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.

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.

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.

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.

An induced pluripotent stem cell line (FHUSTCi002-A) derived from a patient with leucoencephalopathy with brain stem and spinal cord involvement and lactate elevation.

Leucoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL) is commonly induced by DARS2 abnormalities and accompanied by slowly progressing pyramidal and cerebellar dysfunction, as well as concomitant dorsal column dysfunction. In this study, an LBSL induced pluripotent stem cell (iPSC) line was generated from peripheral blood mononuclear cells of a female patient carrying biallelic mutations in DARS2. Pluripotency, differentiation potential, and karyotypic normality of this cell line were confirmed. This iPSC line offers a useful cellular model to investigate LBSL phenotypes, mechanisms, and therapy.

Free Energy Landscape of the Adenylation Reaction of the Aminoacylation Process at the Active Site of Aspartyl tRNA Synthetase.

The process of protein biosynthesis is initiated by the aminoacylation process where a transfer ribonucleic acid (tRNA) is charged by the attachment of its cognate amino acid at the active site of the corresponding aminoacyl tRNA synthetase enzyme. The first step of the aminoacylation process, known as the adenylation reaction, involves activation of the cognate amino acid where it reacts with a molecule of adenosine triphosphate (ATP) at the active site of the enzyme to form the aminoacyl adenylate and inorganic pyrophosphate. In the current work, we have investigated the adenylation reaction between aspartic acid and ATP at the active site of the fully solvated aspartyl tRNA synthetase (AspRS) from Escherichia coli in aqueous medium at room temperature through hybrid quantum mechanical/molecular mechanical (QM/MM) simulations combined with enhanced sampling methods of well-tempered and well-sliced metadynamics. The objective of the present work is to study the associated free energy landscape and reaction barrier and also to explore the effects of active site mutation on the free energy surface of the reaction. The current calculations include finite temperature effects on free energy profiles. In particular, apart from contributions of interaction energies, the current calculations also include contributions of conformational, vibrational, and translational entropy of active site residues, substrates, and also the rest of the solvated protein and surrounding water into the free energy calculations. The present QM/MM metadynamics simulations predict a free energy barrier of 23.35 and 23.5 kcal/mol for two different metadynamics methods used to perform the reaction at the active site of the wild type enzyme. The free energy barrier increases to 30.6 kcal/mol when Arg217, which is an important conserved residue of the wild type enzyme at its active site, is mutated by alanine. These free energy results including the effect of mutation compare reasonably well with those of kinetic experiments that are available in the literature. The current work also provides molecular details of structural changes of the reactants and surroundings as the system dynamically evolves along the reaction pathway from reactant to the product state through QM/MM metadynamics simulations.

A single amino acid substitution alters activity and specificity in Plasmodium falciparum aspartyl & asparaginyl-tRNA synthetases.

The specificity of each aminoacyl-tRNA synthetase (aaRS) for its cognate amino acid ensures correct tRNA esterification and allows fidelity in protein synthesis. The aaRSs discriminate based on the chemical properties of their amino acid substrates and structural features of the binding pockets. In this study, we characterized aspartyl-(DRS) and asparaginyl-tRNA synthetase (NRS) from Plasmodium falciparum to determine the basis of their specificity towards L-asp and L-asn respectively. The negatively charged L-asp and its analogue L-asn differ only in their side-chain groups i.e., -OH and -NH2. Further, the amino acid binding sites are highly conserved within these two enzymes. Analysis of the substrate (L-asp/L-asn) binding sites across species revealed two highly conserved residues in PfDRS (D408 and K372) and PfNRS (E395 and L360) that are involved in recognition of the Oδ2/Nδ2 of L-asp/L-asn respectively. These residues were mutated and swapped between the D408→E in PfDRS and the corresponding E395→D in PfNRS. A similar approach was employed for residue number K372→L in PfDRS and L360→K in PfNRS. The mutated PfDRSD408E retained its enzymatic activity during step 1 of aminoacylation reaction towards L-asp and L-asn and esterified tRNAAsp with L-asp like wild type enzyme, while the PfDRSK372L was rendered enzymatically inactive. The correspondingly mutated PfNRSE395D was enzymatically inactive. The mutated PfNRSL360K had an altered specificity and esterified tRNAAsn with non-cognate amino acid L-asp and not L-asn. These data suggest that the residue K372 is crucial for the enzymatic activity of PfDRS while the residue L360 in PfNRS imparts specificity towards L-asn.

