histidyl-tRNA Synthetase Gene Research Articles

Characterization of a novel heterozygous variant in the histidyl-tRNA synthetase gene associated with Charcot-Marie-Tooth disease type 2W.

Heterozygous pathogenic variants in the histidyl-tRNA synthetase (HARS) gene are associated with Charcot-Marie-Tooth (CMT) type 2W disease, classified as an axonal peripheral neuropathy. To date, at least 60 variants causing CMT symptoms have been identified in seven different aminoacyl-tRNA synthetases, with eight being found in the catalytic domain of HARS. The genetic data clearly show a causative role of aminoacyl-tRNA synthetases in CMT; however, the cellular mechanisms leading to pathology can vary widely and are unknown in the case of most identified variants. Here we describe a novel HARS variant, c.412T>C; p.Y138H, identified through a CMT gene panel in a patient with peripheral neuropathy. To determine the effect of p.Y138H we employed a humanized HARS yeast model and recombinant protein biochemistry, which identified a deficiency in protein dimerization and a growth defect which shows mild but significant improvement with histidine supplementation. This raises the potential for a clinical trial of histidine.

Histamine production in Lactobacillus vaginalis improves cell survival at low pH by counteracting the acidification of the cytosol

Histamine, one of the most toxic and commonly encountered biogenic amines (BA) in food, is produced by the microbial decarboxylation of histidine. It may accumulate at high concentrations in fish and fermented food. Cheese has some of the highest histamine concentrations, the result of the histidine-decarboxylase activity of certain lactic acid bacteria (LAB). The present work describes the nucleotide sequence and transcriptional organization of the gene cluster responsible for histamine biosynthesis (the HDC cluster) in Lactobacillus vaginalis IPLA 11064 isolated from cheese. The influence of histidine availability and pH on histamine production and the expression of the HDC cluster genes is also examined. As expected, the results suggest that the production of histamine under acidic conditions improves cell survival by maintaining the cytosol at an appropriate pH. However, the transcriptional regulation of the HDC cluster is quite different from that described in other dairy histamine-producing LAB, probably due to the lack of a termination-antitermination system in the histidyl-tRNA synthetase gene (hisS).

Overexpression of mitochondrial histidyl-tRNA synthetase restores mitochondrial dysfunction caused by a deafness-associated tRNAHis mutation.

The deafness-associated m.12201T>C mutation affects the A5-U68 base-pairing within the acceptor stem of mitochondrial tRNAHis The primary defect in this mutation is an alteration in tRNAHis aminoacylation. Here, we further investigate the molecular mechanism of the deafness-associated tRNAHis 12201T>C mutation and test whether the overexpression of the human mitochondrial histidyl-tRNA synthetase gene (HARS2) in cytoplasmic hybrid (cybrid) cells carrying the m.12201T>C mutation reverses mitochondrial dysfunctions. Using molecular dynamics simulations, we demonstrate that the m.12201T>C mutation perturbs the tRNAHis structure and function, supported by decreased melting temperature, conformational changes, and instability of mutated tRNA. We show that the m.12201T>C mutation-induced alteration of aminoacylation tRNAHis causes mitochondrial translational defects and respiratory deficiency. We found that the transfer of HARS2 into the cybrids carrying the m.12201T>C mutation raises the levels of aminoacylated tRNAHis from 56.3 to 75.0% but does not change the aminoacylation of other tRNAs. Strikingly, HARS2 overexpression increased the steady-state levels of tRNAHis and of noncognate tRNAs, including tRNAAla, tRNAGln, tRNAGlu, tRNALeu(UUR), tRNALys, and tRNAMet, in cells bearing the m.12201T>C mutation. This improved tRNA metabolism elevated the efficiency of mitochondrial translation, activities of oxidative phosphorylation complexes, and respiration capacity. Furthermore, HARS2 overexpression markedly increased mitochondrial ATP levels and membrane potential and reduced production of reactive oxygen species in cells carrying the m.12201T>C mutation. These results indicate that HARS2 overexpression corrects the mitochondrial dysfunction caused by the tRNAHis mutation. These findings provide critical insights into the pathophysiology of mitochondrial disease and represent a step toward improved therapeutic interventions for mitochondrial disorders.

