Functional enrichment analysis reveals the involvement of DARS2 in multiple biological pathways and its potential as a therapeutic target in esophageal carcinoma.

OBJECTIVE: The enzyme Aspartyl tRNA synthetase 2 (DARS2) is a crucial enzyme in the mitochondrial tRNA synthesis pathway, playing a critical role in maintaining normal mitochondrial function and protein synthesis. However, the role of DARS2 in ESCA is unclear. MATERIALS AND METHODS: Transcriptional data of pan-cancer and ESCA were downloaded from UCSC XENA, TCGA, and GEO databases to analyze the differential expression of DARS2 between tumor samples and normal samples, and its correlation with clinicopathological features of ESCA patients. R was used for GO, KEGG, and GSEA functional enrichment analysis of DARS2 co-expression and to analyze the connection of DARS2 with glycolysis and m6A-related genes. In vitro experiments were performed to assess the effects of interfering with DARS2 expression on ESCA cells. TarBase v.8, mirDIP, miRTarBase, ENCORI, and miRNet databases were used to analyze and construct a ceRNA network containing DARS2. RESULTS: DARS2 was overexpressed in various types of tumors. In vitro experiments confirmed that interfering with DARS2 expression significantly affected the proliferation, migration, apoptosis, cell cycle, and glycolysis of ESCA cells. DARS2 may be involved in multiple biological pathways related to tumor development. Furthermore, correlation and differential analysis revealed that DARS2 may regulate ESCA m6A modification through its interaction with METTL3 and YTHDF1. A ceRNA network containing DARS2, DLEU2/has-miR-30a-5p/DARS2, was successfully predicted and constructed. CONCLUSIONS: Our findings reveal the upregulation of DARS2 in ESCA and its association with clinical features, glycolysis pathway, m6A modification, and ceRNA network. These discoveries provide valuable insights into the molecular mechanisms underlying ESCA.

Novel NARS2 variants in a patient with early-onset status epilepticus: case study and literature review.

BACKGROUND: NARS2 as a member of aminoacyl-tRNA synthetases was necessary to covalently join a specific tRNA to its cognate amino acid. Biallelic variants in NARS2 were reported with disorders such as Leigh syndrome, deafness, epilepsy, and severe myopathy. CASE PRESENTATION: Detailed clinical phenotypes were collected and the NARS2 variants were discovered by whole exome sequencing and verified by Sanger sequencing. Additionally, 3D protein structure visualization was performed by UCSF Chimera. The proband in our study had early-onset status epilepticus with abnormal EEG and MRI results. She also performed global developmental delay (GDD) and myocardial dysfunction. Next-generation sequencing (NGS) and Sanger sequencing revealed compound heterozygous missense variants [NM_024678.6:exon14: c.1352G > A(p.Arg451His); c.707T > C(p.Phe236Ser)] of the NARS2 gene. The proband develops refractory epilepsy with GDD and hyperlactatemia. Unfortunately, she finally died for status seizures two months later. CONCLUSION: We discovered two novel missense variants of NARS2 in a patient with early-onset status epilepticus and myocardial dysfunction. The NGS enables the patient to be clearly diagnosed as combined oxidative phosphorylation deficiency 24 (COXPD24, OMIM:616,239), and our findings expands the spectrum of gene variants in COXPD24.

MicroRNA (let-7b-5p)-targeted DARS2 regulates lung adenocarcinoma growth by PI3K/AKT signaling pathway.