Functional genomics screening identifies aspartyl-tRNA synthetase as a novel prognostic marker and a therapeutic target for gastric cancers.

Efficient molecular targeting therapies for most gastric cancers (GCs) are currently lacking, despite GC being one of the most frequent and often devastating malignancies worldwide. Thus, identification of novel therapeutic targets for GC is in high demand. Recent advancements of high-throughput nucleic acid synthesis methods combined with next-generation sequencing (NGS) platforms have made it feasible to conduct functional genomics screening using large-scale pooled lentiviral libraries aimed at discovering novel cancer therapeutic targets. In this study, we performed NGS-based functional genomics screening for human GC cell lines using an originally constructed 6,399 shRNA library targeting all 2,096 human metabolism genes. Our screening identified aspartyl-tRNA synthetase (DARS) as a possible candidate for a therapeutic target for GC. In-house tissue microarrays containing 346 cases of GC combined with public datasets showed that patients with high expression levels of DARS protein exhibited more advanced clinicopathologic parameters and a worse prognosis, specifically among diffuse-type GC patients. Both in vitro and in vivo experiments concretely evidenced that DARS inhibition achieved robust growth suppression of GC cells. Moreover, RNA sequencing of GC cell lines under shRNA-mediated DARS knockdown suggested that DARS inhibition exerts its effect through the inactivation of multiple p-ERK pathways. This MAPK-related growth suppression by DARS inhibition would also be applicable to other cancers; thus, it is warranted to investigate the expression and clinical significance of DARS in a wide spectrum of malignancies. Taken together, NGS-based high-throughput pooled lentiviral screening showed DARS as a novel prognostic marker and a promising therapeutic target for GC. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Developmental delay and late onset HBSL pathology in hypomorphic Dars1M256L mice.

The leukodystrophy Hypomyelination with Brainstem and Spinal cord involvement and Leg spasticity (HBSL) is caused by recessive mutations of the DARS1 gene, which encodes the cytoplasmic aspartyl-tRNA synthetase. HBSL is a spectrum disorder with disease onset usually during early childhood and no available treatment options. Patients display regression of previously acquired motor milestones, spasticity, ataxia, seizures, nystagmus, and intellectual disabilities. Gene-function studies in mice revealed that homozygous Dars1 deletion is embryonically lethal, suggesting that successful modelling of HBSL requires the generation of disease-causing genocopies in mice. In this study, we introduced the pathogenic DARS1 M256L mutation located on exon nine of the murine Dars1 locus. Despite causing severe illness in humans, homozygous Dars1 M256L mice were only mildly affected. To exacerbate HBSL symptoms, we bred Dars1 M256L mice with Dars1-null 'enhancer' mice. The Dars1 M256L/- offspring displayed increased embryonic lethality, severe developmental delay, reduced body weight and size, hydrocephalus, anophthalmia, and vacuolization of the white matter. Remarkably, the Dars1 M256L/- genotype affected energy metabolism and peripheral organs more profoundly than the nervous system and resulted in reduced body fat, increased respiratory exchange ratio, reduced liver steatosis, and reduced hypocellularity of the bone marrow. In summary, homozygous Dars1 M256L and compound heterozygous Dars1 M256L/- mutation genotypes recapitulate some aspects of HBSL and primarily manifest in developmental delay as well as metabolic and peripheral changes. These aspects of the disease might have been overlooked in HBSL patients with severe neurological deficits but could be included in the differential diagnosis of HBSL in the future.

Anti-HIV Effects of Baculiferins Are Regulated by the Potential Target Protein DARS.

