Production of Tobacco Etch Virus Protease (TEV) Expressed in the Endotoxin-Free Bacillus subtilis and Its Application.

Tobacco Etch virus (TEV) protease is one of the most common tools for removing fusion tags, but no study has shown that TEV can be expressed at high levels in the GRAS host strain Bacillus subtilis and purified for further application. In this study, the fusion protein BsLysSN-TEV C/S-His-TEV consisting of a fusion tag, N-terminal domain of a lysyl-tRNA synthetase (BsLysSN) coded by B. subtilis lysS gene, placed at the N-terminus followed by an endoprotease TEV cleavage site and then the expression of this fusion protein in the cytoplasm of B. subtilis was investigated. The SDS-PAGE and Western-blot analysis demonstrated that His-TEV was overexpressed under the induction of IPTG. This result infers that His-TEV protease showed promising activity in the B. subtilis cytoplasm by the cleavage of the fusion protein. These cleavage products could be purified using the Ni-NTA column, which effectively cleaved the purified recombinant protein substrate, which can be applied in the protein purification process to remove the fusion tag. Significantly, since both His-TEV protease and the fusion recombinant protein substrate are expressed in the endotoxin-free host strain, the tag removal and purified product should be theoretically endotoxin-free, which could be a promising approach for producing therapeutic proteins and also for other relevant biomedical applications.

The polyphyletic origins of glycyl-tRNA synthetase and lysyl-tRNA synthetase and their implications.

I analyzed the polyphyletic origin of glycyl-tRNA synthetase (GlyRS) and lysyl-tRNA synthetase (LysRS), making plausible the following implications. The fact that the genetic code needed to evolve aminoacyl-tRNA synthetases (ARSs) only very late would be in perfect agreement with a late origin, in the main phyletic lineages, of both GlyRS and LysRS. Indeed, as suggested by the coevolution theory, since the genetic code was structured by biosynthetic relationships between amino acids and as these occurred on tRNA-like molecules which were evidently already loaded with amino acids during its structuring, this made possible a late origin of ARSs. All this corroborates the coevolution theory of the origin of the genetic code to the detriment of theories which would instead predict an early intervention of the action of ARSs in organizing the genetic code. Furthermore, the assembly of the GlyRS and LysRS protein domains in main phyletic lineages is itself at least evidence of the possibility that ancestral genes were assembled using pieces of genetic material that coded these protein domains. This is in accordance with the exon theory of genes which postulates that ancestral exons coded for protein domains or modules that were assembled to form the first genes. This theory is exemplified precisely in the evolution of both GlyRS and LysRS which occurred through the assembly of protein domains in the main phyletic lineages, as analyzed here. Furthermore, this late assembly of protein domains of these proteins into the two main phyletic lineages, i.e. a polyphyletic origin of both GlyRS and LysRS, appears to corroborate the progenote evolutionary stage for both LUCA and at least the first part of the evolutionary stages of the ancestor of bacteria and that of archaea. Indeed, this polyphyletic origin would imply that the genetic code was still evolving because at least two ARSs, i.e. proteins that make the genetic code possible today, were still evolving. This would imply that the evolutionary stages involved were characterized not by cells but by protocells, that is, by progenotes because this is precisely the definition of a progenote. This conclusion would be strengthened by the observation that both GlyRS and LysRS originating in the phyletic lineages leading to bacteria and archaea, would demonstrate that, more generally, proteins were most likely still in rapid and progressive evolution. Namely, a polyphyletic origin of proteins which would qualify at least the initial phase of the evolutionary stage of the ancestor of bacteria and that of archaea as stages belonging to the progenote.

Structure-guided conversion from an anaplastic lymphoma kinase inhibitor into Plasmodium lysyl-tRNA synthetase selective inhibitors.

Aminoacyl-tRNA synthetases (aaRSs) play a central role in the translation of genetic code, serving as attractive drug targets. Within this family, the lysyl-tRNA synthetase (LysRS) constitutes a promising antimalarial target. ASP3026, an anaplastic lymphoma kinase (ALK) inhibitor was recently identified as a novel Plasmodium falciparum LysRS (PfLysRS) inhibitor. Here, based on cocrystal structures and biochemical experiments, we developed a series of ASP3026 analogues to improve the selectivity and potency of LysRS inhibition. The leading compound 36 showed a dissociation constant of 15.9 nM with PfLysRS. The inhibitory efficacy on PfLysRS and parasites has been enhanced. Covalent attachment of L-lysine to compound 36 resulted in compound 36K3, which exhibited further increased inhibitory activity against PfLysRS but significantly decreased activity against ALK. However, its inhibitory activity against parasites did not improve, suggesting potential future optimization directions. This study presents a new example of derivatization of kinase inhibitors repurposed to inhibit aaRS.