Background: The aberrant intracellular expression of a mitochondrial aspartyl-tRNA synthetase 2 (DARS2) has been reported in human cancers. Nevertheless, its critical role and detailed mechanism in lung adenocarcinoma (LUAD) remain unexplored. Methods: Initially, The Cancer Genome Atlas (TCGA)-based Gene Expression Profiling Interactive Analysis (GEPIA) database (http://gepia.cancer-pku.cn/) was used to analyze the prognostic relevance of DARS2 expression in LUAD. Further, cell counting kit (CCK)-8, immunostaining, and transwell invasion assays in LUAD cell lines in vitro, as well as DARS2 silence on LUAD by tumorigenicity experiments in vivo in nude mice, were performed. Besides, we analyzed the expression levels of p-PI3K (phosphorylated-Phosphotylinosital3 kinase), PI3K, AKT (Protein Kinase B), p-AKT (phosphorylated-Protein Kinase B), PCNA (proliferating cell nuclear antigen), cleaved-caspase 3, E-cadherin, and N-cadherin proteins using the Western blot analysis. Results: LUAD tissues showed higher DARS2 expression compared to normal tissues. Upregulation of DARS2 could be related to Tumor-Node-Metastasis (TNM) stage, high lymph node metastasis, and inferior prognosis. DARS2 silence decreased the proliferation, migration, and invasion abilities of LUAD cells. In addition, the DARS2 downregulation decreased the PCNA and N-cadherin expression and increased cleaved-caspase 3 and E-cadherin expressions in LUAD cells, coupled with the inactivation of the PI3K/AKT signaling pathway. Moreover, DARS2 silence impaired the tumorigenicity of LUAD in vivo. Interestingly, let-7b-5p could recognize DARS2 through a complementary sequence. Mechanistically, the increased let-7b-5p expression attenuated the promo-oncogenic action of DARS2 during LUAD progression, which were inversely correlated to each other in the LUAD tissues. Conclusion: In summary, let-7b-5p downregulated DARS2 expression, regulating the progression of LUAD cells by the PI3K/AKT signaling pathway.

Reaction hijacking inhibition of Plasmodium falciparum asparagine tRNA synthetase.

Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl-tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure-activity relationship and the selectivity mechanism.

Mitochondrial dysfunction abrogates dietary lipid processing in enterocytes.

Digested dietary fats are taken up by enterocytes where they are assembled into pre-chylomicrons in the endoplasmic reticulum followed by transport to the Golgi for maturation and subsequent secretion to the circulation1. The role of mitochondria in dietary lipid processing is unclear. Here we show that mitochondrial dysfunction in enterocytes inhibits chylomicron production and the transport of dietary lipids to peripheral organs. Mice with specific ablation of the mitochondrial aspartyl-tRNA synthetase DARS2 (ref. 2), the respiratory chain subunit SDHA3 or the assembly factor COX10 (ref. 4) in intestinal epithelial cells showed accumulation of large lipid droplets (LDs) in enterocytes of the proximal small intestine and failed to thrive. Feeding a fat-free diet suppressed the build-up of LDs in DARS2-deficient enterocytes, which shows that the accumulating lipids derive mostly from digested fat. Furthermore, metabolic tracing studies revealed an impaired transport of dietary lipids to peripheral organs in mice lacking DARS2 in intestinal epithelial cells. DARS2 deficiency caused a distinct lack of mature chylomicrons concomitant with a progressive dispersal of the Golgi apparatus in proximal enterocytes. This finding suggests that mitochondrial dysfunction results in impaired trafficking of chylomicrons from the endoplasmic reticulum to the Golgi, which in turn leads to storage of dietary lipids in large cytoplasmic LDs. Taken together, these results reveal a role for mitochondria in dietary lipid transport in enterocytes, which might be relevant for understanding the intestinal defects observed in patients with mitochondrial disorders5.

Integrative lactylation and tumor microenvironment signature as prognostic and therapeutic biomarkers in skin cutaneous melanoma.