Abstract 2728: Circulating levels of Resokine, a soluble modulator of the immune system, are upregulated in both experimental cancer models and in patients across multiple tumor types

Abstract Resokine is a newly identified regulator of immune cell activity, and circulating levels of Resokine in normal individuals may represent a soluble set-point control to modulate T cell activity. Resokine activity is a non-canonical function arising from the tRNA synthetase gene family, and the activity is effected by a 60 amino acid N-terminal domain arising from the gene for histidyl-tRNA synthetase which is present in the full-length protein as well as multiple splice variants that have lost their original tRNA synthetase functionality. Resokine is secreted from cells, including tumor cell lines, and in vitro studies have demonstrated that Resokine can inhibit the activation of immune cells. In vitro, for example, Resokine addition during T cell activation induced by antibodies to CD3 and CD28, can result in reduced levels of inflammatory cytokines such as IL-2, interferon gamma, and TNF alpha; inhibition of the up-regulation of cell-surface activation markers, such as CD69, CD40L and 4-1BB; and inhibition of release of the cytotoxic mediator granzyme B. We have tested levels of circulating Resokine in both mice with syngeneic tumors as well as >300 cancer patients across multiple tumor types. In normal C57Bl6 mice serum levels of Resokine ranged from 70-250pM (n=10) whereas in mice bearing B16F10 tumors, levels were significantly higher (450-3000pM, p<0.001) and correlated with tumor size. Resokine levels in normal human volunteers exhibit a more variable range, from 8pM to >2333pM (n=148), with 18% of individuals having levels <30pM, which was set as the active threshold level based on the concentration required to inhibit T cell activation in vitro. In contrast, samples across >300 cancer patients with different tumor types, exhibited higher circulating levels with only 4% of individuals having levels below the activity threshold of 30pM. This data is consistent with the hypothesis that tumors secrete Resokine as an additional mechanism to down-regulate immune activity, and suggests further investigation of the utility of Resokine levels as a new biomarker of immune activity in patients. Citation Format: Ryan Adams, Elisabeth Mertsching, Leslie Nangle, Kathy Ogilvie, Steven Crampton, John Bruner, Samantha Tyler, Sanna Rosengren, Andrea Cubitt, David King, John Mendlein. Circulating levels of Resokine, a soluble modulator of the immune system, are upregulated in both experimental cancer models and in patients across multiple tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2728.

Serendipity or prepared mind? Recollections of the KOP translocation (1967) and of one form of Perrault syndrome.

The human X/autosome translocation, designated KOP, was discovered by Dr. Philip D. Pallister in Montana in 1967 in a young man with apparent Klinefelter syndrome. Collaboratively it was possible to elucidate the genetic nature of his unprecedented chromosome rearrangement and its developmental effects in mother and son. In retrospect, these clinical and genetic studies at the height of the somatic cell genetics era (Ruddle, Siniscalco, etc.) presented human genetics with a highly productive opportunity to begin gene mapping of autosomes and the X chromosome. The late Victor McKusick considered the discovery of the KOP translocation, as he determined personally in Montana, one of the major transforming events in human genetics. The Perrault syndrome evaluated in two families in Montana and one in Sicily for familial deafness, primary amenorrhea and neurologic impairment (progressive in some), turned out to be heterogeneous. In the "hands" of Dr. M.-C. King of Seattle four forms of Perrault syndrome have been identified. The autosomal recessive mutation present in the P family studied with Dr. Pallister in Helena, turned out to affect the mitochondrial histidyl tRNA synthetase gene present in prokaryotes, annelids, fungi and mammals, hence, must already have been present in LUCA some 3.8 billion years ago.

Interference with histidyl-tRNA synthetase by a CRISPR spacer sequence as a factor in the evolution of Pelobacter carbinolicus

BackgroundPelobacter carbinolicus, a bacterium of the family Geobacteraceae, cannot reduce Fe(III) directly or produce electricity like its relatives. How P. carbinolicus evolved is an intriguing problem. The genome of P. carbinolicus contains clustered regularly interspaced short palindromic repeats (CRISPR) separated by unique spacer sequences, which recent studies have shown to produce RNA molecules that interfere with genes containing identical sequences.ResultsCRISPR spacer #1, which matches a sequence within hisS, the histidyl-tRNA synthetase gene of P. carbinolicus, was shown to be expressed. Phylogenetic analysis and genetics demonstrated that a gene paralogous to hisS in the genomes of Geobacteraceae is unlikely to compensate for interference with hisS. Spacer #1 inhibited growth of a transgenic strain of Geobacter sulfurreducens in which the native hisS was replaced with that of P. carbinolicus. The prediction that interference with hisS would result in an attenuated histidyl-tRNA pool insufficient for translation of proteins with multiple closely spaced histidines, predisposing them to mutation and elimination during evolution, was investigated by comparative genomics of P. carbinolicus and related species. Several ancestral genes with high histidine demand have been lost or modified in the P. carbinolicus lineage, providing an explanation for its physiological differences from other Geobacteraceae.ConclusionsThe disappearance of multiheme c-type cytochromes and other genes typical of a metal-respiring ancestor from the P. carbinolicus lineage may be the consequence of spacer #1 interfering with hisS, a condition that can be reproduced in a heterologous host. This is the first successful co-introduction of an active CRISPR spacer and its target in the same cell, the first application of a chimeric CRISPR construct consisting of a spacer from one species in the context of repeats of another species, and the first report of a potential impact of CRISPR on genome-scale evolution by interference with an essential gene.