Human lysyl-tRNA synthetase phosphorylation promotes HIV-1 proviral DNA transcription.

Human lysyl-tRNA synthetase (LysRS) was previously shown to be re-localized from its normal cytoplasmic location in a multi-aminoacyl-tRNA synthetase complex (MSC) to the nucleus of HIV-1 infected cells. Nuclear localization depends on S207 phosphorylation but the nuclear function of pS207-LysRS in the HIV-1 lifecycle is unknown. Here, we show that HIV-1 replication was severely reduced in a S207A-LysRS knock-in cell line generated by CRISPR/Cas9; this effect was rescued by S207D-LysRS. LysRS phosphorylation up-regulated HIV-1 transcription, as did direct transfection of Ap4A, an upstream transcription factor 2 (USF2) activator that is synthesized by pS207-LysRS. Overexpressing an MSC-derived peptide known to stabilize LysRS MSC binding inhibited HIV-1 replication. Transcription of HIV-1 proviral DNA and other USF2 target genes was reduced in peptide-expressing cells. We propose that nuclear pS207-LysRS generates Ap4A, leading to activation of HIV-1 transcription. Our results suggest a new role for nuclear LysRS in facilitating HIV-1 replication and new avenues for antiviral therapy.

Changes in subcellular localization of Lysyl-tRNA synthetase and the 67-kDa laminin receptor in epithelial ovarian cancer metastases.

BACKGROUND: Although lysyl-tRNA synthetase (KARS1) is predominantly located in the cytosol, it is also present in the plasma membrane where it stabilizes the 67-kDa laminin receptor (67LR). This physical interaction is strongly increased under metastatic conditions. However, the dynamic interaction of these two proteins and the turnover of KARS1 in the plasma membrane has not previously been investigated. OBJECTIVE: Our objective in this study was to identify the membranous location of KARS1 and 67LR and investigate if this changes with the developmental stage of epithelial ovarian cancer (EOC) and treatment with the inhibitor BC-K01. In addition, we evaluated the therapeutic efficacy of BC-K01 in combination with paclitaxel, as the latter is frequently used to treat patients with EOC. METHODS: Overall survival and prognostic significance were determined in EOC patients according to KARS1 and 67LR expression levels as determined by immunohistochemistry. Changes in the location and expression of KARS1 and 67LR were investigated in vitro after BC-K01 treatment. The effects of this compound on tumor growth and apoptosis were evaluated both in vitro and in vivo. RESULTS: EOC patients with high KARS1 and high 67LR expression had lower progression-free survival rates than those with low expression levels of these two markers. BC-K01 reduced cell viability and increased apoptosis in combination with paclitaxel in EOC cell xenograft mouse models. BC-K01 decreased membranous KARS1 expression, causing a reduction in 67LR membrane expression in EOC cell lines. BC-K01 significantly decreased in vivo tumor weight and number of nodules, especially when used in combination with paclitaxel. CONCLUSIONS: Co-localization of KARS1 and 67LR in the plasma membrane contributes to EOC progression. Inhibition of the KARS1-67LR interaction by BC-K01 suppresses metastasis in EOC.

Discovery of novel potent migrastatic Thiazolo[5,4-b]pyridines targeting Lysyl-tRNA synthetase (KRS) for treatment of Cancer metastasis.

Recently, non-canonical roles of Lysyl-tRNA Synthetase (KRS), which is associated with cell migration and cancer metastasis, have been reported. Therefore, KRS has emerged as a promising target for the treatment of cell migration-related diseases, especially cancer metastasis, although the satisfying chemical inhibitors targeting KRS have not yet been identified. Here, we report the discovery of novel, mechanistically unique, and potent cell migration inhibitors targeting KRS, including the chemical and biological studies on the most effective N,N-dialkylthiazolo [5,4-b]pyridin-2-amine (SL-1910). SL-1910 exhibited highly potent migration inhibition (EC50 = 81 nM against the mutant KRS-overexpressed MDA-MB-231 cells) and was superior to the previously reported KRS inhibitor (migration inhibitory EC50 = 8.5 µM against H226 cells). The KRS protein binding study via fluorescence-based binding titration and KRS protein 2D-NMR mapping study, in vitro concentration-dependent cell migration inhibition, and in vivo anti-metastatic activity of SL-1910, which consists of a new scaffold, have been reported in this study. In addition, in vitro absorption, distribution, metabolism, and excretion studies and mouse pharmacokinetics experiments for SL-1910 were conducted.