BACKGROUND: The incidence of skin cutaneous melanoma (SKCM), one of the most aggressive and lethal skin tumors, is increasing worldwide. However, for advanced SKCM, we still lack an accurate and valid way to predict its prognosis, as well as novel theories to guide the planning of treatment options for SKCM patients. Lactylation (LAC), a novel post-translational modification of histones, has been shown to promote tumor growth and inhibit the antitumor response of the tumor microenvironment (TME) in a variety of ways. We hope that this study will provide new ideas for treatment options for SKCM patients, as well as research on the molecular mechanisms of SKCM pathogenesis and development. METHODS: At the level of the RNA sequencing set (TCGA, GTEx), we used differential expression analysis, LASSO regression analysis, and multifactor Cox regression analysis to screen for prognosis-related genes and calculate the corresponding LAC scores. The content of TME cells in the tumor tissue was calculated using the CIBERSORT algorithm, and the TME score was calculated based on its results. Finally, the LAC-TME classifier was established and further analyzed based on the two scores, including the construction of a prognostic model, analysis of clinicopathological characteristics, and correlation analysis of tumor mutation burden (TMB) and immunotherapy. Based on single-cell RNA sequencing data, this study analyzed the cellular composition in SKCM tissues and explored the role of LAC scores in intercellular communication. To validate the functionality of the pivotal gene CLPB in the model, cellular experiments were ultimately executed. RESULTS: We screened a total of six prognosis-related genes (NDUFA10, NDUFA13, CLPB, RRM2B, HPDL, NARS2) and 7 TME cells with good prognosis. According to Kaplan-Meier survival analysis, we found that the LAClow/TMEhigh group had the highest overall survival (OS) and the LAChigh/TMElow group had the lowest OS (p value < 0.05). In further analysis of immune infiltration, tumor microenvironment (TME), functional enrichment, tumor mutational load and immunotherapy, we found that immunotherapy was more appropriate in the LAClow/TMEhigh group. Moreover, the cellular assays exhibited substantial reductions in proliferation, migration, and invasive potentials of melanoma cells in both A375 and A2058 cell lines upon CLPB knockdown. CONCLUSIONS: The prognostic model using the combined LAC score and TME score was able to predict the prognosis of SKCM patients more consistently, and the LAC-TME classifier was able to significantly differentiate the prognosis of SKCM patients across multiple clinicopathological features. The LAC-TME classifier has an important role in the development of immunotherapy regimens for SKCM patients.

Expanding phenotype heterogeneity of NARS2 by presenting subdural hematoma and parenchymal hemorrhage.

BACKGROUND: NARS2 encodes mitochondrial Asparaginyl-tRNA Synthetase 2, which catalyzes the aminoacylation of tRNA-Asn in the mitochondria. To date, 24 variants have been reported in NARS2 gene in 35 patients. The phenotypic variability of NARS2-associated disorder is broad, ranging from neurodevelopmental disorders to hearing loss. In this study, we report some novel imaging findings in an Iranian patient suffering from epileptic encephalopathy, caused by a previously reported variant, c.500A > G; p.(His167Arg), in NARS2. METHODS: The spectrum of clinical manifestations of two Iranian patients was investigated and genetic analysis was performed by Whole-exome sequencing (WES). Additionally, we also reviewed the literature and summarized the phenotypes of previously reported patients with variants in the NARS2 gene. RESULTS: Here, we present the phenotypic and genetic features of 2 unrelated Iranian infants presented with neurodevelopmental delay, seizures, hearing impairment, feeding problems, elevated serum lactate levels in addition to subdural hematoma and cerebral parenchymal hemorrhage in the brain magnetic resonance imaging (MRI) of one of the patients. Genetic analysis revealed a biallelic missense variant in NARS2: c.500A > G; p.(His167Arg). We described the subdural hematoma and cerebral parenchymal hemorrhage of the brain for the first time. CONCLUSIONS: Our study provides new clinical findings, subdural hematoma, and parenchymal hemorrhage, in NARS2-related disorders. Our findings along with previous studies provide more evidence of the clinical presentation of the disease caused by pathogenic variants in NARS2. Expanding the clinical spectrum increases the diagnostic rate of molecular testing and improves the quality of counseling for at-risk couples.

DARS2 promotes the occurrence of lung adenocarcinoma via the ERK/c-Myc signaling pathway.

BACKGROUND: DARS2 expression is upregulated in lung adenocarcinoma (LUAD) which correlates with tumor patient stage and prognosis. The mechanism of DARS2 involvement in LUAD still needs to be further explored. METHODS: In this study, we found that DARS2 expression in LUAD tissue was significantly higher than that in normal tissue. At the same time, the Kaplan-Meier curve showed that the survival prognosis of LUAD patients with high expression of DARS2 was significantly worse than low expression of DARS2. The expression of DARS2 was detected in LUAD and adjacent normal tissues by IHC staining, histochemical scoring and a survival curve was generated. In addition, we demonstrated that the knockdown and overexpression of DARS2 significantly affected the proliferation, invasion, and migration of LUAD cells in vitro and in vivo. Finally, western blot and rescue assay were performed on LUAD cells to further explore and verify the signaling pathway. RESULTS: DARS2 expression was significantly upregulated in LUAD tissues and cell lines. What is more, the increased expression of DARS2 was closely related to proliferation, invasion and metastasis. The tumorigenic assay in nude mice further showed that the tumorigenic ability of nude mice was significantly improved with the increase in DARS2 expression. Finally, we determined that DARS2 plays its role in LUAD by targeting the ERK/c-Myc signaling pathway. CONCLUSION: Our data revealed the oncogenic role of DARS2 in LUAD, indicating that DARS2 may be a predictive biomarker and novel therapeutic target for LUAD.

