Crucial Amino Acid Residues Research Articles

Studies on the antifungal and serotonin receptor agonist activities of the secondary metabolites from piezotolerant deep-sea fungus Ascotricha sp.

ABSTRACTThe potent antifungal agent sesquiterpenes and serotonin 5-HT2C agonist ascotricin were produced by a newly isolated deep-sea fungus Ascotricha sp. This fungus was isolated from deep-sea sediment collected at a depth of 1235 m and characterized. Piezotolerance was successfully tested under high pressure-low temperature (100 bar pressure and 20ºC) microbial cultivation system. Production of secondary metabolites was enhanced at optimized culture conditions. The in-vivo antifungal activity of sesquiterpenes was studied using the Caenorhabditis elegans – Candida albicans model system. The sesquiterpenes affected the virulence of C. albicans and prolonged the life of the host C. elegans. These findings suggest that sesquiterpenes are attractive antifungal drug candidates. The 5-HT2C receptor agonist is a potential target for the development of drugs for a range of central nervous system disorders. The interaction of 5-HT2C agonist ascotricin with the receptor was studied through bioinformatic analysis. The in silico molecular docking and molecular dynamic simulation studies demonstrated that they fit into the serotonin 5-HT2C active site and the crucial amino acid residues involved in the interactions were identified. To our knowledge, this is first report of in vivo antifungal analysis of sesquiterpenes and in silico studies of serotonin 5-HT2C receptor-ascotricin complex.

Characterization of a novel thiol activated phospholipase TAPLB1 from Trichosporon asahii MSR 54

Phospholipases are hydrolytic enzymes that play crucial roles in vivo and also possess immense biotechnological potential. In the present study, the phospholipase B of Trichosporon asahii MSR54 was overexpressed in E. coli and characterized. The 68-kDa enzyme was monomeric in solution and possessed phospholipase, lysophospholipase, esterase and acyltransferase activities. It was maximally active at pH 8.0 and 40 °C. The enzyme retained >50% activity between pH 3.0–8.0 and had a half-life of 30 min at 60 °C. Its activity was not metal dependent and was stable in the presence of most metal ions. Its catalytic efficiency on lysophosphatidyl choline was 1.0 × 103 mM−1 h−1. Site directed mutagenesis revealed R121 (present in the GYRAMV motif), S194 (present in the conserved GLSGG motif) and D420 (present in LVDXGE motif) to be the crucial amino acid residues for esterolytic activity. S194 and D420 were also the catalytic amino acids for lysophospholipase and phospholipase activities of the enzymes, while R121 was not involved in catalysis of phospholipid substrates. Further, it was found that cysteine residues in C61 and C354 were involved in disulphide linkages that imparted the properties of thiol activation and thermostability, respectively.

Purification, characterization and potential applications of a novel halotolerant metalloprotease from marine bacterium Vibrio sp. LA‐05

AbstractBACKGROUNDMany reported proteases showed low activity and stability under harsh operating conditions, e.g. high pH, salinity and surfactants, which hinders expansion of their applications in industry. Here, a novel halotolerant metalloprotease from marine bacterium Vibrio sp. LA‐05 endowed with superior properties was purified and characterized, and its industrial application also evaluated.RESULTSThe protease was active and stable at 25–40°C and pH 6.0–10.0. Moreover, it was remarkably stable in high salt solution, tolerant to denaturing agents, organic solvents, surfactants and bleaching agents, and quite compatible and stable with many commercial detergents. Its residual activities were above 22% even after co‐incubation with 20% NaCl for 480 h. Except Ariel and Tide, the residual activities were all above 55% after co‐incubation with five other liquid detergents for 240 h. The corresponding theoretical analyses show that it is a metalloprotease containing 1 mol of zinc ion per mole of protease, the mature active protease predominantly in α‐helix form consists of 321 amino acids, and the central zinc ion along with 11 other conserved and crucial amino acid residues forms the catalytic domain.CONCLUSIONThese excellent properties indicated that the protease has considerable potential as a bio‐additive in the application of detergent formulations. © 2018 Society of Chemical Industry

UV RESISTANCE LOCUS 8 From Chrysanthemum morifolium Ramat (CmUVR8) Plays Important Roles in UV-B Signal Transduction and UV-B-Induced Accumulation of Flavonoids.