Baculiferins are a group of marine sponge-derived polycyclic alkaloids with anti-HIV (human immunodeficiency virus) activities. To identify additional baculiferin-based congeners for SAR analysis and to investigate the mode of action, a total of 18 new baculiferin-type derivatives were synthesized. The inhibitory activities of the congeners against the HIV-1 virus were evaluated in vitro, and the relevant SAR was discussed. Compound 18 exerted the most potent activity toward VSV-G-pseudotyped HIV-1 (IC50 of 3.44 µM) and HIV-1 strain SF33 (IC50 of 2.80 µM) in vitro. To identify the cellular targets, three photoaffinity baculiferin probes were simultaneously synthesized. Photoaffinity labeling experiments together with LC-MS/MS data identified aspartate-tRNA ligase (DARS) as a putative target protein of 18. The overexpression and knockdown of DARS in HEK293T cells provided additional data to demonstrate that DARS is a potential target protein in the regulation of HIV virus infection. The modes of antiviral baculiferins 13 and 18 binding to DARS were determined by a molecular docking simulation. Thus, baculiferin 18 is considered a promising lead as a new molecular target for the development of anti-HIV agents.

Molecular dynamics simulations of cognate and non-cognate AspRS-tRNAAsp complexes.

Aspartyl tRNA synthetase (AspRS), one of the 20 aminoacyl-tRNA synthetases, plays an important role in protein synthesis by catalyzing the aminoacylation reaction and synthesises Aspartyl-tRNA (tRNAAsp). A typical three-dimensional structure of AspRS comprises three distinct domains for the recognition of cognate tRNA and catalysis, namely, anti-codon binding domain/N-terminal domain, hinge domain and catalytic domain through their interactions with anti-codon loop, D-stem and acceptor arm of cognate tRNA, respectively. In this work, we have studied the structural characteristics of each domain of AspRS to understand the recognition mechanism of tRNAAsp using molecular dynamics simulations. The dynamics of AspRS-tRNAAsp complexes from E.coli (cognate and non-cognate), S.cerevisiae (cognate) and T.thermophilus (non-cognate) were compared to understand the differences in recognition of cognate and non-cognate tRNAs. Our results explain that the conformational changes associated with the recognition of tRNA occur only in the cognate complexes. Among the cognate complexes, the conformational changes in yeast AspRS are highly controlled during tRNAAsp recognition than that of in the E. coli AspRS. Moreover, the functional motions required for the tRNA recognition are observed only in the cognate complexes, and the conformational changes in AspRS and their recognition of tRNAAsp are organism specific.Communicated by Ramaswamy H. Sarma.

Plant-Specific Domains and Fragmented Sequences Imply Non-Canonical Functions in Plant Aminoacyl-tRNA Synthetases.

Aminoacyl-tRNA synthetases (aaRSs) play essential roles in protein translation. In addition, numerous aaRSs (mostly in vertebrates) have also been discovered to possess a range of non-canonical functions. Very few studies have been conducted to elucidate or characterize non-canonical functions of plant aaRSs. A genome-wide search for aaRS genes in Arabidopsis thaliana revealed a total of 59 aaRS genes. Among them, asparaginyl-tRNA synthetase (AsnRS) was found to possess a WHEP domain inserted into the catalytic domain in a plant-specific manner. This insertion was observed only in the cytosolic isoform. In addition, a long stretch of sequence that exhibited weak homology with histidine ammonia lyase (HAL) was found at the N-terminus of histidyl-tRNA synthetase (HisRS). This HAL-like domain has only been seen in plant HisRS, and only in cytosolic isoforms. Additionally, a number of genes lacking minor or major portions of the full-length aaRS sequence were found. These genes encode 14 aaRS fragments that lack key active site sequences and are likely catalytically null. These identified genes that encode plant-specific additional domains or aaRS fragment sequences are candidates for aaRSs possessing non-canonical functions.