Aspartyl-tRNA synthetase 2 orchestrates iron-sulfur metabolism in hematopoietic stem cells via fine-tuning alternative RNA splicing.

Aspartyl-tRNA synthetase 2 (Dars2) is involved in the regulation of mitochondrial protein synthesis and tissue-specific mitochondrial unfolded protein response (UPRmt). The role of Dars2 in the self-renewal and differentiation of hematopoietic stem cells (HSCs) is unknown. Here, we show that knockout (KO) of Dars2 significantly impairs the maintenance of hematopoietic stem and progenitor cells (HSPCs) without involving its tRNA synthetase activity. Dars2 KO results in significantly reduced expression of Srsf2/3/6 and impairs multiple events of mRNA alternative splicing (AS). Dars2 directly localizes to Srsf3-labeled spliceosomes in HSPCs and regulates the stability of Srsf3. Dars2-deficient HSPCs exhibit aberrant AS of mTOR and Slc22a17. Dars2 KO greatly suppresses the levels of labile ferrous iron and iron-sulfur cluster-containing proteins, which dampens mitochondrial metabolic activity and DNA damage repair pathways in HSPCs. Our study reveals that Dars2 plays a crucial role in the iron-sulfur metabolism and maintenance of HSPCs by modulating RNA splicing.

DARS2 overexpression is associated with PET/CT metabolic parameters and affects glycolytic activity in lung adenocarcinoma.

BACKGROUND: This study investigated the correlation between the expression of DARS2 and metabolic parameters of 18F-FDG PET/CT, and explored the potential mechanisms of DARS2 affecting the proliferation and glycolysis of lung adenocarcinoma (LUAD) cells. METHODS: This study used genomics and proteomics to analyze the difference in DARS2 expression between LUAD samples and control samples. An analysis of 62 patients with LUAD who underwent 18F-FDG PET/CT examinations before surgery was conducted retrospectively. The correlation between DARS2 expression and PET/CT metabolic parameters, including SUVmax, SUVmean, MTV, and TLG, was examined by Spearman correlation analysis. In addition, the molecular mechanism of interfering with DARS2 expression in inhibiting LUAD cell proliferation and glycolysis was analyzed through in vitro cell experiments. RESULTS: DARS2 expression was significantly higher in LUAD samples than in control samples (p < 0.001). DARS2 has high specificity (98.4%) and sensitivity (95.2%) in the diagnosis of LUAD. DARS2 expression was positively correlated with SUVmax, SUVmean, and TLG (p < 0.001). At the same time, the sensitivity and specificity of SUVmax in predicting DARS2 overexpression in LUAD were 88.9% and 65.9%, respectively. In vitro cell experiments have shown that interfering with DARS2 expression can inhibit the proliferation and migration of LUAD cells, promote cell apoptosis, and inhibit the glycolytic activity of tumor cells by inhibiting the expression of glycolytic related genes SLC2A1, GPI, ALDOA, and PGAM1. CONCLUSIONS: Overexpression of DARS2 is associated with metabolic parameters on 18F-FDG PET/CT, which can improve LUAD diagnosis accuracy. DARS2 may be a useful biomarker to diagnose, prognosis, and target treatment of LUAD patients.

Aspartyl tRNA-synthetase (AspRS) gene family enhances drought tolerance in poplar through BABA-PtrIBIs-PtrVOZ signaling module.