UV Resistance Locus 8 (UVR8), an ultraviolet-B (UV-B; 280–315 nm) photoreceptor, participates in the regulation of various plant growth and developmental processes. UV-B radiation is an important factor enhancing the production of active components in medicinal plants. To-date, however, studies on UV-B photoreceptors have largely focused on Arabidopsis, and the functions of UVR8 in medicinal plants are still largely unknown. In the present study, a homolog of Arabidopsis UVR8, CmUVR8, was isolated from Chrysanthemum morifolium Ramat, and its structure and function were analyzed in detail. Protein sequence analysis showed that CmUVR8 contained nine conserved regulators of chromosome condensation 1 repeats, seven conserved bladed propellers, one C27 region, three “GWRHT” motifs and several crucial amino acid residues (such as 14 Trps and 2 Args), similar to AtUVR8. 3-D structural analysis of CmUVR8 indicated that its structure was similar to AtUVR8. Heterologous expression of CmUVR8 could rescued the deficient phenotype of uvr8-6, a mutant of UVR8 in Arabidopsis, indicating the role of CmUVR8 in the regulation of hypocotyl elongation and HY5 gene expression under UV-B irradiation. Moreover, CmUVR8 regulates UV-B-induced expression of four flavonoids biosynthesis-related genes and the UV-B-induced accumulation of flavonoids. Furthermore, the interaction between CmUVR8 and CmCOP1 were confirmed using a yeast two-hybrid assay. These results indicated that CmUVR8 plays important roles in UV-B signal transduction and the UV-B-induced accumulation of flavonoids, as a counterpart of AtUVR8.

A Synergistic Transcriptional Regulation of Olfactory Genes Drives Blood-Feeding Associated Complex Behavioral Responses in the Mosquito Anopheles culicifacies.

Decoding the molecular basis of host seeking and blood feeding behavioral evolution/adaptation in the adult female mosquitoes may provide an opportunity to design new molecular strategy to disrupt human-mosquito interactions. Although there is a great progress in the field of mosquito olfaction and chemo-detection, little is known about the sex-specific evolution of the specialized olfactory system of adult female mosquitoes that enables them to drive and manage the complex blood-feeding associated behavioral responses. A comprehensive RNA-Seq analysis of prior and post blood meal olfactory system of An. culicifacies mosquito revealed a minor but unique change in the nature and regulation of key olfactory genes that may play a pivotal role in managing diverse behavioral responses. Based on age-dependent transcriptional profiling, we further demonstrated that adult female mosquito's chemosensory system gradually learned and matured to drive the host-seeking and blood feeding behavior at the age of 5–6 days. A time scale expression analysis of Odorant Binding Proteins (OBPs) unravels unique association with a late evening to midnight peak biting time. Blood meal-induced switching of unique sets of OBP genes and Odorant Receptors (Ors) expression coincides with the change in the innate physiological status of the mosquitoes. Blood meal follows up experiments further provide enough evidence that how a synergistic and concurrent action of OBPs-Ors may drive “prior and post blood meal” associated complex behavioral events. A dominant expression of two sensory appendages proteins (SAP-1 & SAP2) in the legs of An. culicifacies suggests that this mosquito species may draw an extra advantage of having more sensitive appendages than An. stephensi, an urban malarial vector in the Indian subcontinents. Finally, our molecular modeling analysis predicts crucial amino acid residues for future functional characterization of the sensory appendages proteins which may play a central role in regulating multiple behaviors of An. culicifacies mosquito.SIGNIFICANCE Evolution and adaptation of blood feeding behavior not only favored the reproductive success of adult female mosquitoes but also make them important disease-transmitting vectors. An environmental exposure after emergence may favor the broadly tuned olfactory system of mosquitoes to drive complex behavioral responses. But, how these olfactory derived genetic factors manage female specific “pre and post” blood meal associated complex behavioral responses are not well known. Our findings suggest that a synergistic action of olfactory factors may govern an innate to prime learning strategy to facilitate rapid blood meal acquisition and downstream behavioral activities. A species-specific transcriptional profiling and an in-silico analysis predict that “sensory appendages protein” may be a unique target to design disorientation strategy against the mosquito Anopheles culicifacies.

New Insights on Cyclization Specificity of Fungal Type III Polyketide Synthase, PKSIIINc in Neurospora crassa.

