Neuropathy-associated histidyl-tRNA synthetase variants attenuate protein synthesis in vitro and disrupt axon outgrowth in developing zebrafish.

Charcot-Marie-Tooth disease (CMT) encompasses a set of genetically and clinically heterogeneous neuropathies characterized by length-dependent dysfunction of the peripheral nervous system. Mutations in over 80 diverse genes are associated with CMT, and aminoacyl-tRNA synthetases (ARS) constitute a large gene family implicated in the disease. Despite considerable efforts to elucidate the mechanistic link between ARS mutations and the CMT phenotype, the molecular basis of the pathology is unknown. In this work, we investigated the impact of three CMT-associated substitutions (V155G, Y330C, and R137Q) in the cytoplasmic histidyl-tRNA synthetase (HARS1) on neurite outgrowth and peripheral nervous system development. The model systems for this work included a nerve growth factor-stimulated neurite outgrowth model in rat pheochromocytoma cells (PC12), and a zebrafish line with GFP/red fluorescent protein reporters of sensory and motor neuron development. The expression of CMT-HARS1 mutations led to attenuation of protein synthesis and increased phosphorylation of eIF2α in PC12 cells and was accompanied by impaired neurite and axon outgrowth in both models. Notably, these effects were phenocopied by histidinol, a HARS1 inhibitor, and cycloheximide, a protein synthesis inhibitor. The mutant proteins also formed heterodimers with wild-type HARS1, raising the possibility that CMT-HARS1 mutations cause disease through a dominant-negative mechanism. Overall, these findings support the hypothesis that CMT-HARS1 alleles exert their toxic effect in a neuronal context, and lead to dysregulated protein synthesis. These studies demonstrate the value of zebrafish as a model for studying mutant alleles associated with CMT, and for characterizing the processes that lead to peripheral nervous system dysfunction.

Identification of Chemical Compounds That Inhibit the Function of Histidyl-tRNA Synthetase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa histidyl-tRNA synthetase (HisRS) was selected as a target for antibiotic drug development. The HisRS protein was overexpressed in Escherichia coli and kinetically evaluated. The KM values for interaction of HisRS with its three substrates, histidine, ATP, and tRNAHis, were 37.6, 298.5, and 1.5 µM, while the turnover numbers were 8.32, 16.8, and 0.57 s-1, respectively. A robust screening assay was developed, and 800 natural products and 890 synthetic compounds were screened for inhibition of activity. Fifteen compounds with inhibitory activity were identified, and the minimum inhibitory concentration (MIC) was determined for each against a panel of nine pathogenic bacteria. Each compound exhibited broad-spectrum activity. Based on structural similarity and MIC results, four compounds, BT02C02, BT02D04, BT08E04, and BT09C11, were selected for additional analysis. These compounds inhibited the activity of HisRS with IC50 values of 4.4, 9.7, 14.1, and 11.3 µM, respectively. Time-kill studies indicated a bacteriostatic mode of inhibition for each compound. BT02D04 and BT08E04 were noncompetitive with both histidine and ATP, BT02C02 was competitive with histidine but noncompetitive with ATP, and BT09C11 was uncompetitive with histidine and noncompetitive with ATP. These compounds were not observed to be toxic to human cell cultures.

Characterization of aminoacyl-tRNA synthetase stability and substrate interaction by differential scanning fluorimetry.

Differential scanning fluorimetry (DSF) is a fluorescence-based assay to evaluate protein stability by determining protein melting temperatures. Here, we describe the application of DSF to investigate aminoacyl-tRNA synthetase (AARS) stability and interaction with ligands. Employing three bacterial AARS enzymes as model systems, methods are presented here for the use of DSF to measure the apparent temperatures at which AARSs undergo melting transitions, and the effect of AARS substrates and inhibitors. One important observation is that the extent of temperature stability realized by an AARS in response to a particular bound ligand cannot be predicted a priori. The DSF method thus serves as a rapid and highly quantitative approach to measure AARS stability, and the ability of ligands to influence the temperature at which unfolding transitions occur.

