histidyl-tRNA Synthetase 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.

Gcn2 structurally mimics and functionally repurposes the HisRS enzyme for the integrated stress response

Protein kinase Gcn2 attenuates protein synthesis in response to amino acid starvation while stimulating translation of a transcriptional activator of amino acid biosynthesis. Gcn2 activation requires a domain related to histidyl-tRNA synthetase (HisRS), the enzyme that aminoacylates tRNAHis. While evidence suggests that deacylated tRNA binds the HisRS domain for kinase activation, ribosomal P-stalk proteins have been implicated as alternative activating ligands on stalled ribosomes. We report crystal structures of the HisRS domain of Chaetomium thermophilum Gcn2 that reveal structural mimicry of both catalytic (CD) and anticodon-binding (ABD) domains, which in authentic HisRS bind the acceptor stem and anticodon loop of tRNAHis. Elements for forming histidyl adenylate and aminoacylation are lacking, suggesting that Gcn2HisRS was repurposed for kinase activation, consistent with mutations in the CD that dysregulate yeast Gcn2 function. Substituting conserved ABD residues well positioned to contact the anticodon loop or that form a conserved ABD-CD interface impairs Gcn2 function in starved cells. Mimicry in Gcn2HisRS of two highly conserved structural domains for binding both ends of tRNA-each crucial for Gcn2 function-supports that deacylated tRNAs activate Gcn2 and exemplifies how a metabolic enzyme is repurposed to host new local structures and sequences that confer a novel regulatory function.

Clinical features associated with the presence of anti-Ro52 and anti-Ro60 antibodies in Jo-1 antibody-positive anti-synthetase syndrome.

Anti-SSA antibodies target two unrelated proteins, Ro52 (E3 ligase) and Ro60 (RNA binding protein). Previous studies indicate that anti-Ro52 antibodies are frequently associated with various myositis-specific autoantibodies (MSAs)-including anti-tRNA synthetase antibodies-and that the coexistence of MSAs and anti-Ro52 antibodies may portend worse clinical outcomes. Although not well-described in the setting of myositis, work from our animal model of HRS (histidyl-tRNA synthetase)-induced myositis suggests that anti-Ro60 antibodies may also be linked to specific MSAs such as anti-HRS/Jo-1. We therefore aimed to demonstrate the prevalence and clinical characteristics of Ro52 and Ro60 antibody positivity in patients possessing Jo-1 antibodies. To establish the immunological link between anti-synthetase, anti-Ro52, and anti-Ro60 antibodies, we evaluated the relative titers of these antibodies in blood and bronchoalveolar lavage fluid (BALF) of mice following immunization with HRS/Jo-1. In parallel, we used ELISA-based approaches to assess sera from 177 anti-Jo1 antibody-positive patients for the presence of anti-Ro52 and/or anti-Ro60 antibodies. We then determined statistical associations between co-existing anti-Jo-1, anti-Ro52, and/or anti-Ro60 antibodies and clinical manifestations associated with the anti-synthetase syndrome. Mice immunized with HRS had higher levels of anti-Ro52 and anti-Ro60 antibodies in serum and BALF than PBS-immunized mice. In 177 anti-Jo-1 antibody-positive patients, the prevalence of anti-Ro52 and anti-Ro60 antibodies was 36% and 15%, respectively. The frequency of dry eye/dry mouth, interstitial pneumonia, and pulmonary events over time differed between patients with various combinations of anti-Ro52 and anti-Ro60 antibodies. While anti-Ro52 antibodies generally correlated with statistically significant increases in each of these clinical manifestations, the presence of Ro60 antibodies alone was associated with decreased frequency of ILD. Anti-Ro52 and/or anti-Ro60 antibodies are often co-expressed with anti-Jo1 antibodies, defining clinical subsets with different disease course/outcomes.

Antigen-driven T cell-macrophage interactions mediate the interface between innate and adaptive immunity in histidyl-tRNA synthetase-induced myositis.