Synthetic post-translational modifications of elongation factor P using the ligase EpmA.

Canonically, tRNA synthetases charge tRNA. However, the lysyl-tRNA synthetase paralog EpmA catalyzes the attachment of (R)-ß-lysine to the ε-amino group of lysine 34 of the translation elongation factor P (EF-P) in Escherichia coli. This modification is essential for EF-P-mediated translational rescue of ribosomes stalled at consecutive prolines. In this study, we determined the kinetics of EpmA and its variant EpmA_A298G to catalyze the post-translational modification of K34 in EF-P with eight noncanonical substrates. In addition, acetylated EF-P was generated using an amber suppression system. The impact of these synthetically modified EF-P variants on in vitro translation of a polyproline-containing NanoLuc luciferase reporter was analyzed. Our results show that natural (R)-ß-lysylation was more effective in rescuing stalled ribosomes than any other synthetic modification tested. Thus, our work not only provides new biochemical insights into the function of EF-P, but also opens a new route to post-translationally modify proteins using EpmA.

How HIV-1 Integrase Associates with Human Mitochondrial Lysyl-tRNA Synthetase.

Replication of human immunodeficiency virus type 1 (HIV-1) requires the packaging of tRNALys,3 from the host cell into the new viral particles. The GagPol viral polyprotein precursor associates with mitochondrial lysyl-tRNA synthetase (mLysRS) in a complex with tRNALys, an essential step to initiate reverse transcription in the virions. The C-terminal integrase moiety of GagPol is essential for its association with mLysRS. We show that integrases from HIV-1 and HIV-2 bind mLysRS with the same efficiency. In this work, we have undertaken to probe the three-dimensional (3D) architecture of the complex of integrase with mLysRS. We first established that the C-terminal domain (CTD) of integrase is the major interacting domain with mLysRS. Using the pBpa-photo crosslinking approach, inter-protein cross-links were observed involving amino acid residues located at the surface of the catalytic domain of mLysRS and of the CTD of integrase. In parallel, using molecular docking simulation, a single structural model of complex was found to outscore other alternative conformations. Consistent with crosslinking experiments, this structural model was further probed experimentally. Five compensatory mutations in the two partners were successfully designed which supports the validity of the model. The complex highlights that binding of integrase could stabilize the tRNALys:mLysRS interaction.

Inhibition of Plasmodium falciparum Lysyl-tRNA synthetase via an anaplastic lymphoma kinase inhibitor.

Aminoacyl-tRNA synthetases are attractive targets for the development of antibacterial, antifungal, antiparasitic agents and for the treatment of other human diseases. Lysyl-tRNA synthetase (LysRS) from this family has been validated as a promising target for the development of antimalarial drugs. Here, we developed a high-throughput compatible assay and screened 1215 bioactive compounds to identify Plasmodium falciparum cytoplasmic LysRS (PfLysRS) inhibitor. ASP3026, an anaplastic lymphoma kinase inhibitor that was used in clinical trials for the treatment of B-cell lymphoma and solid tumors, was identified as a novel PfLysRS inhibitor. ASP3026 suppresses the enzymatic activity of PfLysRS at nanomolar potency, which is >380-fold more effective than inhibition of the human counterpart. In addition, the compound suppressed blood-stage P. falciparum growth. To understand the molecular mechanism of inhibition by ASP3026, we further solved the cocrystal structure of PfLysRS-ASP3026 at a resolution of 2.49 Å, providing clues for further optimization of the compound. Finally, primary structure-activity relationship analyses indicated that the inhibition of PfLysRS by ASP3026 is highly structure specific. This work not only provides a new chemical scaffold with good druggability for antimalarial development but also highlights the potential for repurposing kinase-inhibiting drugs to tRNA synthetase inhibitors to treat human diseases.

Lysyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization and Identification of Inhibitory Compounds.