Type III polyketide synthases (PKSs) biosynthesize varied classes of metabolites with diverse bio-functionalities. Inherent promiscuous substrate specificity, multiple elongations of reaction intermediates and several modes of ring-closure, confer the proteins with the ability to generate unique scaffolds from limited substrate pools. Structural studies have identified crucial amino acid residues that dictate type III PKS functioning, though cyclization specific residues need further investigation. PKSIIINc, a functionally and structurally characterized type III PKS from the fungus, Neurospora crassa, is known to biosynthesize alkyl-resorcinol, alkyl-triketide- and alkyl-tetraketide-α-pyrone products. In this study, we attempted to identify residue positions governing cyclization specificity in PKSIIINc through comparative structural analysis. Structural comparisons with other type III PKSs revealed a motif with conserved hydroxyl/thiol groups that could dictate PKSIIINc catalysis. Site-directed mutagenesis of Cys120 and Ser186 to Ser and Cys, respectively, altered product profiles of mutant proteins. While both C120S and S186C proteins retained wild-type PKSIIINc product activity, S186C favoured lactonization and yielded higher amounts of the α-pyrone products. Notably, C120S gained new cyclization capability and biosynthesized acyl-phloroglucinol in addition to wild-type PKSIIINc products. Generation of alkyl-resorcinol and acyl-phloroglucinol by a single protein is a unique observation in fungal type III PKS family. Mutation of Cys120 to bulky Phe side-chain abrogated formation of tetraketide products and adversely affected overall protein stability as revealed by molecular dynamics simulation studies. Our investigations identify residue positions governing cyclization programming in PKSIIINc protein and provide insights on how subtle variations in protein cores dictate product profiles in type III PKS family.

A review of the design and modification of lactoferricins and their derivatives.

Lactoferricin (Lfcin), a multifunction short peptide with a length of 25 residues, is derived from the whey protein lactoferrin by acidic pepsin hydrolysis. It has potent nutritional enhancement, antimicrobial, anticancer, antiviral, antiparasitic, and anti-inflammatory activities. This review describes the research advantages of the above biological functions, with attention to the molecular design and modification of Lfcin. In this examination of design and modification studies, research on the identification of Lfcin active derivatives and crucial amino acid residues is also reviewed. Many strategies for Lfcin optimization have been studied in recent decades, but we mainly introduce chemical modification, cyclization, chimera and polymerization of this peptide. Modifications such as incorporation of D-amino acids, acetylation and/or amidation could effectively improve the activity and stability of these compounds. Due to their wide array of bio-functions and applications, Lfcins have great potential to be developed as biological agents with multiple functions involved with nutritional enhancement, as well as disease preventive and therapeutic effects.

Bioluminescence and kinetic aspects of double mutated aequorin variants

Aequorin as an old small calcium-sensitive photoprotein is a blue fluorescence protein which converts coelenterazine (a substrate) to coelenteramide with a flash type emission. The decay kinetics and emission properties of this protein can be changed using directed mutagenesis of crucial amino acid residue. In this work, we prepared three double mutants: Y82F/W86F, Y82F/D153G, and W86F/D153G. According to our results, it seems that presence of Y82F mutation results in shift of emission to longer wavelengths while the W86F mutation shifts the emission to shorter wavelengths. Furthermore, comparison of the variants for light half-life indicated decreased t1/2 for the two variants of Y82F/D153G and W86F/D153G. But in compared to wild type aequorin, the Y82F/W86F variant displayed a 2-fold increase of light half-life. On the other hand, the thermostability properties of double mutants confirmed that only Y82F/D153G variant of apoaequorin is higher stability than others. Also, the single W86F mutant reached the highest stability against thermal shock. Our data suggest that replacement of single or few point mutations in the binding pocket or active site of aequorin affects its bioluminescence and kinetic properties and so could be used for new reporter production of this photoprotein with the feasibility and limited substitutions.

Synergistic effect of eugenol with Colistin against clinical isolated Colistin-resistant Escherichia coli strains

BackgroundBacterial infections have become more challenging to treat due to the emergence of multidrug-resistant pathogenic bacteria. Combined antibiotics prove to be a relatively effective method to control such resistant strains. This study aim to investigate synergistic activity of eugenol combined with colistin against a collection of clinical isolated Escherichia coli (E.coli) strains, and to evaluate potential interaction.MethodsAntimicrobial susceptibility, minimum inhibitory concentration (MIC) and fractional inhibitory concentration index (FICI) of the bacteria were determined by disk diffusion assay, broth microdilution method and checkerboard assay, respectively. The mcr-1 mRNA expression was measured by Real-time PCR. To predict possible interactions between eugenol and MCR-1, molecular docking assay was taken.ResultsFor total fourteen strains including eight colistin-resistant strains, eugenol was determined with MIC values of 4 to 8 μg/mL. Checkerboard dilution test suggested that eugenol exhibited synergistic activity when combined with colistin (FICI ranging from 0.375 to 0.625). Comparison analysis of Real-time PCR showed that synergy could significantly down-regulate expression of mcr-1 gene. A metal ion coordination bond with catalytic zinc atom and a hydrogen bond with crucial amino acid residue Ser284 of MCR-1 were observed after molecular docking, indicating antibacterial activity and direct molecular interactions of eugenol with MCR-1 protein.ConclusionsOur results demonstrated that eugenol exhibited synergistic effect with colistin and enhanced its antimicrobial activity. This might further contribute to the antibacterial actions against colistin-resistant E.coli strains.Graphical abstractSynergistic effect of eugenol with colistin against colistin-resistant Escherichia coli isolates.