Genomic organization, transcriptional mapping, and evolutionary implications of the human bi-directional histidyl-tRNA synthetase locus (HARS/HARSL)

Histidyl-tRNA synthetase catalyses the covalent ligation of histidine to its cognate tRNA as an early step in protein biosynthesis. In humans, the histidyl-tRNA synthetase gene (HARS) is oriented opposite of a synthetase-like gene (HARSL) that bears striking homology to HARS. In this report, we describe the genomic organization of the HARS/HARSL locus and map multiple transcripts originating from a bi-directional promoter controlling the differential expression of these genes. The HARS and HARSL genes each contain 13 exons with strong structural and sequence homology over exons 3–12. HARS transcripts originate from two distinct promoters; a cluster of short transcripts map 15–65 bp upstream of the HARS ORF while a single, longer transcript (352 bp 5 ′-UTR) maps to a distal promoter. Similarly, multiple HARSL transcripts (mapping 10–198 bp upstream of its ORF) are produced by the shared bi-directional promoter. Human and rodent HARS/HARSL loci are homologous and support a model of inverted gene duplication to explain the emergence of HARSL during mammalian evolution.

Myositis induced by naked DNA immunization with the gene for histidyl-tRNA synthetase.

Polymyositis is regarded as an autoimmune inflammatory muscle disease. A major subgroup of patients have autoantibodies to cellular histidyl-transfer RNA synthetase (HRS). We have analyzed the role of the autoantigen HRS in the induction of murine myositis in a comparative study of inoculation of BALB/c mice with recombinant HRS protein versus naked DNA coding for HRS. Adult BALB/c mice produced antibodies to human HRS following inoculation with HRS protein and adjuvant, but myositis was not observed. Alternatively, expression plasmid DNA constructs encoding full-length and truncated human HRS were inoculated intramuscularly in gene transfer studies. DNA-inoculated mice produced relatively low anti-HRS antibody titers. However, in contrast to recombinant HRS protein-inoculated mice, HRS gene transfer induced pathology with evidence of cellular infiltration of perivascular and endomysial regions of the inoculated muscle. Multiple inoculations of a plasmid construct encoding a hybrid molecule consisting of HRS and the transferrin receptor cytoplasmic tail induced the highest levels of antibodies and persisting cellular infiltration. Unlike HRS, expression of influenza virus hemagglutinin (HA) following inoculation of an HA plasmid did not induce myositis. Transfer of naked DNA constructs expressing HRS is likely to provide valuable information on the autoimmune response to this protein and its role in the development of myositis.

Cloning and characterization of the C. elegans histidyl-tRNA synthetase gene.

In this paper, we report the cloning and sequencing of the C. elegans histidyl-tRNA synthetase gene. The complete genomic sequence, and most of the cDNA sequence, of this gene is now determined. The gene size including flanking and coding regions is 2230 nucleotides long. Three small introns (45-50 bp long) are found to interrupt the open reading frame. The open reading frame translates to 523 amino acids. This putative protein sequence shows extensive homology with the human and yeast histidyl-tRNA the histidyl-tRNA synthetase gene is a single copy gene. Hence, it is very likely that it encodes both the cytoplasmic and the mitochondrial histidyl-tRNA synthetases. It is likely to be trans-spliced since it contains a trans-splice site in its 5' untranslated region.

The histidyl-tRNA synthetase from Streptococcus equisimilis: overexpression in Escherichia coli, purification, and characterization.