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial infections and has highly developed systems for acquiring resistance against numerous antibiotics. The gene (lysS) encoding P. aeruginosa lysyl-tRNA synthetase (LysRS) was cloned and overexpressed, and the resulting protein was purified to 98% homogeneity. LysRS was kinetically evaluated, and the Km values for the interaction with lysine, adenosine triphosphate (ATP), and tRNALys were determined to be 45.5, 627, and 3.3 µM, respectively. The kcatobs values were calculated to be 13, 22.8, and 0.35 s-1, resulting in kcatobs/KM values of 0.29, 0.036, and 0.11 s-1µM-1, respectively. Using scintillation proximity assay technology, natural product and synthetic compound libraries were screened to identify inhibitors of function of the enzyme. Three compounds (BM01D09, BT06F11, and BT08F04) were identified with inhibitory activity against LysRS. The IC50 values were 17, 30, and 27 µM for each compound, respectively. The minimum inhibitory concentrations were determined against a panel of clinically important pathogens. All three compounds were observed to inhibit the growth of gram-positive organisms with a bacteriostatic mode of action. However, two compounds (BT06F11 and BT08F04) were bactericidal against cultures of gram-negative bacteria. When tested against human cell cultures, BT06F11 was not toxic at any concentration tested, and BM01D09 was toxic only at elevated levels. However, BT08F04 displayed a CC50 of 61 µg/mL. In studies of the mechanism of inhibition, BM01D09 inhibited LysRS activity by competing with ATP for binding, and BT08F04 was competitive with ATP and uncompetitive with the amino acid. BT06F11 inhibited LysRS activity by a mechanism other than substrate competition.

Using the IPTG-Inducible Pgrac212 Promoter for Overexpression of Human Rhinovirus 3C Protease Fusions in the Cytoplasm of Bacillus subtilis Cells.

Expression and secretion of recombinant proteins in the endotoxin-free bacterium, Bacillus subtilis, has been thoroughly studied, but overexpression in the cytoplasm has been limited to only a few proteins. Here, we used the robust IPTG-inducible promoter, Pgrac212, to overexpress human rhinovirus 3C protease (HRV3C) in the cytoplasm of B. subtilis cells. A novel solubility tag, the N-terminal domain of the lysS gene of B. subtilis coding for a lysyl-tRNA synthetase was placed at the N terminus with a cleavage site for the endoprotease HRV3C, followed by His-HRV3C or His-GST-HRV3C. The recombinant protease was purified by using a Ni-NTA column. In this study, the His-HRV3C and His-GST-HRV3C proteases were overexpressed in the cytoplasm of B. subtilis at 11% and 16% of the total cellular proteins, respectively. The specific protease activities were 8065 U/mg for His-HRV3C and 3623 U/mg for His-GST-HRV3C. The purified enzymes were used to cleave two different substrates followed by purification of the two different protein targets, the green fluorescent protein and the beta-galactosidase. In conclusion, the combination of an inducible promoter Pgrac212 and a solubility tag allowed the overexpression of the HRV3C protease in the cytoplasm of B. subtilis. The resulting fusion protein was purified using a nickel column and was active in cleaving target proteins to remove the fusion tags. This study offers an effective method for producing recombinant proteins in the cytoplasm of endotoxin-free bacteria.

Copper-Triggered Bioorthogonal Cleavage Reactions for Reversible Protein and Cell Surface Modifications.

Temporal and reversible control over protein and cell conjugations holds great potential for traceless release of antibody-drug conjugates (ADCs) on tumor sites as well as on-demand altering or removal of targeting elements on cell surface. We herein developed a bioorthogonal and traceless releasable reaction on proteins and intact cells to fulfill such purposes. A systematic survey of transition metals in catalyzing the bioorthogonal cleavage reactions revealed that copper complexes such as Cu(I)-BTTAA and dual-substituted propargyl (dsPra) or propargyloxycarbonyl (dsProc) moieties offered a bioorthogonal releasable pair for reversible blockage and rescue of primary amines and phenol alcohols on small molecule drugs, protein side chains, as well as intact cell surface. For proof-of-concept, we employed such Cu(I)-BTTAA/dsProc and Cu(I)-BTTAA/dsPra pairs as a "traceless linker" strategy to construct cleavable ADCs to unleash cytotoxic compounds on cancer cells in situ and as a "reversible modification" strategy for cell surface engineering. Furthermore, by coupling with the genetic code expansion strategy, we site-specifically modulated ligand-receptor interactions on live cell membranes. Together, our work expanded the transition-metal-mediated bioorthogonal cleavage tool kit from terminal decaging to internal-linker breakage, which offered a temporal and reversible conjugation strategy on therapeutic proteins and cells.

