleles in human populations and assessing them for a role in neurodegenerative phenotypes are discussed.

Equilibrium Unfolding of Bombyx mori Glycyl-tRNA Synthetase

Unfolding of Bombyx mori glycyl-tRNA synthetase was examined by multiple spectroscopic techniques. Tryptophan fluorescence of wild type enzyme and an N-terminally truncated form (N55) increased at low concentrations of urea or guanidine-HCl followed by a reduction in intensity at intermediate denaturant concentrations; a transition at higher denaturant was detected as decreased fluorescence intensity and a red-shifted emission. Solute quenching of fluorescence indicated that tryptophans become progressively solvent-exposed during unfolding. Wild type enzyme had stronger negative CD bands between 220 and 230 nm than the mutant, indicative of greater alpha-helical content. Urea or guanidine-HCl caused a reduction in ellipticity at 222 nm at low denaturant concentration with the wild type enzyme, a transition that is absent in the mutant; both enzymes exhibited a cooperative transition at higher denaturant concentrations. Both enzymes dissociate to monomers in 1.5 m urea. Unfolding of wild type enzyme is described by a multistate unfolding and a parallel two state unfolding; the two-state component is absent in the mutant. Changes in spectral properties associated with unfolding were largely reversible after dilution to low denaturant. Unfolding of glycyl-tRNA synthetase is complex with a native state, a native-like monomer, partially unfolded states, and the unfolded state.

A Novel Gene Oriented in a Head-to-Head Configuration with the Human Histidyl-tRNA Synthetase (HRS) Gene Encodes an mRNA That Predicts a Polypeptide Homologous to HRS

The human histidyl-tRNA synthetase (HRS) gene encodes an enzyme that catalyzes the esterification of histidine to its cognate tRNA as an early step in protein biosynthesis. Previous reports have described a bidirectional promoter element which coordinates the transcription of both HRS and an unknown mRNA whose gene is oriented in a head-to-head configuration with HRS. We have isolated and characterized a human genomic DNA clone that encodes portions of these oppositely transcribed mRNAs and a putatively full-length cDNA clone (HO3) corresponding to the gene mapping immediately 5′ of HRS. The largest open reading frame within HO3 (1518 bp) shares approximately 75% nucleotide sequence identity with human HRS (1527 bp) and predicts a polypeptide with extensive amino acid sequence homology with the HRS protein (72%). Moreover, amino acid sequence motifs characteristic of class II aminoacyl-tRNA synthetases are conserved within HO3. Despite their similarity, HRS and HO3 have divergent amino-terminal domains which correspond to the first two exons of each gene. RNA blot analysis revealed that HRS (2.0 kb) and HO3 (2.5 kb) exhibit distinct patterns of steady-state mRNA expression among multiple human tissues.

A motif in human histidyl-tRNA synthetase which is shared among several aminoacyl-tRNA synthetases is a coiled-coil that is essential for enzymatic activity and contains the major autoantigenic epitope.

In myositis, disease-specific autoantibodies may be directed against an aminoacyl-tRNA synthetase, usually histidyl-tRNA synthetase. To explore the basis for this phenomenon, we have made recombinant histidyl-tRNA synthetase in the baculovirus system. It was enzymatically active and recognized by human autoantibodies. A truncated protein lacking the first 60 amino acids was inactive as an antigen and as an enzyme. This region is within the first two exons, is predicted to have a coiled-coil configuration, and is found in some other synthetases but not in Escherichia coli or yeast histidyl-tRNA synthetase. Circular dichroism showed that the peptides from this region (amino acids 1-60 and 1-47) have the predicted high alpha-helical content, but smaller fragments (1-30, 14-45, and 31-60) do not. The peptides with a high alpha-helical content could inhibit autoantibodies almost completely, whereas the smaller peptides were unable to do so. The amino acid sequence of this coiled-coil domain in human histidyl-tRNA synthetase resembles the sequence of the extended this coiled-coil arm near the NH2 terminus of bacterial seryl-tRNA synthetase as well as similar regions in some eukaryotic aminoacyl-tRNA synthetases, raising the possibility that this domain serves a similar tRNA-stabilizing role and has been preserved from a common ancestor.

Transcriptional analyses of the gene region that encodes human histidyl-tRNA synthetase: identification of a novel bidirectional regulatory element

A recombinant phage clone containing the 5' end of the gene HRS encoding human histidyl-tRNA synthetase (HRS) has been isolated. Primer extension analyses indicated that there are two types of HRS transcripts. The longer transcripts were initiated from a single transcription start point ( tsp) located approximately 455 bp upstream and the shorter transcripts were initiated from multiple tsp located approximately 38 to 82 bp upstream from the HRS ATG start codon. Functionally, we have identified two regions (+1 to −122; −185 to −502), each of which when placed 5' of a promoterless cat construct can initiate transcription in both orientations after transfection into HeLa cells. A pair of imperfect inverted repeats ( IIR) was located within the region +1 to −122. Using mobility shift assays, we have identified a nuclear factor that binds specifically to each half of the IIR. However, this pair of IIR (−73 to −110) was not sufficient for bidirectional transcription activity. At least one copy of a 27-bp oligodeoxyribonucleotide (oligo), which spans − 94 to−120, was required in order to facilitate bidirectional transcription activity. From mobility shift assays using HeLa cell nuclear extracts and this 27-bp oligo, we have identified two DNA-protein complexes, both of which are presumably required to initiate bidirectional transcription.

A test of human cDNA synthesis by the polymerase chain reaction

A fast and efficient method to test the quality of human cDNA synthesis based on the polymerase chain reaction (PCR) is described. An aliquot of the cDNA reaction is used as a template for PCR amplification of a segment of the human histidyl-tRNA synthetase gene. The presence of an intron of ∼300 bp in that region of the gene permits the identification of any genomic contamination in the cDNA sample. The same protocol has also been used with other mammalian DNAs with similar results.

Stimulation and partial stabilization of human histidyl-tRNA synthetase by hemoglobin

Histidyl-tRNA synthetase, an enzyme against which antibodies are directed in some patients with polymyositis, has been purified 5000-fold from HeLa cells, but was extremely labile to dilution or on storage at −80°C. In order to facilitate study of the biochemical and immunological properties of the enzyme, a stabilizer was sought. Hemoglobin at 2 mg/ml was found to stimulate the enzyme and also partially preserved the activity of the enzyme stored at a low concentration (< 10μg/ml). Hematin, but not the globin protein, could substitute for hemoglobin in stimulating the enzyme.