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To find potential inhibitors of Naegleria fowleri S-adenosyl-L-homocysteine hydrolase (NfSAHH), a brain-eating parasite, structure-based drug design was adopted. N. fowlerica causes primary amebic meningoencephalitis (PAM), a fatal central nervous system (CNS) disorder if untreated. NfSAHH protein (PDB ID: 5v96), involved in parasite growth and gene regulation, was targeted and screened against 163 metabolites from Gossypium hirsutum (cotton plant). With the aid of different software and web tools, the metabolites were subjected to several analyses. The RMSD was evaluated to validate our molecular docking strategy. Neplanocin A, a common anti-parasitic medication, was used as a reference to select top ligands for post-docking studies. Significant interactions were observed with residues THR-198, HIS-395, and MET-400. The drug-likeness of the top fifty hits was analyzed using Lipinski, Ghose, Veber, Egan, and Muegge rules. The top ten compounds following Lipinski’s RO5 were studied regarding medicinal chemistry, pharmacokinetic simulation, and Swiss target prediction. Advanced strategies, including molecular dynamic simulations, binding energy calculations, and principal component analysis, were employed for the top three hits, namely curcumin, heliocide H2, and piceid, which indicated that heliocide H2 is the most promising candidate, while curcumin and piceid may need further optimization to improve their stability. Overall, the top ten phytochemicals, dotriacontanol, melissic acid, curcumin, 6,6′-dimethoxygossypol, phytosphingosine 2, methyl stearate, stearic acid, piceid, heliocide H2, and 6-methoxygossypol, reported in our study, are worthy enough to be subjected to in vivo and in vitro experimentation to find a novel drug to treat PAM.

Neplanocin A (NPA) is a natural carbocyclic analogue of adenosine that was isolated from Ampullariella regularis, which is known for its antibacterial, antiviral, and anticancer activity. Although the activity of this compound has been demonstrated in many biological models, the mechanism of its anticancer activity is not fully understood. In the current work, we present the comparison of the biological activity of two enantiomers of neplanocin A in the series of cancerous and non-cancerous cell types. In all tested cell lines, the compound with natural stereochemistry, (-)-NPA, was found to be more cytotoxic than its synthetic (+)-NPA derivative; however, sensitivity to neplanocins A varied between cell types. To determine possible reasons for the observed differences in individual cancer cell types, the expression level and effects of individual genes of adenosine-interacting enzymes were analyzed. Bioinformatic analysis of the interaction between (-)-NPA and (+)-NPA with major adenosine-interacting enzymes, such as adenosine kinase (ADK), adenosine deaminases (ADA and ADA2), and S-adenosylhomocysteine hydrolase (SAHH, AHCY), was performed. The molecular docking results revealed differences in the binding energy of the individual enantiomers of neplanocin A with the targets, which sheds new light on the mechanism of action of these adenosine analogues.

Tyrosine nitration is recognized as an important post-translational protein modification in animal cells that can be used as an indicator of a nitrosative process. However, in plant systems, there is scant information on proteins that undergo this process. In sunflower hypocotyls, the content of tyrosine nitration (NO2-Tyr) and the identification of nitrated proteins were studied by high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) and proteomic approaches, respectively. In addition, the cell localization of nitrotyrosine proteins and peroxynitrite were analysed by confocal laser-scanning microscopy (CLSM) using antibodies against 3-nitrotyrosine and 3′-(p-aminophenyl) fluorescein (APF) as the fluorescent probe, in that order. The concentration of Tyr and NO2-Tyr in hypocotyls was 0.56 μmol mg−1 protein and 0.19 pmol mg−1 protein, respectively. By proteomic analysis, a total of 21 nitrotyrosine-immunopositive proteins were identified. These targets include proteins involved in photosynthesis, and in antioxidant, ATP, carbohydrate, and nitrogen metabolism. Among the proteins identified, S-adenosyl homocysteine hydrolase (SAHH) was selected as a model to evaluate the effect of nitration on SAHH activity using SIN-1 (a peroxynitrite donor) as the nitrating agent. When the hypocotyl extracts were exposed to 0.5 mM, 1 mM, and 5 mM SIN-1, the SAHH activity was inhibited by some 49%, 89%, and 94%, respectively. In silico analysis of the barley SAHH sequence, characterized Tyr448 as the most likely potential target for nitration. In summary, the present data are the first in plants concerning the content of nitrotyrosine and the identification of candidates of protein nitration. Taken together, the results suggest that Tyr nitration occurs in plant tissues under physiological conditions that could constitute an important process of protein regulation in such a way that, when it is overproduced in adverse circumstances, it can be used as a marker of nitrosative stress.