Probing the Single Key Amino Acid Responsible for the Novel Catalytic Function of ent-Kaurene Oxidase Supported by NADPH-Cytochrome P450 Reductases in Tripterygium wilfordii.

Tripterygium wilfordii produces not only ent-kaurene, which is an intermediate of gibberellin (GA) biosynthesis in flowering plants, but also 16α-hydroxy-ent-kaurane, whose physiological role has not been characterized. The two compounds are biosynthesized from the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) by diterpene synthases, which have been discovered and functionally characterized in T. wilfordii. Here, we described the functional characterization of four cytochrome P450 reductases (TwCPR) and one ent-kaurene oxidase (TwKO). Four TwCPRs were found to have relatively ubiquitous expression in T. wilfordii root, stem, leaf, and flower tissues. Co-expression of both a TwCPR and TwKO in yeast showed that TwCPR3 has a slightly better activity for providing the electrons required for these reactions, indicating that TwCPR3 is more suitable for use in the functional analysis of other cytochrome P450 monooxygenases. TwKO catalyzed the three-step oxidation of the C4α methyl of the tetracyclic diterpene intermediate ent-kaurene to form ent-kaurenoic acid as an early step in GA biosynthesis. Notably, TwKO could also convert 16α-hydroxy-ent-kaurane to 16α-hydroxy-ent-kaurenoic acid, indicating an important function of 16α-hydroxy-ent-kaurane in the anti-HIV principle tripterifordin biosynthetic pathway in planta. Homology modeling and molecular docking were used to investigate the unknown crucial active amino acid residue involved in the catalytic reaction of TwKO, and one key residue (Leu387) contributed to the formation of 16α-hydroxy-ent-kaurenoic acid, most likely by forming hydrogen bonds with the hydroxyl group (-OH) of 16α-hydroxy-ent-kaurane, which laid the basis for further investigation of the multifunctional nature of KO catalysis. Also, our findings paved the way for the complete biosynthesis of the anti-HIV principle tripterifordin.

Structural investigation of vesnarinone at the pore domains of open and open-inactivated states of hERG1 K+ channel

In this study, the dynamics of vesnarinone bounded hERG1 K+ channels are investigated using in silico approaches such as molecular docking, molecular dynamics (MD) simulations, MM/PBSA (Molecular Mechanics/Poisson Boltzmann Surface Area) calculations and Principal Component Analysis (PCA). Vesnarinone (a cardiotonic agent) falls into a category of drugs that inhibit phosphodiesterase 3-type (PDE3) enzymes. PDE3 enzymes have specific roles in the dehydyrolysis of intracellular second messengers 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP). Thus, PDE3 inhibitors elevate the intracellular concentrations of these substrates. However, it is also known that vesnarinone inhibits the human ether-à-go-go-related gene (hERG) channels. Since inhibition of hERG channels may cause life-threatening arrhythmias, leading to Torsades de pointes (TdP) and long QT syndrome (LQTS), it is important to understand the particular residue-drug interactions and hERG channel dynamics. Applying the computational approaches in this study, have helped to elucidate the possible binding patterns and time evaluation dynamics of this drug at hERG1 channel models (both in its open and open-inactivated states) together with the crucial amino acid residues that mostly contribute in binding processes via interaction binding energy decomposition analysis.