Previous work in humans has demonstrated that both innate and adaptive immune signaling pathways contribute to the pathogenesis of idiopathic inflammatory myopathy (IIM), a systemic autoimmune disease targeting muscle as well as extra-muscular organs. To better define interactive signaling networks in IIM, we characterized the cellular phenotype and transcriptomic profiles of muscle-infiltrating cells in our established murine model of histidyl-tRNA synthetase (HRS)-induced myositis. Myositis was induced in wild type (WT) and various congenic/mutant strains of C57BL/6 mice through intramuscular immunization with recombinant HRS. Histopathological, immunohistochemical, flow cytometric, and transcriptomic assessments were used to characterize the functional relationship between muscle-infiltrating cell populations in these strains lacking different components of innate and/or adaptive immune signaling. RAG1 KO mice developed markedly reduced muscle inflammation relative to WT mice, demonstrating a key requirement for T cells in driving HRS-induced myositis. While the reduction of mononuclear cell infiltrates in CD4-Cre.MyD88fl/fl conditional knockout mice and OT-II TCR transgenic mice highlighted roles for both innate and TCR-mediated/adaptive immune signaling in T cells, diminished inflammation in Lyz2-Cre.MyD88fl/fl conditional knockout mice underscored the importance of macrophage/myeloid cell populations in supporting T cell infiltration. Single cell RNA sequencing-based clustering of muscle-infiltrating subpopulations and associated pathway analyses showed that perturbations of T cell signaling/function alter the distribution and phenotype of macrophages, fibroblasts, and other non-lymphoid cell populations contributing to HRS-induced myositis. Overall, HRS-induced myositis reflects the complex interplay between multiple cell types that collectively drive a TH1-predominant, pro-inflammatory tissue phenotype requiring antigen-mediated activation of both MyD88- and TCR-dependent T cell signaling pathways.

Highly Sensitive Colorimetric Detection of Histidine Using Histidyl-tRNA Synthetase as the Bioreceptor

This study investigated the enzymatic detection of histidine among the 20 amino acids using histidyl-tRNA synthetase (HisRS) as the histidine recognition element. Previously, we studied the binding ability of HisRS and used it as the recognition element in a biosensor; however, in this study, we focused on improving the sensitivity and expanding the dynamic range of histidine analysis using HisRS, as well as analyzing the histidine concentration in real food samples for practical application. Previous studies, as well as our own, have indicated that the binding of HisRS, is strongly affected by the concentrations of 5′-triphosphate (ATP) and magnesium chloride (MgCl2) in the HisRS reaction buffer. Ammonium molybdate or Trinder’s reagent was used for the coloring reaction. Upon optimization of the reaction conditions, we found that 40 mM ATP and 50 mM MgCl2 yielded a linear relationship from 1 to 150 μM histidine (correlation coefficient: r = 0.960). Compared to our previous methods, this method detected histidine over a wider range and with higher sensitivity. Moreover, in real food samples, the plots of spiked histidine were in good agreement with the histidine calibration curve, with a measurement error range of within ± 4.9%.

Efzofitimod: a novel anti-inflammatory agent for sarcoidosis.

Efzofitimod is a first-in-class biologic based on a naturally occurring splice variant of histidyl-tRNA synthetase (HARS) that downregulates immune responses via selective modulation of neuropilin-2 (NRP2). Preclinical data found high expression of NRP2 in sarcoidosis granulomas. Treatment with efzofitimod reduced the granulomatous inflammation induced by P. acnes in an animal model of sarcoidosis. A dose escalating trial of efzofitimod in sarcoidosis with chronic symptomatic pulmonary disease found that treatment with efzofitimod was associated with improved quality of life with a trend towards reduced glucocorticoid use and stable to improved pulmonary function. These studies have led to a large Phase 3 trial of efzofitimod in symptomatic pulmonary sarcoidosis.

Evaluation of immunomodulatory potential of recombinant histidyl-tRNA synthetase (rLdHisRS) protein of Leishmania donovani as a vaccine candidate against visceral leishmaniasis

