Integration of ligand and structure-based pharmacophore screening for the identification of novel natural leads against Euchromatic histone lysine methyltransferase 2 (EHMT2/G9a).

Herein, we report a blended ligand and structure-based pharmacophore screening approach to identify new natural leads against the Protein Lysine Methyltransferase 2 (EHMT2/G9a). The EHMT2/G9a has been associated with Cancer, Alzheimer's, and aging and is considered an emerging drug target having no clinically passed inhibitor. Purposefully, we developed the ligand-based pharmacophore (Pharmacophore-L) based on the common features of known inhibitors and the structure-based pharmacophore (Pharmacophore-S) based on the interaction profile of available crystal structures. The Pharmacophore-L and Pharmacophore-S were subjected to multiple tiers of validations and utilized in combination for the screening of total 741543 compounds coming from multiple databases. Additional layers of stringency were applied in the screening process to test drug-likeness (using Lipinski's rule, Veber's rule, SMARTS and ADMET filtration), to rule out any toxicity (TOPKAT analysis). The interaction profiles, stabilities, and comparative analysis against the reference were carried out by flexible docking, MD simulation, and MM-GBSA analysis, which finally led to three leads as potential inhibitors of G9a.Communicated by Ramaswamy H. Sarma.

Structural analysis of human ATE1 isoforms and their interactions with Arg-tRNAArg.

Posttranslational protein arginylation has been shown as a key regulator of cellular processes in eukaryotes by affecting protein stability, function, and interaction with macromolecules. Thus, the enzyme Arginyltransferase and its targets, are of immense interest to modulate cellular processes in the normal and diseased state. While the study on the effect of this posttranslational modification in mammalian systems gained momentum in the recent times, the detail structures of human ATE1 (hATE1) enzymes has not been investigated so far. Thus, the purpose of this study was to predict the overall structure and the structure function relationship of hATE1 enzyme and its four isoforms. The structure of four ATE1 isoforms were modelled and were docked with 3'end of the Arg-tRNAArg which acts as arginine donor in the arginylation reaction, followed by MD simulation. All the isoforms showed two distinct domains. A compact domain and a somewhat flexible domain as observed in the RMSF plot. A distinct similarity in the overall structure and interacting residues were observed between hATE1-1 and X4 compared to hATE1-2 and 5. While the putative active sites of all the hATE1 isoforms were located at the same pocket, differences were observed in the active site residues across hATE1 isoforms suggesting different substrate specificity. Mining of nsSNPs showed several nsSNPs including cancer associated SNPs with deleterious consequences on hATE1 structure and function. Thus, the current study for the first time shows the structural differences in the mammalian ATE1 isoforms and their possible implications in the function of these proteins.Communicated by Ramaswamy H. Sarma.

3D QSAR pharmacophore based lead identification of G9a lysine methyltransferase towards epigenetic therapeutics.

The G9a, Lysine Methyltransferase that methylates the histone 3 lysine 9 (H3K9) of the nucleosome, is an excellent epigenetic target having no clinically passed inhibitor currently owing to adverse in vivo ADMET toxicities. In this work, we have carried out detailed computational investigations to find novel and safer lead against the target using advanced 3 D QSAR pharmacophore screening of databases containing more than 400000 entrees of natural compounds. The screening was conducted at different levels at increasing stringencies by employing pharmacophore mapping, druglikenesses and interaction profiles of the selected to identify potential hit compounds. The potential hits were further screened by advanced flexible docking, ADME and toxicity analysis to eight hit compounds. Based on the comparative analysis of the hits with the reference inhibitor, we identified one lead inhibitor against the G9a, having better binding efficacy and a safer ADMET profile than the reference inhibitor. Finally, the results were further verified using robust molecular dynamics simulation and MM-GBSA binding energy calculation. The natural compounds are generally considered benign due to their long human uses and this is the first attempt of in silico screening of a large natural compound library against G9a to our best knowledge. Therefore, the finding of this study may add value towards the development of epigenetic therapeutics against the G9a.Communicated by Ramaswamy H. Sarma.

Heat stress induced arginylation of HuR promotes alternative polyadenylation of Hsp70.3 by regulating HuR stability and RNA binding.