We describe the high-level expression of the Streptococcus equisimilis histidyl-tRNA synthetase gene (hisS) in Escherichia coli and the purification and characterization of the gene product. Due to a lack of an efficient E. coli ribosome binding sequence in the hisS gene, the coding region was fused in-frame to the expression vector pT7-7, thereby creating a fusion gene construct (pT7-7recIII), which is under the control of a strong bacteriophage T7 promoter. Another construct (pT-7recII) was used for low level expression of the native histidyl-tRNA synthetase (HisRS). The plasmids were electroporated into E. coli HB101, which already contained pGP1-2. After temperature induction, the fusion HisRS, which has an extra 15 amino acids between the initiator Met and the second amino acid, Lys, was expressed at a level of approximately 18% of total cell protein (approximately 50 mg/liter of bacterial culture). The fusion HisRS was purified to > 99% by a combination of anion exchange and cation exchange chromatography of the S100 fraction. The predicted MWs of the native and fusion proteins are 47,932 and 49,717, respectively. The mass of the active fusion HisRS was estimated to be 94,000 Da by Sephacryl S-200 gel filtration chromatography and 108,200 Da by nondenaturing PAGE. Both methods show that the functional enzyme is a dimer of two identical subunits. SDS-PAGE analysis of purified fusion HisRS with or without reduction showed a single band of M(r) = 53.7 kDa.

Molecular cloning, sequence, structural analysis and expression of the histidyl-tRNA synthetase gene from Streptococcus equisimilis.

The histidyl-tRNA synthetase gene (hisS) from Streptococcus equisimilis was cloned and sequenced. The gene for this aminoacyl-tRNA synthetase has an open reading frame of 1278 nucleotides. The deduced amino acid sequence encodes a protein of 426 amino acids with MW = 47,932. The protein is predicted to be soluble with a pl = 5.27. The protein sequence has extensive overall identity/similarity with the Escherichia coli and the yeast histidyl-tRNA synthetases (approximately 58% and approximately 20%, respectively). A putative promoter for gene transcription lies within two hundred nucleotides of the polypeptide start codon. The enzyme was overexpressed, to a level of about 18% of total cellular protein, as a fusion protein (containing an additional 15 amino acids) in E. coli using the pT7 expression system containing the T7 RNA polymerase/promoter (Tabor and Richardson, Proc. Natl. Acad. Sci. U.S.A. 82:1074-1078, 1985). The predicted MW for the hisS gene product is in good agreement with the size of the fusion protein determined by SDS-PAGE (M(r) = 53,700). Amino acid sequencing of the intact fusion protein and proteolytic fragments confirmed the deduced sequence of the synthetase at many positions throughout the protein. The expressed protein catalyzed the specific aminoacylation of tRNA(His) in vitro.

Sequence and characterization of thegcpEgene ofEscherichia coli

In Escherichia coli, the gene gcpE encodes a protein of unknown function and lies immediately upstream of the hisS gene for histidyl-tRNA synthetase. The nucleic acid sequence of gcpE predicts an open reading frame for a protein of 40 681 Da. The probable transcription terminator of gcpE overlaps the hisS promoter. Haploid cells containing a disrupted gcpE could not be obtained, suggesting that the gcpE gene is essential.

Sequence and characterization of the gcpE gene of Escherichia coli

In Escherichia coli, the gene gcpE encodes a protein of unknown function and lies immediately upstream of the hisS gene for histidyl-tRNA synthetase. The nucleic acid sequence of gcpE predicts an open reading frame for a protein of 40 681 Da. The probable transcription terminator of gcpE overlaps the hisS promoter. Haploid cells containing a disrupted gcpE could not be obtained, suggesting that the gcpE gene is essential.

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.

Anti-Jo-1 Autoantibodies and the Immunopathogenesis of Autoimmune Myositis

Polymyositis and dermatomyositis are inflammatory myopathies characterized by proximal muscle weakness and myopathic electromyographic and histological findings. While the causes of myositis are not known, the close association of these disorders with a spectrum of autoantibodies suggests an etiologic and/or pathogenetic role for autoimmune processes. Of particular interest in this regard are antibodies directed against histidyl as well as other tRNA synthetases which are almost uniquely associated with myositis and may define a distinct subset of patients. Recently we isolated the histidyl tRNA synthetase gene which encodes the autoantigen representing the most frequent target of the myositis autoimmune response. The isolation and expression of this gene has allowed us to investigate both the autoreactive epitopes on histidyl-tRNA synthetase and the extent to which these correlate with functional epitopes on the molecule. As described here, the results of these studies as well as other recent data pertaining to the immunopathogenesis of myositis, provide a framework for delineating the mechanisms which render synthetases and other translation-related proteins autoantigenic in myositis, and allow one to examine the significance of such autoimmune responses in the etiology and pathogenesis of inflammatory myopathy.