Visceral leishmaniasis is neglected tropical protozoan disease caused by Leishmania donovani and are associated with high fatality rate in developing countries since prophylactic vaccines are not available. In the present study, we evaluated the immunomodulatory potential of L. donovani histidyl-tRNA synthetase (LdHisRS) and predicted the epitopes using immunoinformatic tools. Histidyl-tRNA synthetase (HisRS) is a class IIa aminoacyl t-RNA synthetase enzyme (aaRS) required for histidine incorporation into proteins during protein synthesis. The recombinant LdHisRS protein (rLdHisRS) was expressed in E coli BL-21cells, and its immunomodulatory role was assessed in J774A.1 murine macrophage and in BALB/c mice, respectively. LdHisRS specifically stimulated and triggered enhance cell proliferation, nitric oxide release and IFN-γ (70%; P < 0.001), and IL-12 (55.37%; P < 0.05) cytokine release in vitro, whereas BALB/c mice immunized with rLdHisRS show higher NO release (80.95%; P<0.001), higher levels of Th1 cytokines IFN-γ (14%; P < 0.05), TNF-α (34.93%; P < 0.001), and IL-12 (28.49%; P < 0.001) and robust IgG (p<0.001) and IgG2a (p<0.001) production. We also identified 20 Helper T-lymphocytes (HTLs), 30 cytotoxic T lymphocytes (CTLs), and 18 B-cell epitopes from HisRS protein of L. donovani. All these epitopes can be further used to make a multiepitope vaccine against L. donovani.

Recognition of tRNAHis in an RNase P-Free Nanoarchaeum.

The 5' extra guanosine with 5'-monophosphate at position -1 (G-1) of tRNAHis (p-tRNAHis) is a nearly universal feature that establishes tRNAHis identity. G-1 is either genome encoded and retained after processing by RNase P (RNase P) or posttranscriptionally incorporated by tRNAHis guanylyltransferase (Thg1) after RNase P cleavage. However, RNase P is not found in the hyperthermophilic archaeum Nanoarchaeum equitans; instead, all of its tRNAs, including tRNAHis, are transcribed as leaderless tRNAs with 5'-triphosphate (ppp-tRNAs). How N. equitans histidyl-tRNA synthetase (NeHisRS) recognizes its cognate tRNA (NetRNAHis) is of particular interest. In this paper, we show that G-1 serves as the major identity element of NetRNAHis, with its anticodon performing a similar role, though to a lesser extent. Moreover, NeHisRS distinctly preferred p-tRNAHis over ppp-tRNAHis (~5-fold difference). Unlike other prokaryotic HisRSs, which strongly prefer tRNAHis with C73, this enzyme could charge tRNAsHis with A73 and C73 with nearly equal efficiency. As a result, mutation at the C73-recognition amino acid residue Q112 had only a minor effect (<2-fold reduction). This study suggests that NeHisRS has evolved to disregard C73, but it still maintains its evolutionarily preserved preference toward tRNAHis with 5'-monophosphate. IMPORTANCE Mature tRNAHis has, at its 5'-terminus, an extra guanosine with 5'-monophosphate, designated G-1. G-1 is the major recognition element for histidyl-tRNA synthetase (HisRS), regardless of whether it is of eukaryotic or prokaryotic origin. However, in the hyperthermophilic archaeum Nanoarchaeum equitans, all its tRNAs, including tRNAHis, are transcribed as leaderless tRNAs with 5'-triphosphate. This piqued our curiosity about whether N. equitans histidyl-tRNA synthetase (NeHisRS) prefers tRNAHis with 5'-triphosphate. We show herein that G-1 is still the major recognition element for NeHisRS. However, unlike other prokaryotic HisRSs, which strongly prefer tRNAHis with C73, this enzyme shows almost the same preference for C73 and A73. Most intriguingly, NeHisRS still prefers 5'-monophosphate over 5'-triphosphate. It thus appears that the preference of HisRS for tRNAHis with 5'-monophosphate emerged very early in evolution.

Towards a Cure for HARS Disease.

Histidyl-tRNA synthetase (HARS) ligates histidine to its cognate transfer RNA (tRNAHis). Mutations in HARS cause the human genetic disorders Usher syndrome type 3B (USH3B) and Charcot-Marie-Tooth syndrome type 2W (CMT2W). Treatment for these diseases remains symptomatic, and no disease specific treatments are currently available. Mutations in HARS can lead to destabilization of the enzyme, reduced aminoacylation, and decreased histidine incorporation into the proteome. Other mutations lead to a toxic gain-of-function and mistranslation of non-cognate amino acids in response to histidine codons, which can be rescued by histidine supplementation in vitro. We discuss recent advances in characterizing HARS mutations and potential applications of amino acid and tRNA therapy for future gene and allele specific therapy.

Histidine supplementation can escalate or rescue HARS deficiency in a Charcot-Marie-Tooth disease model.