A binding hotspot in Trypanosoma cruzi histidyl-tRNA synthetase revealed by fragment-based crystallographic cocktail screens.

American trypanosomiasis, commonly known as Chagas disease, is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. The chronic form of the infection often causes debilitating morbidity and mortality. However, the current treatment for the disease is typically inadequate owing to drug toxicity and poor efficacy, necessitating a continual effort to discover and develop new antiparasitic therapeutic agents. The structure of T. cruzi histidyl-tRNA synthetase (HisRS), a validated drug target, has previously been reported. Based on this structure and those of human cytosolic HisRS, opportunities for the development of specific inhibitors were identified. Here, efforts are reported to identify small molecules that bind to T. cruzi HisRS through fragment-based crystallographic screening in order to arrive at chemical starting points for the development of specific inhibitors. T. cruzi HisRS was soaked into 68 different cocktails from the Medical Structural Genomics of Pathogenic Protozoa (MSGPP) fragment library and diffraction data were collected to identify bound fragments after soaking. A total of 15 fragments were identified, all bound to the same site on the protein, revealing a fragment-binding hotspot adjacent to the ATP-binding pocket. On the basis of the initial hits, the design of reactive fragments targeting the hotspot which would be simultaneously covalently linked to a cysteine residue present only in trypanosomatid HisRS was initiated. Inhibition of T. cruzi HisRS was observed with the resultant reactive fragments and the anticipated binding mode was confirmed crystallographically. These results form a platform for the development of future generations of selective inhibitors for trypanosomatid HisRS.

Síndrome por anticuerpos antisintetasa / Anti-synthetase syndrome

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Affinity labeling of Escherichia coli histidyl-tRNA synthetase with reactive ATP analogues. Identification of labeled amino acid residues by matrix assisted laser desorption-ionization mass spectrometry.

Recent affinity labeling studies have revealed that dimeric histidyl-tRNA synthetase from Escherichia coli displayed half-of-the-sites reactivity toward labeling with pyridoxal 5'-phosphate [Kalogerakos, T., Hountondji, C., Berne, P. F., Dutka, S. & Blanquet, S. (1994) Biochimie (Paris) 76, 33-44]. In the present report, affinity labeling studies were conducted by using other ATP analogues such as pyridoxal 5'-diphospho-5'-adenosine (pyridoxal-ppAdo), pyridoxal 5'-triphospho-5'-adenosine (pyridoxal-pppAdo), pyridoxal 5'-diphosphate (pyridoxal-P2) and 5'-p-fluorosulfonylbenzoyladenosine (FSO2BzAdo). The histidine-dependent isotopic [32P]PP/ATP exchange activity of His-tRNA synthetase was rapidly and completely lost upon incubation with either pyridoxal-ppAdo, pyridoxal-pppAdo or pyridoxal-P2, followed by reduction with sodium borohydride. Complete inactivation of His-tRNA synthetase corresponded to the incorporation of 2.8 mol of either pyridoxal-ppAdo or pyridoxal-P2/mol dimeric synthetase. Incubation of His-tRNA synthetase with FSO2BzAdo also resulted in a complete inactivation of the synthetase. However, contrasting with the pyridoxal derivatives, the plot of the residual enzymatic activity against the amount of covalently bound FSO2BzAdo appeared biphasic. In the early stages of inactivation, the relationship between the amount of residual activity and FSO2BzAdo incorporation was linear and extrapolated to a stoichiometry of 1.1 mol reagent/mol His-tRNA synthetase, suggesting that the labeling of one subunit was sufficient to inactivate one dimeric His-tRNA synthetase molecule. At longer incubation periods, additional reagent incorporation occurred and culminated at 2.5 mol label/mol His-tRNA synthetase. Excess of MgATP protected the enzyme against inactivation by either studied reagent. The labeled amino acid residues were identified by matrix-assisted-laser-desorption-ionization mass spectrometry, by measuring the peptide mass increase caused by the reagents. An identical set of four lysyl residues (Lys2, Lys118, Lys369 and Lys370 of His-tRNA synthetase) was found attached to pyridoxal-ppAdo or pyridoxal-P2. In addition, pyridoxal-ppAdo labeled the alpha-amino group of the N-terminal alanine. In a His-tRNA synthetase sample having incorporated 2.5 mol FSO2BzAdo/mol), the labeled amino acid residues were Lys118, Lys196, Tyr262 (or Tyr263), Lys369 and Lys377. Whatever the used reagent, Lys118 appeared to be the predominantly labeled residue, Lys118 belongs to fragment 112-124 (RHERPQK-GRYRQF) corresponding to motif 2 of class 2 aminoacyl-tRNA synthetases. The other modified lysyl residues (lysines 369, 370 and 377) are close to the catalytic motif 3, in the C-terminal region of the synthetase. Tyr262 and Tyr263 belong to a fragment 256-263 (LVRGLDYY) highly conserved among all known His-tRNA synthetase primary structures. Examination of the recently solved structure of crystalline E. coli His-tRNA synthetase [Amez, J. G., Harris, D. C., Mitschler, A., Rees, B., Francklyn, C. S. & Moras, D. (1995) EMBO J. 14, 4143-4155] shows that, with the exception of lysines 369, 370 and 377, the location of which may account for peculiar accessibility and reactivity, all the amino acid residues identified in this study map near the enzyme nucleotide-binding site, at the N-terminal catalytic domain of the synthetase.