Stabilization of Intrinsically Disordered DKK2 Protein by Fusion to RNA-Binding Domain.

Intrinsic disorders are a common feature of hub proteins in eukaryotic interactomes controlling the signaling pathways. The intrinsically disordered proteins (IDPs) are prone to misfolding, and maintaining their functional stability remains a major challenge in validating their therapeutic potentials. Considering that IDPs are highly enriched in RNA-binding proteins (RBPs), here we reasoned and confirmed that IDPs could be stabilized by fusion to RBPs. Dickkopf2 (DKK2), Wnt antagonist and a prototype IDP, was fused with lysyl-tRNA synthetase (LysRS), with or without the fragment crystallizable (Fc) domain of an immunoglobulin and expressed predominantly as a soluble form from a bacterial host. The functional competence was confirmed by in vitro Wnt signaling reporter and tube formation in human umbilical vein endothelial cells (HUVECs) and in vivo Matrigel plug assay. The removal of LysRS by site-specific protease cleavage prompted the insoluble aggregation, confirming that the linkage to RBP chaperones the functional competence of IDPs. While addressing to DKK2 as a key modulator for cancer and ischemic vascular diseases, our results suggest the use of RBPs as stabilizers of disordered proteinaceous materials for acquiring and maintaining the structural stability and functional competence, which would impact the druggability of a variety of IDPs from human proteome.

Genetic Code Expansion Method for Temporal Labeling of Endogenously Expressed Proteins.

We here present a method that combines genetic code expansion with CRISPR/Cas9 genome engineering to label endogenously expressed proteins with high spatiotemporal resolution. The method exploits the use of an orthogonal tRNA/tRNA synthetase pair in conjugation with noncanonical amino acids to create stop codon read through events. To demonstrate the functionality of the method, we pulse labeled endogenous ß-actin and tumor protein p53 with a minimally invasive HA tag at their C-termini. Targeting the protein label with a proximity ligation assay plus real time imaging facilitates seamless quantification of the protein synthesis rate and spatial localization at the single cell level. The presented approach does not interfere with any physiological control of cellular expression, nor did we observe any perturbation of endogenous protein functions.

Lysyl-tRNA synthetase-expressing colon spheroids induce M2 macrophage polarization to promote metastasis.

Lysyl-tRNA synthetase (KRS) functions canonically in cytosolic translational processes. However, KRS is highly expressed in colon cancer, and localizes to distinct cellular compartments upon phosphorylations (i.e., the plasma membranes after T52 phosphorylation and the nucleus after S207 phosphorylation), leading to probably alternative noncanonical functions. It is unknown how other subcellular KRSs crosstalk with environmental cues during cancer progression. Here, we demonstrate that the KRS-dependent metastatic behavior of colon cancer spheroids within 3D gels requires communication between cellular molecules and extracellular soluble factors and neighboring cells. Membranous KRS and nuclear KRS were found to participate in invasive cell dissemination of colon cancer spheroids in 3D gels. Cancer spheroids secreted GAS6 via a KRS-dependent mechanism and caused the M2 polarization of macrophages, which activated the neighboring cells via secretion of FGF2/GROα/M-CSF to promote cancer dissemination under environmental remodeling via fibroblast-mediated laminin production. Analyses of tissues from clinical colon cancer patients and Krs-/+ animal models for cancer metastasis supported the roles of KRS, GAS6, and M2 macrophages in KRS-dependent positive feedback between tumors and environmental factors. Altogether, KRS in colon cancer cells remodels the microenvironment to promote metastasis, which can thus be therapeutically targeted at these bidirectional KRS-dependent communications of cancer spheroids with environmental cues.

KARS-related diseases: progressive leukoencephalopathy with brainstem and spinal cord calcifications as new phenotype and a review of literature.