S -adenosyl- L -homocysteine hydrolase (SAH hydrolase or SAHH) is a highly conserved enzyme that catalyses the reversible hydrolysis of SAH to L -homocysteine (HCY) and adenosine (ADO). High-resolution crystal structures have been reported for bacterial and plant SAHHs, but not mammalian SAHHs. Here, we report the first high-resolution crystal structure of mammalian SAHH (mouse SAHH) in complex with a reaction product (ADO) and with two reaction intermediate analogues—3’-keto-aristeromycin (3KA) and noraristeromycin (NRN)—at resolutions of 1.55, 1.55 and 1.65 Å. Each of the three structures constitutes a structural snapshot of one of the last three steps of the five-step process of SAH hydrolysis by SAHH. In the NRN complex, a water molecule, which is an essential substrate for ADO formation, is structurally identified for the first time as the candidate donor in a Michael addition by SAHH to the 3’-keto-4’,5’-didehydroadenosine reaction intermediate. The presence of the water molecule is consistent with the reaction mechanism proposed by Palmer & Abeles in 1979. These results provide insights into the reaction mechanism of the SAHH enzyme.

Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) are IP₃-gated$Ca^{2+}$channels that are located on intracellular Ca₃⁺ stores. We previously identified an IP₃R binding protein, termed IP₃R binding protein released with IP₃ (IRBIT). Because IRBIT is released from IP₃ by physiological concentrations of IP₃, we hypothesized that IRBIT is a signaling molecule that is released from IP₃R and regulates downstream target molecules in response to the production of IP₃. Therefore, in this study, we attempted to identify the target molecules of IRBIT, and we succeeded in identifying Na⁺/HCO₃⁻ cotransporter 1 (NBC1) as an IRBIT binding protein. Of the two major splicing variants of NBC1, pancreas-type NBC1 (pNBC1) and kidney-type NBC1 (kNBC1), IRBIT was found to bind specifically to pNBC1 and not to bind to kNBC1. IRBIT binds to the N-terminal pNBC1-specific domain, and its binding depends on the phosphorylation of multiple serine residues of IRBIT. Also, an electrophysiological analysis in Xenopus oocytes revealed that pNBC1 requires coexpression of IRBIT to manifest substantial activity comparable with that of kNBC1, which displays substantial activity independently of IRBIT. These results strongly suggest that pNBC1 is the target molecule of IRBIT and that IRBIT has an important role in pH regulation through pNBC1. Also, our findings raise the possibility that the regulation through IRBIT enables NBC1 variants to have different physiological roles.

An antioxidant molecule namely, adenosyl homocysteinase (AHc) was identified from the earlier constructed transcriptome database of Spirulina, where it was cultured in a sulphur deprived condition. From the AHc protein, a small peptide NL13 was identified using bioinformatics tools and was predicted to have antioxidant property. Further, the peptide was synthesised and its antioxidant mechanism was addressed at molecular level. NL13 was subjected to various antioxidant assays including DPPH assay, HARS assay, SARS Assay, NO assay and ABTS assay, where NL13 exhibited significant (P < 0.05) potential antioxidant activity compared to its antioxidant control, Trolox. Cytotoxicity was performed on Human whole blood and the cell viability was performed on VERO fibroblast cells. In both assays, it was found that NL13 did not exhibit any cytotoxic effect towards the cells. Further, the intracellular ROS was performed on Multimode reader followed by imaging on fluorescence microscope which showed scavenging activity even at lower concentration of NL13 (31.2 µM). An effective wound healing property of NL13 on VERO cells was confirmed by analysing the cell migration rate at two different time intervals (24 and 48 h). Overall, the study shows that NL13 peptide scavenges the intracellular oxidative stress.