BACKGROUND: Drought stress is a prevalent abiotic stress that significantly hinders the growth and development of plants. According to studies, ß-aminobutyric acid (BABA) can influence the ABA pathway through the AtIBI1 receptor gene to enhance cold resistance in Arabidopsis. However, the Aspartate tRNA-synthetase (AspRS) gene family, which acts as the receptor for BABA, has not yet been investigated in poplar. Particularly, it is uncertain how the AspRS gene family (PtrIBIs)r can resist drought stress after administering various concentrations of BABA to poplar. RESULTS: In this study, we have identified 12 AspRS family genes and noted that poplar acquired four PtrIBI pairs through whole genome duplication (WGD). We conducted cis-action element analysis and found a significant number of stress-related action elements on different PtrIBI genes promoters. The expression of most PtrIBI genes was up-regulated under beetle and mechanical damage stresses, indicating their potential role in responding to leaf damage stress. Our results suggest that a 50 mM BABA treatment can alleviate the damage caused by drought stress in plants. Additionally, via transcriptome sequencing, we observed that the partial up-regulation of BABA receptor genes, PtrIBI2/4/6/8/11, in poplars after drought treatment. We hypothesize that poplar responds to drought stress through the BABA-PtrIBIs-PtrVOZ coordinated ABA signaling pathway. Our research provides molecular evidence for understanding how plants respond to drought stress through external application of BABA. CONCLUSIONS: In summary, our study conducted genome-wide analysis of the AspRS family of P. trichocarpa and identified 12 PtrIBI genes. We utilized genomics and bioinformatics to determine various characteristics of PtrIBIs such as chromosomal localization, evolutionary tree, gene structure, gene doubling, promoter cis-elements, and expression profiles. Our study found that certain PtrIBI genes are regulated by drought, beetle, and mechanical damage implying their crucial role in enhancing poplar stress tolerance. Additionally, we observed that external application of low concentrations of BABA increased plant drought resistance under drought stress. Through the BABA-PtrIBIs-PtrVOZ signaling module, poplar plants were able to transduce ABA signaling and regulate their response to drought stress. These results suggest that the PtrIBI genes in poplar have the potential to improve drought tolerance in plants through the topical application of low concentrations of BABA.

Mutations in DARS2 result in global dysregulation of mRNA metabolism and splicing.

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a rare neurological disorder caused by the mutations in the DARS2 gene, which encodes the mitochondrial aspartyl-tRNA synthetase. The objective of this study was to understand the impact of DARS2 mutations on cell processes through evaluation of LBSL patient stem cell derived cerebral organoids and neurons. We generated human cerebral organoids (hCOs) from induced pluripotent stem cells (iPSCs) of seven LBSL patients and three healthy controls using an unguided protocol. Single cells from 70-day-old hCOs were subjected to SMART-seq2 sequencing and bioinformatic analysis to acquire high-resolution gene and transcript expression datasets. Global gene expression analysis demonstrated dysregulation of a number of genes involved in mRNA metabolism and splicing processes within LBSL hCOs. Importantly, there were distinct and divergent gene expression profiles based on the nature of the DARS2 mutation. At the transcript level, pervasive differential transcript usage and differential spliced exon events that are involved in protein translation and metabolism were identified in LBSL hCOs. Single-cell analysis of DARS2 (exon 3) showed that some LBSL cells exclusively express transcripts lacking exon 3, indicating that not all LBSL cells can benefit from the "leaky" nature common to splice site mutations. At the gene- and transcript-level, we uncovered that dysregulated RNA splicing, protein translation and metabolism may underlie at least some of the pathophysiological mechanisms in LBSL. To confirm hCO findings, iPSC-derived neurons (iNs) were generated by overexpressing Neurogenin 2 using lentiviral vector to study neuronal growth, splicing of DARS2 exon 3 and DARS2 protein expression. Live cell imaging revealed neuronal growth defects of LBSL iNs, which was consistent with the finding of downregulated expression of genes related to neuronal differentiation in LBSL hCOs. DARS2 protein was downregulated in iNs compared to iPSCs, caused by increased exclusion of exon 3. The scope and complexity of our data imply that DARS2 is potentially involved in transcription regulation beyond its canonical role of aminoacylation. Nevertheless, our work highlights transcript-level dysregulation as a critical, and relatively unexplored, mechanism linking genetic data with neurodegenerative disorders.