Arginylation was previously found to promote stabilization of heat shock protein 70.3 (Hsp70.3) mRNA and cell survival in mouse embryonic fibroblasts (MEFs) on exposure to heat stress (HS). In search of a factor responsible for these phenomena, the current study identified human antigen R (HuR) as a direct target of arginylation. HS induced arginylation of HuR affected its stability and RNA binding activity. Arginylated HuR failed to bind Hsp70.3 3' UTR, allowing the recruitment of cleavage stimulating factor 64 (CstF64) in the proximal poly-A-site (PAS), generating transcripts with short 3'UTR. However, HuR from Ate1 knock out (KO) MEFs bound to proximal PAS region with higher affinity, thus excluded CstF64 recruitment. This inhibited the alternative polyadenylation (APA) of Hsp70.3 mRNA and generated the unstable transcripts with long 3'UTR. The inhibition of RNA binding activity of HuR was traced to arginylation-coupled phosphorylation of HuR, by check point kinase 2 (Chk2). Arginylation of HuR occurred at the residue D15 and the arginylation was needed for the phosphorylation. Accumulation of HuR also decreased cell viability upon HS. In conclusion, arginylation dependent modifications of HuR maintained its cellular homeostasis, and promoted APA of Hsp70.3 pre-mRNA, during early HS response.

Comparative analysis of Naja kaouthia venom from North-East India and Bangladesh and its cross reactivity with Indian polyvalent antivenoms.

Naja kaouthia is one of the most prevalent medically important snakes of North East India and Bangladesh responsible for most of the bite cases. In this study, an attempt was made to decipher venom variation of Naja kaouthia venom from North East India and Bangladesh. Using multidimensional methods including reverse phase HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-PAGE) and two-dimensional gel electrophoresis (2D-PAGE), the quantitative differences in venom composition have been revealed. Moreover, tested in-vitro biochemical and biological activities also exhibited differences which could be due to venom variability. Furthermore, neutralization efficacy of commercially available Indian polyvalent antivenoms (Vins, Bharat Serum, Haffkine) was evaluated and the results displayed significant differences in neutralizing efficacy between the antivenoms. Immunoblotting experiments showed antivenom molecules cross reacted with high molecular mass components while poorly reacted towards low molecular mass proteins. Immuno-depletion study demonstrated that Vins polyvalent antivenom was poor in immunocapturing the venom proteins of both North East Indian and Bangladesh origin Naja kaouthia at the ratio of 1:16 (venom: antivenom).

Loss of ATE1-mediated arginylation leads to impaired platelet myosin phosphorylation, clot retraction, and in vivo thrombosis formation.

Protein arginylation by arginyl-transfer RNA protein transferase (ATE1) is emerging as a regulator protein function that is reminiscent of phosphorylation. For example, arginylation of ß-actin has been found to regulate lamellipodial formation at the leading edge in fibroblasts. This finding suggests that similar functions of ß-actin in other cell types may also require arginylation. Here, we have tested the hypothesis that ATE1 regulates the cytoskeletal dynamics essential for in vivo platelet adhesion and thrombus formation. To test this hypothesis, we generated conditional knockout mice specifically lacking ATE1 in their platelets and in their megakaryocytes and analyzed the role of arginylation during platelet activation. Surprisingly, rather than finding an impairment of the actin cytoskeleton structure and its rearrangement during platelet activation, we observed that the platelet-specific ATE1 knockout led to enhanced clot retraction and in vivo thrombus formation. This effect might be regulated by myosin II contractility since it was accompanied by enhanced phosphorylation of the myosin regulatory light chain on Ser19, which is an event that activates myosin in vivo. Furthermore, ATE1 and myosin co-immunoprecipitate from platelet lysates. This finding suggests that these proteins directly interact within platelets. These results provide the first evidence that arginylation is involved in phosphorylation-dependent protein regulation, and that arginylation affects myosin function in platelets during clot retraction.

Small molecule inhibitors of arginyltransferase regulate arginylation-dependent protein degradation, cell motility, and angiogenesis.

Posttranslational arginylation mediated by arginyltransferase (ATE1) is an emerging major regulator of embryogenesis and cell physiology. Impairments of ATE1 are implicated in congenital heart defects, obesity, cancer, and neurodegeneration making this enzyme an important therapeutic target, whose potential has been virtually unexplored. Here we report the development of a biochemical assay for identification of small molecule inhibitors of ATE1 and application of this assay to screen a library of 3280 compounds. Our screen identified two compounds which specifically affect ATE1-regulated processes in vivo, including tannic acid, which has been previously shown to inhibit protein degradation and angiogenesis and to act as a therapeutic agent in heart disease and cancer. Our data suggest that these actions of tannic acid are mediated by its direct effect on ATE1, which regulates protein degradation and angiogenesis in vivo.