Aminoacyl-tRNA synthetases are essential enzymes responsible for charging amino acids onto cognate tRNAs during protein synthesis. In histidyl-tRNA synthetase (HARS), autosomal dominant mutations V133F, V155G, Y330C and S356N in the HARS catalytic domain cause Charcot-Marie-Tooth disease type 2W (CMT2W), while tRNA-binding domain mutation Y454S causes recessive Usher syndrome type IIIB. In a yeast model, all human HARS variants complemented a genomic deletion of the yeast ortholog HTS1 at high expression levels. CMT2W associated mutations, but not Y454S, resulted in reduced growth. We show mistranslation of histidine to glutamine and threonine in V155G and S356N but not Y330C mutants in yeast. Mistranslating V155G and S356N mutants lead to accumulation of insoluble proteins, which was rescued by histidine. Mutants V133F and Y330C showed the most significant growth defect and decreased HARS abundance in cells. Here, histidine supplementation led to insoluble protein aggregation and further reduced viability, indicating histidine toxicity associated with these mutants. V133F proteins displayed reduced thermal stability in vitro, which was rescued by tRNA. Our data will inform future treatment options for HARS patients, where histidine supplementation may either have a toxic or compensating effect depending on the nature of the causative HARS variant.

Human Thg1 displays tRNA-inducible GTPase activity

tRNAHis guanylyltransferase (Thg1) catalyzes the 3′-5′ incorporation of guanosine into position -1 (G-1) of tRNAHis. G-1 is unique to tRNAHis and is crucial for recognition by histidyl-tRNA synthetase (HisRS). Yeast Thg1 requires ATP for G-1 addition to tRNAHis opposite A73, whereas archaeal Thg1 requires either ATP or GTP for G-1 addition to tRNAHis opposite C73. Paradoxically, human Thg1 (HsThg1) can add G-1 to tRNAsHis with A73 (cytoplasmic) and C73 (mitochondrial). As N73 is immediately followed by a CCA end (positions 74–76), how HsThg1 prevents successive 3′-5′ incorporation of G-1/G-2/G-3 into mitochondrial tRNAHis (tRNAmHis) through a template-dependent mechanism remains a puzzle. We showed herein that mature native human tRNAmHis indeed contains only G-1. ATP was absolutely required for G-1 addition to tRNAmHis by HsThg1. Although HsThg1 could incorporate more than one GTP into tRNAmHisin vitro, a single-GTP incorporation prevailed when the relative GTP level was low. Surprisingly, HsThg1 possessed a tRNA-inducible GTPase activity, which could be inhibited by ATP. Similar activity was found in other high-eukaryotic dual-functional Thg1 enzymes, but not in yeast Thg1. This study suggests that HsThg1 may downregulate the level of GTP through its GTPase activity to prevent multiple-GTP incorporation into tRNAmHis.

The Antiepileptic Valproic Acid Ameliorates Charcot-Marie-Tooth 2W (CMT2W) Disease-Associated HARS1 Mutation-Induced Inhibition of Neuronal Cell Morphological Differentiation Through c-Jun N-terminal Kinase.

Hereditary peripheral neuropathies called Charcot-Marie-Tooth (CMT) disease affect the sensory nerves as well as motor neurons. CMT diseases are composed of a heterogeneous group of diseases. They are characterized by symptoms such as muscle weakness and wasting. Type 2 CMT (CMT2) disease is a neuropathy with blunted or disrupted neuronal morphological differentiation phenotypes including process formation of peripheral neuronal axons. In the early stages of CMT2, demyelination that occurs in Schwann cells (glial cells) is rarely observed. CMT2W is an autosomal-dominant disease and is responsible for the gene encoding histidyl-tRNA synthetase 1 (HARS1), which is a family molecule of cytoplasmic aminoacyl-tRNA synthetases and functions by ligating histidine to its cognate tRNA. Despite increasing knowledge of the relationship of mutations on responsible genes with diseases, it still remains unclear how each mutation affects neuronal differentiation. Here we show that in neuronal N1E-115 cells, a severe Asp364-to-Tyr (D364Y) mutation of HARS1 leads to formation of small aggregates of HARS1 proteins; in contrast, wild type proteins are distributed throughout cell bodies. Expression of D364Y mutant proteins inhibited process formation whereas expression of wild type proteins possessed the normal differentiation ability to grow processes. Pretreatment with the antiepileptic valproic acid recovered inhibition of process formation by D364Y mutant proteins through the c-Jun N-terminal kinase signaling pathway. Taken together, these results indicate that the D364Y mutation of HARS1 causes HARS1 proteins to form small aggregates, inhibiting process growth, and that these effects are recovered by valproic acid. This could be a potential therapeutic drug for CMT2W at the cellular levels.