L-histidinol in experimental cancer chemotherapy: improving the selectivity and efficacy of anticancer drugs, eliminating metastatic disease and reversing the multidrug-resistant phenotype.

Human cancer chemotherapy is limited by two major problems: the failure of commonly used anticancer drugs to act against tumor cells in a specific manner and the ability of malignant cells to resist killing by antineoplastic agents. Experimentally, both of these problems can be solved by using L-histidinol in combination with conventional anticancer drugs. A structural analogue of the essential amino acid L-histidine and an inhibitor of protein biosynthesis. L-histidinol improves the selectivity and the efficacy of a variety of cancer drugs in several transplantable murine tumors. Furthermore, L-histidinol circumvents the drug-resistant traits of a variety of cancer cells, including those showing multidrug resistance. This review will summarize these properties of L-histidinol, present new evidence on its ability to increase the vulnerability of both drug-sensitive and drug-resistant human leukemia cells to various anticancer drugs, and show that, in addition to inhibiting protein synthesis, L-histidinol acts as an intracellular histamine antagonist. The establishment of a connection between the latter mechanism and the capacity to modulate anticancer drug action has resulted in a clinical trial in the treatment of human cancer.

Purification of mammalian histidyl-tRNA synthetase and its interaction with myositis-specific anti-Jo-1 antibodies.

Histidyl-tRNA synthetase is purified to near homogeneity from rat liver. The subunit molecular weight of histidyl-tRNA synthetase is 50,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The Stokes radius and the sedimentation coefficient of histidyl-tRNA synthetase are 38 A and 6.0 S, respectively. The native molecular weight of histidyl-tRNA synthetase is calculated to be 96,000 on the basis of its hydrodynamic properties. The purified histidyl-tRNA synthetase reacts with the myositis-specific anti-Jo-1 antibodies. Anti-Jo-1 immunoglobulin G reacts with the native form of histidyl-tRNA synthetase and does not react or only weakly reacts with the denatured form. The anti-Jo-1 antibodies exhibit stronger inhibition toward histidyl-tRNA synthetase that has been preincubated with tRNA than that without preincubation. Anti-Jo-1 antibodies behave as a noncompetitive inhibitor with respect to tRNA in the aminoacylation reaction catalyzed by histidyl-tRNA synthetase. The structural features of the antigen of the anti-Jo-1 antibodies in light of these results are discussed.

Measurement of antibody to Jo-1 by ELISA and comparison to enzyme inhibitory activity.