BACKGROUND: KARS encodes lysyl- transfer ribonucleic acid (tRNA) synthetase, which catalyzes the aminoacylation of tRNA-Lys in the cytoplasm and mitochondria. Eleven families/sporadic patients and 16 different mutations in KARS have been reported to date. The associated clinical phenotype is heterogeneous ranging from early onset encephalopathy to isolated peripheral neuropathy or nonsyndromic hearing impairment. Recently additional presentations including leukoencephalopathy as predominant cerebral involvement or cardiomyopathy, isolated or associated with muscular and cerebral involvement, have been reported. A progressive Leukoencephalopathy with brainstem and spinal cord calcifications was previously described in a singleton patient and in two siblings, without the identification of the genetic cause. We reported here about a new severe phenotype associated with biallelic KARS mutations and sharing some common points with the other already reported phenotypes, but with a distinct clinical and neuroimaging picture. Review of KARS mutant patients published to date will be also discussed. RESULTS: Herein, we report the clinical, biochemical and molecular findings of 2 unreported Italian patients affected by developmental delay, acquired microcephaly, spastic tetraparesis, epilepsy, sensory-neural hypoacusia, visual impairment, microcytic hypochromic anaemia and signs of hepatic dysfunction. MRI pattern in our patients was characterized by progressive diffuse leukoencephalopathy and calcifications extending in cerebral, brainstem and cerebellar white matter, with spinal cord involvement. Genetic analysis performed on these 2 patients and in one subject previously described with similar MRI pattern revealed the presence of biallelic mutations in KARS in all 3 subjects. CONCLUSIONS: With our report we define the molecular basis of the previously described Leukoencephalopathy with Brainstem and Spinal cord Calcification widening the spectrum of KARS related disorders, particularly in childhood onset disease suggestive for mitochondrial impairment. The review of previous cases does not suggest a strict and univocal genotype/phenotype correlation for this highly heterogeneous entity. Moreover, our cases confirm the usefulness of search for common brain and spine MR imaging pattern and of broad genetic screening, in syndromes clinically resembling mitochondrial disorders in spite of normal biochemical assay.

Characterization of association of human mitochondrial lysyl-tRNA synthetase with HIV-1 Pol and tRNA3Lys.

BACKGROUND: An important step in human immunodeficiency virus type 1 (HIV-1) replication is the packaging of tRNA3Lys from the host cell, which plays the role of primer RNA in the process of initiation of reverse transcription. The viral GagPol polyprotein precursor, and the human mitochondrial lysyl-tRNA synthetase (mLysRS) from the host cell, have been proposed to be involved in the packaging process. More specifically, the catalytic domain of mLysRS is supposed to interact with the transframe (TF or p6*) and integrase (IN) domains of the Pol region of the GagPol polyprotein. RESULTS: In this work, we report a quantitative characterization of the protein:protein interactions between mLysRS and its viral partners, the Pol polyprotein, and the isolated integrase and transframe domains of Pol. A dissociation constant of 1.3 ± 0.2 nM was determined for the Pol:mLysRS interaction, which exemplifies the robustness of this association. The protease and reverse transcriptase domains of GagPol are dispensable in this association, but the TF and IN domains have to be connected by a linker polypeptide to recapitulate a high affinity partner for mLysRS. The binding of the viral proteins to mLysRS does not dramatically enhance the binding affinity of mLysRS for tRNA3Lys. CONCLUSIONS: These data support the conclusion that the complex formed between GagPol, mLysRS and tRNA3Lys, which involves direct interactions between the IN and TF domains of Pol with mLysRS, is more robust than suggested by the previous models supposed to be involved in the packaging of tRNA3Lys into HIV-1 particles.

Identification of Major Enzymes Involved in the Synthesis of Diadenosine Tetraphosphate and/or Adenosine Tetraphosphate in Myxococcus xanthus.

Myxococcus xanthus generates diadenosine tetraphosphates (Ap4A) and diadenosine pentaphosphates (Ap5A) under various stress conditions. M. xanthus lysyl-tRNA synthetase (LysS) efficiently synthesizes Ap4A from ATP, Ap5A from ATP and adenosine tetraphosphate (Ap4), and Ap4 from ATP and triphosphate. To identify other M. xanthus enzymes that can catalyze Ap4A and Ap4 synthesis, 15 M. xanthus aminoacyl-tRNA synthetases (aaRSs), four acyl-CoA synthetases (Acys), three acetyl-CoA synthetases (Aces), phosphoglycerate kinase (Pgk), and adenylate kinase (Adk) were expressed in Escherichia coli and examined for Ap4A or Ap4 synthetase activity using ATP or ATP and triphosphate as substrates. Among the tested enzymes, LysS had the highest Ap4A synthetase activity. AlaRS, SerRS, and LeuRS1 showed high ADP synthetase activity with ATP as a substrate in the presence of pyrophosphatase, and also demonstrated the ability to produce Ap4 from ATP and triphosphate in the absence of pyrophosphatase. Ap4 formation by AlaRS, SerRS, and LeuRS1 was approximately 4- to 13-fold higher compared with that of Ap4A, suggesting that these enzymes prefer triphosphate over ATP as a substrate in the second reaction. Some of the recombinant M. xanthus Acys and Aces also synthesized Ap4 from ATP and triphosphate. However, Pgk was capable of catalyzing the production of Ap4 from ATP and 3-phosphoglycerate in the presence of Mg2+ and did not require triphosphate, suggesting that this enzyme is mainly responsible for Ap4 synthesis in M. xanthus.