Are sputum autoantibodies more clinically relevant in idiopathic pulmonary fibrosis than serum autoantibodies?

Background:The adaptive immune system plays a role in the pathogenesis of idiopathic pulmonary fibrosis (IPF) has been reported previously. However, the association between airway and circulating autoantibodies (AAbs) levels is unclear. The aim of this study is to investigate the link between the AAb levels in airway and circulation in stable patients with IPF.Materials and Methods:From June 2016 to March 2017, 21 stable IPF patients and 22 healthy volunteers were recruited. We established Luminex interacting AAbs with bead-antigen complex to detect the immunoglobulin G antibodies levels of ten autoantigens which were matched serum (Se) and sputum (Sp) samples collected from recruited subjects, including Smith (Sm), Anti-ribosomal P antibody (P0), Sjögren syndrome type A antigen (SSA), La/Sjögren syndrome type B antigen (SSB), DNA topoisomerase (Scl-70), histidyl-tRNA synthetase (Jo-1), U1 small nuclear ribonucleoprotein (U1-SnRNP), thyroid peroxidase, Proteinase 3, and Myeloperoxidase. Spearman's rank correlation matrix was applied to explore the associations of Ab profiles between Se and Sp.Results:For IPF patients, Spearman's correlation matrix showed multiple intercorrelations among Sp-AAbs and Sp-AAbs (P < 0.05), while only the levels of AAb against Sm and anti-La in Se were correlated with those Sp-AAb counterparts (P < 0.05). For healthy individuals, only anti-La in Se was associated with those Sp-AAb counterparts (P < 0.05). For IPF patients, there was a positive correlation between carbon monoxide diffusing capacity (DLCO)% predicted and Sp-anti-P0 level (r = 0.464, P = 0.034). Forced vital capacity% predicted was positively correlated with Sp-anti-Scl-70 level (r = 0.466, P = 0.033).Conclusion:Comparing to Se-AAbs, Sp-AAbs are more associated with clinical parameters in the patients with IPF. In order to better understand the role of autoimmunity in the pathogenesis of IPF, detection of Sp-AAbs for local autoimmune responses may be a good choice.

Disruption of Hars2 in Cochlear Hair Cells Causes Progressive Mitochondrial Dysfunction and Hearing Loss in Mice

Mutations in a number of genes encoding mitochondrial aminoacyl-tRNA synthetases lead to non-syndromic and/or syndromic sensorineural hearing loss in humans, while their cellular and physiological pathology in cochlea has rarely been investigated in vivo. In this study, we showed that histidyl-tRNA synthetase HARS2, whose deficiency is associated with Perrault syndrome 2 (PRLTS2), is robustly expressed in postnatal mouse cochlea including the outer and inner hair cells. Targeted knockout of Hars2 in mouse hair cells resulted in delayed onset (P30), rapidly progressive hearing loss similar to the PRLTS2 hearing phenotype. Significant hair cell loss was observed starting from P45 following elevated reactive oxygen species (ROS) level and activated mitochondrial apoptotic pathway. Despite of normal ribbon synapse formation, whole-cell patch clamp of the inner hair cells revealed reduced calcium influx and compromised sustained synaptic exocytosis prior to the hair cell loss at P30, consistent with the decreased supra-threshold wave I amplitudes of the auditory brainstem response. Starting from P14, increasing proportion of morphologically abnormal mitochondria was observed by transmission electron microscope, exhibiting swelling, deformation, loss of cristae and emergence of large intrinsic vacuoles that are associated with mitochondrial dysfunction. Though the mitochondrial abnormalities are more prominent in inner hair cells, it is the outer hair cells suffering more severe cell loss. Taken together, our results suggest that conditional knockout of Hars2 in mouse cochlear hair cells leads to accumulating mitochondrial dysfunction and ROS stress, triggers progressive hearing loss highlighted by hair cell synaptopathy and apoptosis, and is differentially perceived by inner and outer hair cells.

Retinal Gene Therapy for Usher Syndrome: Current Developments, Challenges, and Perspectives.