A study of comparative modelling, simulation and molecular dynamics of CXCR3 receptor with lipid bilayer

The G-coupled receptors seen on the cell surface are composites with a lipid bilayer. The chemokines are kind of G-coupled receptor which majorly involved in the activation and downstream signalling of the cell. In general, many G-coupled receptors lack their 3D structures which become a hurdle in the drug designing process. In this study, comparative modelling of the CXCR3 receptor was carried out, structure evaluation was done using various tools and softwares. Additionally, molecular dynamics and docking were performed to prove the structural quality and architecture. Interestingly, the studies like toggle switch mechanism, lipid dynamics, virtual screening were carried out to find the potent antagonist for the CXCR3 receptor. During virtual screening 14,303 similar molecules were retrieved among them only four compounds have an ability to interact with a crucial amino acid residue of an antagonist. Hence, these screened compounds can serve as a drug candidate for a CXCR3 receptor, but further in vitro and in vivo studies are ought to do to prove its same efficacy.

Nonsynonymous changes of equine lentivirus receptor-1 (ELR1) gene in amino acids involved in the interaction with equine infectious anemia virus (EIAV)

Equine lentivirus receptor-1 (ELR1) has been characterized as the specific functional receptor that mediates equine infectious anemia virus (EIAV) entrance to horse macrophages. This receptor is tumor necrosis factor receptor superfamily member 14 (TNFRSF14). The aim of this study was to investigate the occurrence of allelic variants in the coding sequence of equine TNFRSF14 gene by screening for single-nucleotide polymorphisms (SNPs) in different equine populations. Forty seven horse samples were randomly selected from a reservoir of EIAV-seropositive and seronegative samples collected from different outbreaks and regions of Argentina. DNA samples were scanned via PCR and direct sequencing of exon 3 and exon 5 of TNFRSF14 gene. A total of 21 SNPs were identified, of which 11 were located in coding sequences. Within exon 5, four SNPs caused nonsynonymous substitutions, while two other SNPs caused synonymous substitutions in crucial residues (Ser112 and Thr114) implicated in the interaction with EIAV. Despite some of exon 5 variants occurred exclusively in EIAV-positive or EIAV-negative horses, critical residues for the function of the mature protein were conserved, accounting for selective pressures in favor of preserving the specific function of TNFRSF members and the host immune response. To our knowledge, this is the first report of the existence of allelic variations involving some crucial amino acid residues in horse ELR1. Further, it could be an initial step to test the possible functional relevance and relationship of these variants with EIAV infection and disease progression as well as to develop preventive strategies.

Structural insights into the production of 3-hydroxypropionic acid by aldehyde dehydrogenase from Azospirillum brasilense

3-Hydroxypropionic acid (3-HP) is an important platform chemical to be converted to acrylic acid and acrylamide. Aldehyde dehydrogenase (ALDH), an enzyme that catalyzes the reaction of 3-hydroxypropionaldehyde (3-HPA) to 3-HP, determines 3-HP production rate during the conversion of glycerol to 3-HP. To elucidate molecular mechanism of 3-HP production, we determined the first crystal structure of a 3-HP producing ALDH, α-ketoglutarate-semialdehyde dehydrogenase from Azospirillum basilensis (AbKGSADH), in its apo-form and in complex with NAD+. Although showing an overall structure similar to other ALDHs, the AbKGSADH enzyme had an optimal substrate binding site for accepting 3-HPA as a substrate. Molecular docking simulation of 3-HPA into the AbKGSADH structure revealed that the residues Asn159, Gln160 and Arg163 stabilize the aldehyde- and the hydroxyl-groups of 3-HPA through hydrogen bonds, and several hydrophobic residues, such as Phe156, Val286, Ile288, and Phe450, provide the optimal size and shape for 3-HPA binding. We also compared AbKGSADH with other reported 3-HP producing ALDHs for the crucial amino acid residues for enzyme catalysis and substrate binding, which provides structural implications on how these enzymes utilize 3-HPA as a substrate.

Defining the crucial domain and amino acid residues in bacterial Lon protease for DNA binding and processing of DNA-interacting substrates

Lon protease previously has been shown to interact with DNA, but the role of this interaction for Lon proteolytic activity has not been characterized. In this study, we used truncated Escherichia coli Lon constructs, bioinformatics analysis, and site-directed mutagenesis to identify Lon domains and residues crucial for Lon binding with DNA and effects on Lon proteolytic activity. We found that deletion of Lon's ATPase domain abrogated interactions with DNA. Substitution of positively charged amino acids in this domain in full-length Lon with residues conferring a net negative charge disrupted binding of Lon to DNA. These changes also affected the degradation of nucleic acid-binding protein substrates of Lon, intracellular localization of Lon, and cell morphology. In vivo tests revealed that Lon-DNA interactions are essential for Lon activity in cell division control. In summary, we demonstrate that the ability of Lon to bind DNA is determined by its ATPase domain, that this binding is required for processing protein substrates in nucleoprotein complexes, and that Lon may help regulate DNA replication in response to growth conditions.