DARS-AS1 promotes clear cell renal cell carcinoma by sequestering miR-194-5p to up-regulate DARS.

Clear cell renal cell carcinoma (ccRCC), the most frequent subtype of renal cell carcinoma (RCC), is characterized by high relapse rate and mortality. Long non-coding RNAs (lncRNAs) are critical participants during cancer development. LncRNA DARS antisense RNA 1 (DARS-AS1), a newly-found lncRNA, is not specifically reported in ccRCC. However, Gene Expression Profiling Interactive Analysis (GEPIA) and starBase databases revealed the up-regulation of DARS-AS1 in ccRCC. Current study investigated the function and mechanism of DARS-AS1 in ccRCC. Functional assays including colony formation assay, EdU assay, caspase-3 activity detection, flow cytometry analysis and JC-1 assay were implemented to identify the role of DARS-AS1 in ccRCC. As a result, silencing of DARS-AS1 retarded proliferation and facilitated apoptosis in ccRCC cells. Moreover, mainly a cytoplasmic localization of lncRNA DARS-AS1 was verified in ccRCC cells. Then, we demonstrated that DARS-AS1 positively regulated its nearby gene, aspartyl-tRNA synthetase (DARS), by sequestering miR-194-5p. Moreover, DARS was testified as the oncogene in ccRCC and DARS-AS1 worked as a tumor-facilitator in ccRCC through miR-194-5p/DARS signaling. In a summary, this study firstly uncovered that DARS-AS1 boosted DARS expression via absorbing miR-194-5p, therefore contributing to malignancy in ccRCC. Our findings may be helpful for opening new strategies for ccRCC treatment.

Mutations in aARS genes revealed by targeted next-generation sequencing in patients with mitochondrial diseases.

Mitochondrial diseases are a clinically heterogeneous group of multisystemic disorders that arise as a result of various mitochondrial dysfunctions. Autosomal recessive aARS deficiencies represent a rapidly growing group of severe rare inherited mitochondrial diseases, involving multiple organs, and currently without curative option. They might be related to defects of mitochondrial aminoacyl t-RNA synthetases (mtARS) that are ubiquitous enzymes involved in mitochondrial aminoacylation and the translation process. Here, using NGS analysis of 281 nuclear genes encoding mitochondrial proteins, we identified 4 variants in different mtARS in three patients from unrelated Tunisian families, with clinical features of mitochondrial disorders. Two homozygous variants were found in KARS (c.683C>T) and AARS2 (c.1150-4C>G), respectively in two patients, while two heterozygous variants in EARS2 (c.486-7C>G) and DARS2 (c.1456C>T) were concomitantly found in the third patient. Bio-informatics investigations predicted their pathogenicity and deleterious effects on pre-mRNA splicing and on protein stability. Thus, our results suggest that mtARS mutations are common in Tunisian patients with mitochondrial diseases.

Anticodon-binding domain swapping in a nondiscriminating aspartyl-tRNA synthetase reveals contributions to tRNA specificity and catalytic activity.

The nondiscriminating aspartyl-tRNA synthetase (ND-AspRS), found in many archaea and bacteria, covalently attaches aspartic acid to tRNAAsp and tRNAAsn generating a correctly charged Asp-tRNAAsp and an erroneous Asp-tRNAAsn . This relaxed tRNA specificity is governed by interactions between the tRNA and the enzyme. In an effort to assess the contributions of the anticodon-binding domain to tRNA specificity, we constructed two chimeric enzymes, Chimera-D and Chimera-N, by replacing the native anticodon-binding domain in the Helicobacter pylori ND-AspRS with that of a discriminating AspRS (Chimera-D) and an asparaginyl-tRNA synthetase (AsnRS, Chimera-N), both from Escherichia coli. Both chimeric enzymes showed similar secondary structure compared to wild-type (WT) ND-AspRS and maintained the ability to form dimeric complexes in solution. Although less catalytically active than WT, Chimera-D was more discriminating as it aspartylated tRNAAsp over tRNAAsn with a specificity ratio of 7.0 compared to 2.9 for the WT enzyme. In contrast, Chimera-N exhibited low catalytic activity toward tRNAAsp and was unable to aspartylate tRNAAsn . The observed catalytic activities for the two chimeras correlate with their heterologous toxicity when expressed in E. coli. Molecular dynamics simulations show a reduced hydrogen bond network at the interface between the anticodon-binding domain and the catalytic domain in Chimera-N compared to Chimera-D or WT, explaining its lower stability and catalytic activity.