Use of dual genomic sequencing to screen mitochondrial diseases in pediatrics: a retrospective analysis.

Mitochondrial diseases (MDs) were a large group multisystem disorders, attributable in part to the dual genomic control. The advent of massively sequencing has improved diagnostic rates and speed, and was increasingly being used as a first-line diagnostic test. Paediatric patients (aged < 18 years) who underwent dual genomic sequencing were enrolled in this retrospective multicentre study. We evaluated the mitochondrial disease criteria (MDC) and molecular diagnostic yield of dual genomic sequencing. Causative variants were identified in 177 out of 503 (35.2%) patients using dual genomic sequencing. Forty-six patients (9.1%) had mitochondria-related variants, including 25 patients with nuclear DNA (nDNA) variants, 15 with mitochondrial DNA (mtDNA) variants, and six with dual genomic variants (MT-ND6 and POLG; MT-ND5 and RARS2; MT-TL1 and NARS2; MT-CO2 and NDUFS1; MT-CYB and SMARCA2; and CHRNA4 and MT-CO3). Based on the MDC, 15.2% of the patients with mitochondria-related variants were classified as "unlikely to have mitochondrial disorder". Moreover, 4.5% of the patients with non-mitochondria-related variants and 1.43% with negative genetic tests, were classified as "probably having mitochondrial disorder". Dual genomic sequencing in suspected MDs provided a more comprehensive and accurate diagnosis for pediatric patients, especially for patients with dual genomic variants.

Pharmacoinformatics-based screening of active compounds from Vitex negundo against lymphatic filariasis by targeting asparaginyl-tRNA synthetase.

CONTEXT: Lymphatic filariasis, generally called as elephantiasis, is a vector-borne infectious disease caused by the filarial nematodes, mainly Wuchereria bancrofti, Brugia malayi, and Brugia timori, which are transmitted through mosquitoes. The infection affects the normal flow of lymph leading to abnormal enlargement of body parts, severe pain, permanent disability, and social stigma. Due to the development of resistance as well as toxic effects, existing medicines for lymphatic filariasis are becoming ineffective in killing the adult worms. It is essential to search novel filaricidal drugs with new molecular targets. Asparaginyl-tRNA synthetase (PDB ID: 2XGT) belongs to the group of aminoacyl-tRNA synthetases that catalyze specific attachment of amino acids to their tRNA during protein biosynthesis. Plants and their extracts are well-known medicinal practice for the management of several parasitic infectious diseases including filarial infections. METHODS: In this study, asparaginyl-tRNA synthetase of Brugia malayi was used as a target to perform virtual screening of plant phytoconstituents of Vitex negundo from IMPPAT database, which exhibits anti-filarial and anti-helminthic properties. A total of sixty-eight compounds from Vitex negundo were docked against asparaginyl-tRNA synthetase using Autodock module of PyRx tool. Among the 68 compounds screened, 3 compounds, negundoside, myricetin, and nishindaside, exhibited a higher binding affinity compared to standard drugs. The pharmacokinetic and physicochemical prediction, stability of ligand-receptor complexes via molecular dynamics simulation, and density functionality theory were done further for the top-scored ligands with receptor.

A Multiple Proton Transfer Mechanism for the Charging Step of the Aminoacylation Reaction at the Active Site of Aspartyl tRNA Synthetase.