Antibody to the Jo-1 antigen (histidyl-tRNA synthetase) is found almost exclusively in myositis patients, usually those with adult PM, but has been found in only 30% of that group by immunodiffusion or other techniques thus far reported. We have reexamined the prevalence of antibody to Jo-1 in sera from 130 patients and 82 controls by using the sensitive ELISA technique. The ELISA used affinity-purified, enzymatically active bovine Jo-1 antigen. A wide range of antibody level by ELISA was found among 24 immunodiffusion positive sera. Six myositis and two control sera had apparent specific antibody detectable only by ELISA. Overall, however, the antibody continued to show high myositis specificity with predominance in adult PM (35.8% in that group). Because the antibody inhibits enzymatic activity of the synthetase antigen, we also studied the quantitative inhibitory activity of these sera to compare with the antibody activity as determined by ELISA. Twenty-four immunodiffusion-positive sera, 29 immunodiffusion-negative sera, and 15 normal sera were tested at 1/50 dilution in the reaction mixture. There was background inhibition by all normal sera tested that averaged 30.5%. All but one immunodiffusion negative myositis sera (a high binder by ELISA) inhibited less than 50% of the average with normal serum. Twenty-three of 24 immunodiffusion positive sera inhibited greater than 80% of this normal average; the other inhibited 66%. The serum dilution giving 50% inhibition was highly correlated (R = 0.83) with the ELISA activity. Thus, inhibition of histidyl-tRNA synthetase activity is a relatively accurate measure of Jo-1 antibody. This method should be applicable to measuring antibody to other aminoacyl-tRNA synthetases.

Rat liver histidyl-tRNA synthetase. Purification and inhibition by the myositis-specific anti-Jo-1 autoantibody.

Myositis is an autoimmune inflammatory muscle disease of unknown etiology. We demonstrate directly that the antigen to the myositis-specific anti-Jo-1 antibody is histidyl-tRNA synthetase. The anti-Jo-1 antibody inhibits human HeLa and rat liver histidyl-tRNA synthetase. Using conventional and immunoaffinity chromatography with immobilized anti-Jo-1 antibody, we have purified rat liver histidyl-tRNA synthetase which has a subunit Mr 64,000 and an estimated native Mr suggesting an alpha 2 structure. The evidence indicates that the Jo-1 antigen is histidyl-tRNA synthetase, and that some of the histidyl-tRNA synthetase structure are conserved across species.

Purification and characterization of histidyl-transfer RNA synthetase from Neurospora crassa.

Histidyl-tRNA synthetase (L-histidine:tRNAHis ligase (AMP-forming), EC 6.1.1.21) has been purified 921-fold from crude extracts of lyophilized mycelia of Neurospora crassa. Sodium dodecyl sulfate gel electrophoresis at pH 8.9 of the purified enzyme yields one band with an apparent Mr of 62 500. The estimated Mr by Sephadex gel filtration is 125 000. Thus the native histidyl-tRNA synthetase of N. crassa is a dimer, composed of two identical subunits. The Km values determined in the enzyme-catalyzed esterification of [14C]-histidine to tRNAHis are: for histidine, 5.8 x 10(-6 M, for ATP, 5.9 x 10(-4) M, and for tRNAHis, 1.2 x 10(-7) M. Effects of various intermediates of the histidine, tryptophan and arginine biosynthetic pathways on histidyl-tRNA synthetase activity were studied. The Ki values for imidazoleglycerol phosphate and histidinol (histidine intermediates and competitive inhibitors of the enzyme) are 1.1 x 10(-2) M, 1.3 x 10(-6) M, respectively. The Ki for indoleglycerol phosphate (a tryptophan intermediate and non-competitive inhibitor) is 1.2 x 10(-3) M.

Histidine transfer RNA levels in Friend leukemia cells: stimulation by histidine deprivation.

Friend leukemia cells incubated with sublethal concentrations of histidinol for 5 to 6 days show up to twofold increases in their relative concentrations of histidine transfer RNA and no change in the relative concentrations of leucine transfer RNA. A similar effect is seen when cells are grown to stationary phase in the presence of 0.2 times the amount of histidine in Eagle's minimum essential medium. These observations support the theory that the concentrations of specific transfer RNA's are regulated by a mechanism that is sensitive to the extent of their aminoacylation.