Leishmanial aspartyl-tRNA synthetase: Biochemical, biophysical and structural insights.

Aminoacyl tRNA synthetases (aaRSs) are integral components of protein biosynthesis along with several non-canonical cellular processes. Inhibition studies of aaRSs presented these enzymes as promising drug targets in many pathogens, however aspartyl tRNA synthetase has not been studied in trypanosomatids despite its essentiality. Hence, full-length ORF of Leishmania donovani aspartyl tRNA synthetase (LdaspRS) was cloned and purified to homogeneity followed by molecular mass determination. The aminoacylation assay established that the purified protein performs its function optimally at physiological pH and temperature. The kinetic parameters of LdaspRS revealed the affinity of l-aspartate towards the enzyme to be very much lower than the cofactor. Our study also highlights the moonlighting function of LdaspRS to stimulate the pro-inflammatory cytokines and nitric oxide generation by host macrophage. Furthermore, CD and intrinsic tryptophan fluorescence measurements showed the changes in structural conformation at varying pH, denaturants and ligands. The modelled LdaspRS structure presented all the specific characteristics of class II aaRSs, while in silico study suggested binding of pyrimidine-derived inhibitors in its cofactor binding site with high affinity followed by validation using MD simulation. Altogether, this study could provide a platform for exploring LdaspRS to develop potential therapeutics against leishmaniasis.

Structural Insights into the Binding of Natural Pyrimidine-Based Inhibitors of Class II Aminoacyl-tRNA Synthetases.

The pyrimidine-containing Trojan horse antibiotics albomycin and a recently discovered cytidine-containing microcin C analog target the class II seryl- and aspartyl-tRNA synthetases (serRS and aspRS), respectively. The active components of these compounds are competitive inhibitors that mimic the aminoacyl-adenylate intermediate. How they effectively substitute for the interactions mediated by the canonical purine group is unknown. Employing nonhydrolyzable aminoacyl-sulfamoyl nucleosides substituting the base with cytosine, uracil, and N3-methyluracil the structure-activity relationship of the natural compounds was evaluated. In vitro using E. coli serRS and aspRS, the best compounds demonstrated IC50 values in the low nanomolar range, with a clear preference for cytosine or N3-methyluracil over uracil. X-ray crystallographic structures of K. pneumoniae serRS and T. thermophilus aspRS in complex with the compounds showed the contribution of structured waters and residues in the conserved motif-2 loop in defining base preference. Utilizing the N3-methyluracil bound serRS structure, MD simulations of the fully modified albomycin base were performed to identify the interacting network that drives stable association. This analysis pointed to key interactions with a methionine in the motif-2 loop. Interestingly, this residue is mutated to a glycine in a second serRS (serRS2) found in albomycin-producing actinobacteria possessing self-immunity to this antibiotic. A comparative study demonstrated that serRS2 is poorly inhibited by the pyrimidine-containing intermediate analogs, and an equivalent mutation in E. coli serRS significantly decreased the affinity of the cytosine congener. These findings highlight the crucial role of dynamics and solvation of the motif-2 loop in modulating the binding of the natural antibiotics.

Symmetric Assembly of a Decameric Subcomplex in Human Multi-tRNA Synthetase Complex Via Interactions between Glutathione Transferase-Homology Domains and Aspartyl-tRNA Synthetase.