S-adenosylhomocysteine hydrolase-like protein 1 (AHCYL1), also known as IP(3) receptor-binding protein released with IP(3) (IRBIT), regulates IP(3)-induced Ca(2+) release into the cytoplasm of cells. AHCYL1 is a critical regulator of early developmental stages in zebrafish, but little is known about the function of AHCYL1 or hormonal regulation of expression of the AHCYL1 gene in avian species. Therefore, we investigated differential expression profiles of the AHCYL1 gene in various adult organs and in oviducts from estrogen-treated chickens. Chicken AHCYL1 encodes for a protein of 540 amino acids that is highly conserved and has considerable homology to mammalian AHCYL1 proteins (>94% identity). AHCYL1 mRNA was expressed abundantly in various organs of chickens. Further, the synthetic estrogen agonist induced AHCYL1 mRNA and protein predominantly in luminal and glandular epithelial cells of the chick oviduct. In addition, estrogen activated AHCYL1 through the ERK1/2 signal transduction cascade and that activated expression of AHCYL1 regulated genes affecting oviduct development in chicks as well as calcium release in epithelial cells of the oviduct. Also, microRNAs, miR-124a, miR-1669, miR-1710 and miR-1782 influenced AHCYL1 expression in vitro via its 3'-UTR which suggests that post-transcriptional events are involved in the regulation of AHCYL1 expression in the chick oviduct. In conclusion, these results indicate that AHCYL1 is a novel estrogen-stimulated gene expressed in epithelial cells of the chicken oviduct that likely affects growth, development and calcium metabolism of the mature oviduct of hens via an estrogen-mediated ERK1/2 MAPK cell signaling pathway.

Mycobacterium tuberculosis modulates expression of various metabolism-related genes to adapt in the adverse host environment. The gene coding for M. tuberculosis S-adenosylhomocysteine hydrolase ( Mtb -SahH) is essential for optimal growth and the protein product is involved in intermediary metabolism. However, the relevance of SahH in mycobacterial physiology is unknown. In this study, we analyze the role of Mtb -SahH in regulating homocysteine concentration in surrogate host Mycobacterium smegmatis . Mtb -SahH catalyzes reversible hydrolysis of S-adenosylhomocysteine to homocysteine and adenosine and we demonstrate that the conserved His363 residue is critical for bi-directional catalysis. Mtb -SahH is regulated by serine/threonine phosphorylation of multiple residues by M. tuberculosis PknB. Major phosphorylation events occur at contiguous residues Thr219, Thr220 and Thr221, which make pivotal contacts with cofactor NAD + . Consequently, phosphorylation negatively modulates affinity of enzyme towards NAD + as well as SAH-synthesis. Thr219, Thr220 and Thr221 are essential for enzyme activity and therefore, responsible for SahH-mediated regulation of homocysteine.

Abstract S-adenosylhomocysteine (SAH), formed after donation of the methyl group of S-adenosylmethionine (SAM) to a methyl acceptor, is reversibly hydrolyzed to adenosine (ADO) and homocysteine (HCY) by S-adenosylhomocysteine hydrolase (SAHH). In chestnut blight fungus (Cryphonectria parasitica), sahh, a hypovirus-regulated gene that encodes a deduced SAHH protein was shown to have an SAHH enzymatic activity in vitro. Deletion of sahh resulted in the increased accumulation of intracellular SAH and SAM but decreased ADO, and a remarkably increased accumulation of transcripts that encode adenosine kinase, methionine adenosyltransferase, and an O-methyltransferase, key components of the methylation pathway. The Δsahh knockout mutants showed a phenotype of slower growth rate, fewer aerial hyphae, loss of orange pigment, absence of asexual fruiting bodies and conidia, and a significant reduction in virulence. Deletion of sahh significantly reduced the accumulation level of transcripts of the cyp1 that encodes cyclophilin A as well as genes of the heterotrimeric G-protein signaling pathways including cpga1, cpgb1, and cpgc1 and ste12, a target activated by the MAP kinase cascade. Taken together, we demonstrated that SAHH is required for virulence and multiple traits of phenotype in C. parasitica, by regulation of the expression of genes involved in key process of the cell.

The trans-sulfuration pathway is a biochemical mechanism that links methionine metabolism to the biosynthesis of cellular redox-controlling molecules, like cysteine, glutathione, and taurine. While there is some knowledge about the metabolic intermediates and enzymes that participate in trans-sulfuration, little is known about the physiological importance of this mechanism. Deficiencies within the trans-sulfuration pathway induces (i) the generation of reactive species of oxygen (ROS) and halogens (RHS), (ii) homocyst(e)ine accumulation, and (iii) the synthesis of proinflammatory molecules by macrophages, and contribute to humans pathologies like atherosclerosis and tumor development. In this review we outline the role of this biochemical pathway in tumor development and analyze current findings on the role of trans-sulfuration in mammalian physiology. The potential relationship between chronic inflammation, and tumor and atherosclerotic development are discussed.