Arginyltransferase is an ATP-independent self-regulating enzyme that forms distinct functional complexes in vivo.

Posttranslational arginylation mediated by arginyl transferase (ATE1) plays an important role in cardiovascular development, cell motility, and regulation of cytoskeleton and metabolic enzymes. This protein modification was discovered decades ago, however, the arginylation reaction and the functioning of ATE1 remained poorly understood because of the lack of good biochemical models. Here, we report the development of an in vitro arginylation system, in which ATE1 function and molecular requirements can be tested using purified recombinant ATE1 isoforms supplemented with a controlled number of components. Our results show that arginylation reaction is a self-sufficient, ATP-independent process that can affect different sites in a polypeptide and that arginyl transferases form different molecular complexes in vivo, associate with components of the translation machinery, and have distinct, partially overlapping subsets of substrates, suggesting that these enzymes play different physiological functions.

Differential arginylation of actin isoforms is regulated by coding sequence-dependent degradation.

The mammalian cytoskeletal proteins ß- and γ-actin are highly homologous, but only ß-actin is amino-terminally arginylated in vivo, which regulates its function. We examined the metabolic fate of exogenously expressed arginylated and nonarginylated actin isoforms. Arginylated γ-actin, unlike ß-, was highly unstable and was selectively ubiquitinated and degraded in vivo. This instability was regulated by the differences in the nucleotide coding sequence between the two actin isoforms, which conferred different translation rates. γ-actin was translated more slowly than ß-actin, and this slower processing resulted in the exposure of a normally hidden lysine residue for ubiquitination, leading to the preferential degradation of γ-actin upon arginylation. This degradation mechanism, coupled to nucleotide coding sequence, may regulate protein arginylation in vivo.

Arginylation-dependent neural crest cell migration is essential for mouse development.

Coordinated cell migration during development is crucial for morphogenesis and largely relies on cells of the neural crest lineage that migrate over long distances to give rise to organs and tissues throughout the body. Recent studies of protein arginylation implicated this poorly understood posttranslational modification in the functioning of actin cytoskeleton and in cell migration in culture. Knockout of arginyltransferase (Ate1) in mice leads to embryonic lethality and severe heart defects that are reminiscent of cell migration-dependent phenotypes seen in other mouse models. To test the hypothesis that arginylation regulates cell migration during morphogenesis, we produced Wnt1-Cre Ate1 conditional knockout mice (Wnt1-Ate1), with Ate1 deletion in the neural crest cells driven by Wnt1 promoter. Wnt1-Ate1 mice die at birth and in the first 2-3 weeks after birth with severe breathing problems and with growth and behavioral retardation. Wnt1-Ate1 pups have prominent defects, including short palate and altered opening to the nasopharynx, and cranial defects that likely contribute to the abnormal breathing and early death. Analysis of neural crest cell movement patterns in situ and cell motility in culture shows an overall delay in the migration of Ate1 knockout cells that is likely regulated by intracellular mechanisms rather than extracellular signaling events. Taken together, our data suggest that arginylation plays a general role in the migration of the neural crest cells in development by regulating the molecular machinery that underlies cell migration through tissues and organs during morphogenesis.

Identification and characterization of a virus-inducible non-coding RNA in mouse brain.

Infection of mice with Japanese encephalitis virus or Rabies virus results in the activation of a gene encoding a novel, non-coding RNA (ncRNA) in the mouse central nervous system. This transcript, named virus-inducible ncRNA (VINC), is identical to a 3.18 kb transcript expressed in mouse neonate skin (GenBank accession no. AK028745) that, together with a number of unannotated cDNAs and expressed sequence tags, is grouped in the mouse unigene cluster Mm281895. VINC is expressed constitutively in early mouse embryo and several adult non-neuronal mouse tissues, as well as a murine renal adenocarcinoma (RAG) cell line. Northern blotting of nuclear and cytoplasmic RNAs revealed that VINC is localized primarily in the nucleus of RAG cells and is thus a novel member of the nuclear ncRNA family.