Usher syndrome (USH) represents a group of genetically heterogenous autosomal recessive disorders, characterized by combined vision and sensorineural hearing loss, and in some cases vestibular dysfunction.1–3 It primarily affects the light-sensitive photoreceptor cells in the retina and the auditory hair cells in the cochlea. Vision loss in all cases is progressive, and manifests as retinitis pigmentosa (RP), characterized by the gradual degeneration of the photoreceptor cells.4 Peripheral rod function is lost first, leading to night blindness and constricted visual fields, followed by the death of cones and severe visual impairment. There are currently no drugs or biological therapies proven to be effective in treating USH syndrome. Cochlear implantation, which bypasses the damaged hair cells and stimulates the primary auditory neurons directly, is an effective approach to alleviate the hearing impairment in patients. However, there is no remedy for the progressive loss of the photoreceptor neurons in the retina, and thus a critical unmet need exists to develop therapeutic strategies to prevent blindness in USH syndrome. The generation of animal models that faithfully mimic the human USH disorder, the timing of gene therapy interventions, the identification of correct target cells and patient selection are key factors in successfully reaching this goal. USH syndrome was named after Dr Charles Howard Usher, a Scottish ophthalmologist who described 69 patients with the disease in 1914.5 As is commonly the case in medicine, the disease was first described much earlier (1858) in brothers suffering from blindness and deafness by von Grafe6 and was further characterized by his student, Liebreich,7 3 years later in a larger population of patients. From its discovery until recently, USH syndrome and its subgroups were delineated by clinical characteristics.8 Now the disease is recognized by symptomology and classified by genotyping. To date, there are at least 10 causative genes that are associated with USH syndrome, summarized in previous reviews.1,2,9 In general, based on its diverse clinical symptoms, particularly the onset and severity of the sensorineural hearing impairment and the presence of vestibular dysfunction, USH syndrome is grouped into 3 clinical subtypes. Type I disease is associated with severe-to-profound prelingual hearing loss, vestibular abnormalities and prepubescent onset of RP symptoms.10,11 USH1 results from mutations in MYO7A (USH1B), HARMONIN (USH1C), CDH23 (USH1D), PCDH15 (USH1F), SANS (USH1G), and CIB2 (USH1J). Patients with type II disease display moderate-to-severe congenital hearing loss without vestibular dysfunction, and account for >50% of all USH cases. Their RP symptoms generally begin in the second decade.12 USH2 is caused by mutations in USH2A (USH2A), ADGRV1 (USH2C), and WHRN (USH2D). Patients with type III disease caused by mutations in CLRN1 (USH3A) gene have postlingual progressive hearing loss, variable vestibular dysfunction, and variable onset of RP that rapidly progresses to legal blindness by the fourth decade of life.13–17 Mutations in the HARS gene (Histidyl-tRNA synthetase) are associated with the ultra-rare form USH3B.18,19 Other genes have been described in cases of atypical USH, including: CEP250 and CEP78, encoding members of the CEP family of centrosome-associated proteins; ESPN, encoding the F-actin cross-linker espin; ARSG, encoding the arylsulfatase G enzyme.20

Benzopyrazine-Based Small Molecule Inhibitors As Trypanocidal and Leishmanicidal Agents: Green Synthesis, In Vitro, and In Silico Evaluations.

World Health Organization (WHO) identified twenty tropical disease categories as neglected tropical diseases (NTDs)1. Chagas’ disease (also known as American trypanosomiasis) and leishmaniasis are two major classes of NTDs. The total number of mortality, morbidity, and disability attributed each year due to these two categories of diseases in magnitudes is much higher than the so-called elite diseases like cancer, diabetes, AIDS, cardiovascular and neurodegenerative diseases. Impoverished communities around the world are the major victim of NTDs. The development of new and novel drugs in the battle against Chagas’ disease and leishmaniasis is highly anticipated. An easy and straightforward on-water green access to synthesize benzopyrazines is reported. This ultrasound-assisted procedure does not require any catalyst/support/additive/hazardous solvents and maintains a high atom economy. A series of eleven benzopyrazines has been synthesized, and most of the synthesized compounds possess the drug-likeness following Lipinski’s “Rule of 5”. Benzopyrazines 3 and 4 demonstrated moderate leishmanicidal activity against L. mexicana (M378) strain. The selective lead compound 1 showed good leishmanicidal, and trypanocidal activities (in vitro) against both L. mexicana (M378) and T. cruzi (NINOA) strains compared to the standard controls. The in vitro trypanocidal and leishmanicidal activities of the lead compound 1 have been validated by molecular docking studies against four biomolecular drug targets viz. T. cruzi histidyl-tRNA synthetase, T. cruzi trans-sialidase, leishmanial rRNA A-site, and leishmania major N-myristoyl transferase.