HIV-1 Exploits a Dynamic Multi-aminoacyl-tRNA Synthetase Complex To Enhance Viral Replication.

A hallmark of retroviruses such as human immunodeficiency virus type 1 (HIV-1) is reverse transcription of genomic RNA to DNA, a process that is primed by cellular tRNAs. HIV-1 recruits human tRNALys3 to serve as the reverse transcription primer via an interaction between lysyl-tRNA synthetase (LysRS) and the HIV-1 Gag polyprotein. LysRS is normally sequestered in a multi-aminoacyl-tRNA synthetase complex (MSC). Previous studies demonstrated that components of the MSC can be mobilized in response to certain cellular stimuli, but how LysRS is redirected from the MSC to viral particles for packaging is unknown. Here, we show that upon HIV-1 infection, a free pool of non-MSC-associated LysRS is observed and partially relocalized to the nucleus. Heat inactivation of HIV-1 blocks nuclear localization of LysRS, but treatment with a reverse transcriptase inhibitor does not, suggesting that the trigger for relocalization occurs prior to reverse transcription. A reduction in HIV-1 infection is observed upon treatment with an inhibitor to mitogen-activated protein kinase that prevents phosphorylation of LysRS on Ser207, release of LysRS from the MSC, and nuclear localization. A phosphomimetic mutant of LysRS (S207D) that lacked the capability to aminoacylate tRNALys3 localized to the nucleus, rescued HIV-1 infectivity, and was packaged into virions. In contrast, a phosphoablative mutant (S207A) remained cytosolic and maintained full aminoacylation activity but failed to rescue infectivity and was not packaged. These findings suggest that HIV-1 takes advantage of the dynamic nature of the MSC to redirect and coopt cellular translation factors to enhance viral replication.IMPORTANCE Human tRNALys3, the primer for reverse transcription, and LysRS are essential host factors packaged into HIV-1 virions. Previous studies found that tRNALys3 packaging depends on interactions between LysRS and HIV-1 Gag; however, many details regarding the mechanism of tRNALys3 and LysRS packaging remain unknown. LysRS is normally sequestered in a high-molecular-weight multi-aminoacyl-tRNA synthetase complex (MSC), restricting the pool of free LysRS-tRNALys Mounting evidence suggests that LysRS is released under a variety of stimuli to perform alternative functions within the cell. Here, we show that HIV-1 infection results in a free pool of LysRS that is relocalized to the nucleus of target cells. Blocking this pathway in HIV-1-producing cells resulted in less infectious progeny virions. Understanding the mechanism by which LysRS is recruited into the viral assembly pathway can be exploited for the development of specific and effective therapeutics targeting this nontranslational function.

lysX gene is differentially expressed among Mycobacterium tuberculosis strains with different levels of virulence.

Antimicrobial peptides (AMPs) are mainly produced by epithelial cells and macrophages to eliminate infecting mycobacteria through direct antimicrobial activity and immunomodulation. Indeed, it has been described that this line of defense is essential to control infection. However, Mycobacterium tuberculosis (Mtb) has developed mechanisms to avoid AMPs activity, for instance lysX adds lysine residues to surface phospholipids changing their net charge, leading to the repelling of the AMPs. In the present study, we determined that lysX gene is differentially expressed among Mtb strains. To achieve this aim we used several well-characterized Mtb clinical isolates, lysX mutated strains and reference strains. Our results showed that in the presence of AMPs, lysX expression increased significantly. Strains with higher lysX expression showed increased levels of intracellular survival in vivo and in vitro and induced more severe lesion related with pneumonia. Results showed that ability of Mtb to replicate intracellularly was directly correlated to the level of lysX expression showing that the amount of lysX produced by the bacterial cell is an important variable for the modulation of Mtb virulence.