Aspartyl-tRNA synthetase catalyzes the attachment of aspartic acid to its cognate tRNA by the aminoacylation reaction during the initiation of the protein biosynthesis process. In the second step of the aminoacylation reaction, known as the charging step, the aspartate moiety is transferred from aspartyl-adenylate to the 3'-OH of A76 of tRNA through a proton transfer process. We have investigated different pathways for the charging step through three separate QM/MM simulations combined with the enhanced sampling method of well-sliced metadynamics and found out the most feasible pathway for the reaction at the active site of the enzyme. In the charging reaction, both the phosphate group and the ammonium group after deprotonation can potentially act as a base for proton transfer in the substrate-assisted mechanism. We have considered three possible mechanisms involving different pathways of proton transfer, and only one of them is determined to be enzymatically feasible. The free energy landscape along reaction coordinates where the phosphate group acts as the general base showed that, in the absence of water, the barrier height is 52.6 kcal/mol. The free energy barrier is reduced to 39.7 kcal/mol when the active site water molecules are also treated quantum mechanically, thus allowing a water mediated proton transfer. The charging reaction involving the ammonium group of the aspartyl adenylate is found to follow a path where first a proton from the ammonium group moves to a water in the vicinity forming a hydronium ion (H3O+) and NH2 group. The hydronium ion subsequently passes the proton to the Asp233 residue, thus minimizing the chance of back proton transfer from hydronium to the NH2 group. The neutral NH2 group subsequently takes the proton from the O3' of A76 with a free energy barrier of 10.7 kcal/mol. In the next step, the deprotonated O3' makes a nucleophilic attack to the carbonyl carbon forming a tetrahedral transition state with a free energy barrier of 24.8 kcal/mol. Thus, the present work shows that the charging step proceeds through a multiple proton transfer mechanism where the amino group formed after deprotonation acts as the base to capture a proton from O3' of A76 rather than the phosphate group. The current study also shows the important role played by Asp233 in the proton transfer process.

Leigh Syndrome: Spectrum of Molecular Defects and Clinical Features in Russia.

Leigh syndrome (LS), also known as infantile subacute necrotizing encephalopathy, is the most frequent mitochondrial disorder in children. Recently, more than 80 genes have been associated with LS, which greatly complicates the diagnosis. In this article, we present clinical and molecular findings of 219 patients with LS and give the detailed description of three cases with rare findings in nuclear genes MORC2, NARS2 and VPS13D, demonstrating wide genetic heterogeneity of this mitochondrial disease. The most common cause of LS in Russian patients are pathogenic variants in the SURF1 gene (44.3% of patients). The most frequent pathogenic variant is c.845_846delCT (66.0% of mutant alleles; 128/192), which is also widespread in Eastern Europe. Five main LS genes, SURF1, SCO2, MT-ATP6, MT-ND5 and PDHA1, account for 70% of all LS cases in the Russian Federation. Using next generation sequencing (NGS) technique, we were able to detect pathogenic variants in other nuclear genes: NDUFV1, NDUFS2, NDUFS8, NDUFAF5, NDUFAF6, NDUFA10, SUCLG1, GFM2, COX10, PMPCB, NARS2, PDHB and SLC19A3, including two genes previously associated with Leigh-like phenotypes-MORC2 and VPS13D. We found 49 previously undescribed nucleotide variants, including two deep intronic variants which affect splicing.

Prognostic biomarker DARS2 correlated with immune infiltrates in bladder tumor.

Background: DARS2 is a pivotal member of the Aminoacyl-tRNA synthetases family that is critical for regulating protein translation. However, the biological role of DARS2 in bladder cancer remains elusive. Methods: We analyzed the correlation between DARS2 expression and prognosis, tumor stage, and immune infiltration in bladder cancer using The Cancer Genome Atlas (TCGA) database. We validated findings in clinical samples from The First Affiliated Hospital of Nanchang University and explored the biological functions of DARS2 using cell and animal models. Results: We found DARS2 to be upregulated in bladder cancer, associated with tumor progression and poor prognosis. Immune infiltration analysis suggested that DARS2 may facilitate immune evasion by modulating PD-L1. Cell and animal experiments validated that DARS2 knockdown and overexpress can inhibit or increase cancer cell proliferation, metastasis, tumorigenesis, immune escape, and PD-L1 levels. Conclusions: Our study reveals DARS2 as a potential prognostic biomarker and immunotherapy target in BLCA.

Splicing variants in NARS2 are associated with milder phenotypes and intra-familial variability.