Chromosomal localization of human gene for histidyl-tRNA synthetase: clustering of genes encoding aminoacyl-tRNA synthetases on human chromosome 5.

The gene encoding histidyl-tRNA synthetase was localized to human chromosome 5 using human-Chinese hamster ovary cell hybrids. Seven of the functionally related genes encoding the 20 different aminoacyl-tRNA synthetases have not been mapped in humans and four are located on a single chromosome, number 5, which represents less than 7% of the total genome. Thus, there appears to be significant clustering of this group of genes which may reflect their evolutionary relatedness or common factors involved in regulating their expression.

Amplification of the Gene for Histidyl-tRNA Synthetase in Histidinol-Resistant Chinese Hamster Ovary Cells

Histidinol-resistant (HisOHR) mutants with up to a 30-fold increase in histidyl-tRNA synthetase activity have been isolated by stepwise adaptation of wild-type Chinese hamster ovary (CHO) cells to increasing amounts of histidinol in the medium. Immunoprecipitation of [35S]methionine-labeled cell lysates with antibodies to histidyl-tRNA synthetase showed increased synthesis of the enzyme in histidinol-resistant cells. The histidinol-resistant cell lines had an increase in translatable polyadenylated mRNA for histidyl-tRNA synthetase. A cDNA for CHO histidyl-tRNA synthetase has been cloned, using these histidyl-tRNA synthetase-overproducing mutants as the source of mRNA. Southern blot analysis of wild-type and histidinol-resistant cells with this cDNA showed that the histidyl-tRNA synthetase DNA bands were amplified in the resistant cells. These HisOHR cells owed their resistance to histidinol to amplification of the gene for histidyl-tRNA synthetase.

Amplification of the gene for histidyl-tRNA synthetase in histidinol-resistant Chinese hamster ovary cells.

Histidinol-resistant (HisOHR) mutants with up to a 30-fold increase in histidyl-tRNA synthetase activity have been isolated by stepwise adaptation of wild-type Chinese hamster ovary (CHO) cells to increasing amounts of histidinol in the medium. Immunoprecipitation of [35S]methionine-labeled cell lysates with antibodies to histidyl-tRNA synthetase showed increased synthesis of the enzyme in histidinol-resistant cells. The histidinol-resistant cell lines had an increase in translatable polyadenylated mRNA for histidyl-tRNA synthetase. A cDNA for CHO histidyl-tRNA synthetase has been cloned, using these histidyl-tRNA synthetase-overproducing mutants as the source of mRNA. Southern blot analysis of wild-type and histidinol-resistant cells with this cDNA showed that the histidyl-tRNA synthetase DNA bands were amplified in the resistant cells. These HisOHR cells owed their resistance to histidinol to amplification of the gene for histidyl-tRNA synthetase.

The nucleotide sequence of the promoter region of hisS, the structural gene for histidyl-tRNA synthetase

A plasmid has been constructed which carries hisS, the structural gene for histidyl-tRNA synthetase of E. coli, on a 1.6-kb fragment bounded by PvuII and BstEII sites. The DNA sequence of both ends of this fragment was determined. The amino-terminal sequence of histidyl-tRNA synthetase was also determined to locate the promoter proximal coding region and the frame in which it is read. Three promoters were identified by consensus criteria. The region surrounding these promoters contains extensive twofold symmetry.

Strains of Escherichia coli carrying the structural gene for histidyl-tRNA synthetase on a high copy-number plasmid.

That portion of the Escherichia coli chromosome carried by a number of lambda transducing phages, all of which carry the gua operon, was mapped using restriction endonucleases. The DNA from one of these transducing phages was subcloned onto pBR322. We have identified two recombinant plasmids which carry the Escherichia coli gene hisS, the structural gene for histidyl-tRNA synthetase. The two plasmids, pSE301 and pSE401, have in common a 3,540 bp fragment of E. coli DNA which is bounded by BglII and SalI restriction endonuclease recognition sites. Strains carrying these plasmids overproduce histidyl-tRNA synthetase 20 to 30 fold. The growth rate of these strains is not affected although the histidine biosynthetic enzymes are derepressed. This derepression seems to be in addition to that caused by introduction of a hisT mutation on the chromosome.