Aminoacyl-tRNA synthetases (AARSs) ligate amino acids to their cognate tRNAs during protein synthesis. In humans, eight AARSs and three non-enzymatic AARS-interacting multifunctional proteins (AIMP1-3), which are involved in various biological processes, form a multi-tRNA synthetase complex (MSC). Elucidation of the structures and multiple functions of individual AARSs and AIMPs has aided current understanding of the structural arrangement of MSC components and their assembly processes. Here, we report the crystal structure of a complex comprising a motif from aspartyl-tRNA synthetase (DRS) and the glutathione transferase (GST)-homology domains of methionyl-tRNA synthetase (MRS), glutamyl-prolyl-tRNA synthetase (EPRS), AIMP2, and AIMP3. In the crystal structure, the four GST domains are assembled in the order of MRS-AIMP3-EPRS-AIMP2, and the GST domain of AIMP2 binds DRS through the ß-sheet in the GST domain. The C-terminus of AIMP3 enhances the binding of DRS to the tetrameric GST complex. A DRS dimer and two GST tetramers binding to the dimer with 2-fold symmetry complete a decameric complex. The formation of this complex enhances the stability of DRS and enables it to retain its reaction intermediate, aspartyl adenylate. Since the catalytic domains of MRS and EPRS are connected to the decameric complex through their flexible linker peptides, and lysyl-tRNA synthetase and AIMP1 are also linked to the complex via the N-terminal region of AIMP2, the DRS-GST tetramer complex functions as a frame in the MSC.

Spectroscopic Studies of Asparaginyl-tRNA Synthetase from Entamoeba histolytica.

BACKGROUND: Aminoacyl-tRNA synthetases play an important role in catalyzing the first step in protein synthesis by attaching the appropriate amino acid to its cognate tRNA which then transported to the growing polypeptide chain. Asparaginyl-tRNA Synthetase (AsnRS) from Brugia malayi, Leishmania major, Thermus thermophilus, Trypanosoma brucei have been shown to play an important role in survival and pathogenesis. Entamoeba histolytica (Ehis) is an anaerobic eukaryotic pathogen that infects the large intestines of humans. It is a major cause of dysentery and has the potential to cause life-threatening abscesses in the liver and other organs making it the second leading cause of parasitic death after malaria. Ehis-AsnRS has not been studied in detail, except the crystal structure determined at 3 Å resolution showing that it is primarily α-helical and dimeric. It is a homodimer, with each 52 kDa monomer consisting of 451 amino acids. It has a relatively short N-terminal as compared to its human and yeast counterparts. OBJECTIVE: Our study focusses to understand certain structural characteristics of Ehis-AsnRS using biophysical tools to decipher the thermodynamics of unfolding and its binding properties. METHODS: Ehis-AsnRS was cloned and expressed in E. coli BL21DE3 cells. Protein purification was performed using Ni-NTA affinity chromatography, following which the protein was used for biophysical studies. Various techniques such as steady-state fluorescence, quenching, circular dichroism, differential scanning fluorimetry, isothermal calorimetry and fluorescence lifetime studies were employed for the conformational characterization of Ehis-AsnRS. Protein concentration for far-UV and near-UV circular dichroism experiments was 8 µM and 20 µM respectively, while 4 µM protein was used for the rest of the experiments. RESULTS: The present study revealed that Ehis-AsnRS undergoes unfolding when subjected to increasing concentration of GdnHCl and the process is reversible. With increasing temperature, it retains its structural compactness up to 45ºC before it unfolds. Steady-state fluorescence, circular dichroism and hydrophobic dye binding experiments cumulatively suggest that Ehis-AsnRS undergoes a two-state transition during unfolding. Shifting of the transition mid-point with increasing protein concentration further illustrate that dissociation and unfolding processes are coupled indicating the absence of any detectable folded monomer. CONCLUSION: This article indicates that GdnHCl induced denaturation of Ehis-AsnRS is a two - state process and does not involve any intermediate; unfolding occurs directly from native dimer to unfolded monomer. The solvent exposure of the tryptophan residues is biphasic, indicating selective quenching. Ehis-AsnRS also exhibits a structural as well as functional stability over a wide range of pH.