Amino acid activation analysis of primitive aminoacyl-tRNA synthetases encoded by both strands of a single gene using the malachite green assay

The Rodin-Ohno hypothesis postulates that two classes of aminoacyl-tRNA synthetases were encoded complementary to double-stranded DNA. Particularly, Geobacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS, belonging to class I) and Escherichia coli histidyl-tRNA synthetase (HisRS, belonging to class II) show high complementarity of the middle base of the codons in the mRNA sequence encoding each ATP binding site. Here, for the reported 46-residue peptides designed from the three-dimensional structures of TrpRS and HisRS, amino acid activation analysis was performed using the malachite green assay, which detects the pyrophosphate departing from ATP in the forward reaction of the first step of tRNA aminoacylation. A maltose-binding protein fusion with the 46 residues of TrpRS (TrpRS46mer) exhibited high activation capacity for several amino acids in the presence of ATP and amino acids, but the activity of an alanine substitution mutant of the first histidine in the HIGH motif (TrpRS46merH15A) was largely reduced. In contrast, pyrophosphate release by HisRS46mer in the histidine activation step was lower than that in the case of TrpRS46mer. Both HisRS46mer and the alanine mutant at the 113th arginine (HisRS46merR113A) showed slightly higher levels of pyrophosphate release than the maltose-binding protein alone. These results do not rule out the Rodin-Ohno hypothesis, but may suggest the necessity of establishing unique evolutionary models from different perspectives.

Epidemiology of Idiopathic Inflammatory Myopathies in Africa: A Contemporary Systematic Review.

The epidemiology of idiopathic inflammatory myopathies (IIMs) has been extensively studied in America, Europe, and Asia, but remains unclear in Africa. The aim of this review was to summarize available data on the epidemiology of IIMs in Africa. We searched MEDLINE, EMBASE, and African Journals Online for studies published up to December 30, 2020, and reporting epidemiological data on IIMs in Africa. Data were combined through narrative synthesis. The review protocol was registered with PROSPERO, CRD42020186781. We included 39 studies reporting 683 cases (71.7% adults) of IIMs. Incidence rates of ~7.5/1,000,000 person-years and 1.2/1,000,000 person-years were estimated for dermatomyositis (DM), whereas polymyositis (PM) had an incidence rate of 8.8/1,000,000 person-years. Prevalence estimates of 11.49/100,000 and 11/100,000 (95% confidence interval, 0-32) were provided for IIMs and the PM subtype, respectively. Mean age at diagnosis ranged from 7.9 to 57.2 years, and 50% to 100% of the patients were females. Main subtypes of adult-onset IIMs were DM (21%-93%) and PM (12%-79%), whereas the commonest juvenile subtype was juvenile DM (5.8%-9%). Skeletal muscle involvement (56%-100%) was the main disease feature, and esophagus was the most commonly affected internal organ (6%-65.2%). Anti-Jo1/histidyl tRNA synthetase (7%-100%) and anti-Mi2 (17%-45%) antibodies were the most frequent myositis specific antibodies. Early mortality was high (7.8%-45%), and main death causes were infections, cancers and organ damage in respiratory and cardiovascular domains. Apart from a potential younger age at onset of adult IIMs in Africa, current sparse data mostly suggest a similar epidemiology between Africa and other regions. Further high-quality studies are required to validate these findings.