Biallelic rare variants in NARS2 that encode the mitochondrial asparaginyl-tRNA synthetase are associated with a wide spectrum of clinical phenotypes ranging from severe neurodegenerative disorders to isolated mitochondrial myopathy or deafness. To date, only a small number of patients with NARS2 variants have been reported, and possible genotype-phenotype correlations are still lacking. Here, we present three siblings who had an early-onset hearing loss, while one developed severe symptoms in adulthood associated with early intellectual impairment, refractory seizures, moderate axonal sensorimotor neuropathy, and atypical psychiatric symptoms. Biochemical analysis revealed impairment of the activity and assembly of the respiratory chain complexes in this patient's muscle and fibroblasts. Whole Exome Sequencing allowed identification of a heterozygous variant NM_024678.5(NARS2):c.822G > C (p.Gln274His) that is known to be pathogenic and to affect splicing of the NARS2 gene, but was unable to detect a second variant in this gene. Coverage analysis and Sanger sequencing led to identification of a novel intronic deletion NM_024678.5(NARS2):c.922-21_922-19del in the three siblings in trans with the c.822G > C. Functional analysis by RT-PCR showed that this deletion was causing aberrant splicing and led to exon 9 skipping in NARS2 mRNA in patient fibroblasts. Our work expands the phenotype and genotype spectrum of NARS2-related disorders. We provide evidence of the pathogenic effect of a novel intronic deletion in the NARS2 gene and report on additional adult patients with a large intrafamilial variability associated with splice variants in this gene. More specifically, we detail the phenotype of the oldest living patient to date with NARS2 variants and, for the first time, we report the psychiatric symptoms associated with this gene. Our work confirms the complexity of genotype-phenotype correlation in patients with pathogenic NARS2 variants.

New candidates in the differential diagnosis of malignant mesothelioma from benign mesothelial hyperplasia and adenocarcinoma; DARS2 and suprabasin.

OBJECTIVES: Malignant mesothelioma (MM) is a primary malignant tumour with a very bad prognosis, which develops from the mesothelial cells lining the serosal surfaces. Because MM can show a wide variety of histological patterns and its cytomorphological features are quite extensive, it is often confused with lung adenocarcinomas (LAC) and reactive mesothelial hyperplasia (RMH). The immunohistochemical examination method is the most useful method for discrimination. In this study, we aimed to determine the value of suprabasin and DARS2 markers in the differential diagnosis of RMH, MM and LAC. METHODS: Thirty MM, 30 LAC and 30 RMH samples selected from the archive of Firat University Hospital Pathology Department Laboratory were included in this study. Suprabasin and DARS2 markers were applied to the samples immunohistochemically and their place in the differential diagnosis was examined. RESULTS: Although DARS2 expression was observed in RMH, MM and adenocarcinoma samples, Suprabasin expression was only observed in adenocarcinoma. There was a significant difference between the groups in terms of DARS2 and Suprabasin expression. No suprabasin expression was detected in MM and RMH. CONCLUSION: Suprabasin and DARS2 may be proposed as new biomarkers to differentiate MM from LAC.

High expression of DARS2 indicates poor prognosis in lung adenocarcinoma.

BACKGROUND: DARS2 was overexpressed in multiple tumor types, but the biological role of DARS2 in lung adenocarcinoma (LUAD) have not been elucidated. METHODS: Firstly, the DARS2 expression in LUAD was explored using The Cancer Genome Atlas (TCGA). Then, qRT-PCR and Western blot were performed to confirm DARS2 expression in LUAD. Next, Cox regression and Kaplan-Meier methods were utilized to evaluate whether DARS2 expression can affect the overall survival. The relationships between DARS2 expression and clinicopathological characteristics were investigated by TCGA database. Moreover, we utilized Gene Set Enrichment Analysis (GSEA) to detect DARS2-related signaling pathways in LUAD. Finally, the special function of DARS2 in cell proliferation, invasion and apoptosis was assessed in vitro. RESULTS: The higher expression of DARS2 was found in LUAD compared to para-carcinoma tissues and significantly related to tumor stage, T stage, and M stage. The survival analysis indicated that DARS2 overexpression was related to poor prognosis in LUAD. Multivariate analysis suggested that DARS2 expression was a prognostic indicator. GSEA revealed that DARS2 was primarily involved in cell cycle-related pathways. In addition, upregulation of DARS2 facilitated LUAD cell proliferation, migration, invasion and inhabited apoptosis, DARS2 knockdown showed an opposite result. CONCLUSION: DARS2 modulates the proliferation, invasion and apoptosis of LUAD cells, and sever as a promising therapeutic target for LUAD.