TRANSFER RNA QUEUOSINE MODIFICATION ENZYME MANIPULATES TIGHT JUNCTION PROTEINS IN INFLAMMATORY BOWEL DISEASE

Abstract Background Transfer RNA (tRNA) queuosine (Q)-modification occur at the wobble anticodon position of four special cellular tRNAs. In eukaryotes, tRNA-Q modification relies on the intestinal microbial product queuine and eukaryotic tRNA-guanine transglycosylase complex contains of Q tRNA ribosyltransferase catalytic subunit 1 (QTRT1). Q-tRNA modification is critical for the fidelity and accuracy to translate RNA to protein. It is known that dysfunction of Q-tRNA associates with cancer proliferation and malignancy. However, mechanisms of how Q and Q-tRNA modifications influence intestinal epithelial biology and chronic inflammation are unknown. Methods We explored the expression of QTRT1 in patients with IBD by investigating human biopsy tissues and re-analyzing gene expression datasets (with both UC and CD patients). To further investigate the QTRT1 role in IBD, the dextran sodium sulfate (DSS) induced colitis mouse model and IL10-deficient mouse model were used to evaluate the molecular mechanism. We further investigated the impact of QTRT1 reduction on intestinal cells by generating QTRT1 knockdown HCT116 and Caco-2 BBE cells using a Double Nickase Plasmid. Results We found that the QTRT1 expression in IHC staining was significantly downregulated in the colon of both Ulcerative Colitis (UC) and Crohn’s Disease (CD) patients compared with normal controls. After extracting and analyzing the datasets, we found the gene expression of QTRT1 was significantly suppressed in colonic mucosa of UC and intestinal mucosa of CD, compared with controls. Moreover, the four tRNA-Q-related tRNA synthetase (asparaginyl-tRNA synthetase, aspartyl-tRNA synthetase, histidyl-tRNA synthetase and tyrosyl-tRNA synthetase) were also decreased in IBD patients. The decreased QTRT1-expression was further confirmed in both DSS-induced colitis mice and IL10-defecient mice. Meanwhile, the significantly downregulated beta-catenin and Claudin-5, and upregulated Caludin-2 were identified in these two colitis models. All these molecular alterations, which are important in cell adhesion and tight junction formation, were further verified in protein expression and mRNA level using QTRT1 reduction HCT116 and Caco-2 BBE cells in vitro. Conclusion Our results demonstrate that the QTRT1 plays a novel role in human IBD by altering intestinal cell adhesion and junctions. Microbiome-dependent Q supply can be altered during disease development or when a limited variety of food-types are ingested. Investigations on the gut microbiome-derived tRNA modification in IBD will uncover novel molecular mechanisms for potential prevention and therapy.

Jo-1 autoantigen-specific B cells are skewed towards distinct functional B cell subsets in anti-synthetase syndrome patients

BackgroundAnti-Jo-1 autoantibodies which recognize histidyl-tRNA synthetase identify patients with the rare rheumatologic disease, anti-histidyl-tRNA synthetase syndrome (Jo-1 ARS), a phenotypically distinct subset of idiopathic inflammatory myopathies (IIM). Jo-1-binding B cells (JBCs) are implicated in disease pathogenesis, yet they have not been studied directly. We therefore aimed to characterize JBCs to better understand how they expand and function in Jo-1 ARS.MethodsWe enrolled 10 IIM patients diagnosed with Jo-1 ARS, 4 patients with non-Jo-1 IIM, and 8 age- and sex-matched healthy controls. We phenotypically characterized peripheral blood mononuclear cells (PBMCs) ex vivo using flow cytometry to define the B cell subsets in which JBCs reside. We further tested their ability to differentiate into antibody-secreting cells following stimulation in vitro.ResultsThe majority of JBCs were IgM+ (not class-switched). Compared to non-JBCs in the same donors, JBCs contained a higher percentage of autoimmune-prone CD21lo cells and were increased in the CD21lo IgM+ IgD− CD27+ memory subset relative to healthy donor B cells. Whereas non-JBCs were present in the anergic BND B cell subset, JBCs were nearly absent from this compartment. JBCs were detected among plasmablasts in some donors, but a reduced frequency of JBCs differentiated into CD38hi24− plasmablasts compared to non-JBCs present in the same wells following in vitro stimulation.ConclusionsJBCs are enriched for autoimmune-prone CD21lo B cells, some of which exhibit a memory phenotype in the peripheral repertoire of Jo-1 ARS patients. JBCs undergo limited class switch and show reduced capacity to differentiate into antibody-secreting cells. This suggests complex B cell biology exists beyond class-switched cells that differentiate to secrete anti-Jo-1 autoantibody (i.e., what is captured through serum autoantibody studies). New Jo-1 ARS therapies should thus ideally target non-class-switched JBCs in addition to those that have undergone IgG class-switching to most effectively block cross-talk with autoreactive T cells.