SYNTHESIS, CRYSTAL STUDIES AND PHARMACOLOGICAL ROLE PREDICTION OF 3-IODO-2- METHYL-1 PHENYL SULFONYL-1H INDOLE

ABSTRACTObjective: Indole-based compounds have established many pharmacological applications. Indole acts as a starting compound for various medicinalpreparations. Our objective is to determine the possible pharmacological roles of our crystallized indole-based ligand using computational approach.The activities studied are antibacterial, antitubercular, and antimelanoma.Methods: The structure of the indole compound, CR2 was studied using single crystal X-ray diffraction technique. To predict the pharmacologicalactivities, the structure-based docking method was followed. The protein targets were selected based on their important biological role in eachactivity mentioned above. The interactions between the targets and the crystal ligand were studied using a generic based docking algorithm. Thesignificant pharmacophore features of the ligand were also reported.Results: The ligand showed bonded and non-bonded interactions with the crucial amino acid residues of the active site of each target. The interactionsand the binding energies were quite comparable to the targets’ natural ligands.Conclusion: We suggest through the computational approaches that the ligand may have antitubercular, antibacterial, and antimelanoma activitieswith respect to the targets considered. The new insights of this compound as predicted by the computational methods are believed to provide aplatform for the futuristic pharmacological activities of this compound that can be further considered for wet lab techniques.Keywords: Indole, Dihydrofolate reductase, DNA gyrase, Human B-Raf kinase, Docking, Pharmacophore, Single crystal X-ray diffraction.

Identification of Crucial Amino Acid Residues Involved in Agonist Signaling at the GPR55 Receptor.

GPR55 is a newly deorphanized class A G-protein-coupled receptor that has been implicated in inflammatory pain, neuropathic pain, metabolic disorder, bone development, and cancer. Few potent GPR55 ligands have been identified to date. This is largely due to an absence of information about salient features of GPR55, such as residues important for signaling and residues implicated in the GPR55 signaling cascade. The goal of this work was to identify residues that are key for the signaling of the GPR55 endogenous ligand, l-α-lysophosphatidylinositol (LPI), as well as the signaling of the GPR55 agonist, ML184 {CID 2440433, 3-[4-(2,3-dimethylphenyl)piperazine-1-carbonyl]-N,N-dimethyl-4-pyrrolidin-1-ylbenzenesulfonamide}. Serum response element (SRE) and serum response factor (SRF) luciferase assays were used as readouts for studying LPI and ML184 signaling at the GPR55 mutants. A GPR55 R* model based on the recent δ-opioid receptor (DOR) crystal structure was used to interpret the resultant mutation data. Two residues were found to be crucial for agonist signaling at GPR55, K2.60 and E3.29, suggesting that these residues form the primary interaction site for ML184 and LPI at GPR55. Y3.32F, H(170)F, and F6.55A/L mutation results suggested that these residues are part of the orthosteric binding site for ML184, while Y3.32F and H(170)F mutation results suggest that these two residues are part of the LPI binding pocket. Y3.32L, M3.36A, and F6.48A mutation results suggest the importance of a Y3.32/M3.36/F6.48 cluster in the GPR55 signaling cascade. C(10)A and C(260)A mutations suggest that these residues form a second disulfide bridge in the extracellular domain of GPR55, occluding ligand extracellular entry in the TMH1-TMH7 region of GPR55. Taken together, these results provide the first set of discrete information about GPR55 residues important for LPI and ML184 signaling and for GPR55 activation. This information should aid in the rational design of next-generation GPR55 ligands and the creation of the first high-affinity GPR55 radioligand, a tool that is sorely needed in the field.

Cinnamide Derivatives as Mammalian Arginase Inhibitors: Synthesis, Biological Evaluation and Molecular Docking.

Arginases are enzymes that are involved in many human diseases and have been targeted for new treatments. Here a series of cinnamides was designed, synthesized and evaluated in vitro and in silico for their inhibitory activity against mammalian arginase. Using a microassay on purified liver bovine arginase (b-ARG I), (E)-N-(2-phenylethyl)-3,4-dihydroxycinnamide, also named caffeic acid phenylamide (CAPA), was shown to be slightly more active than our natural reference inhibitor, chlorogenic acid (IC50 = 6.9 ± 1.3 and 10.6 ± 1.6 µM, respectively) but it remained less active that the synthetic reference inhibitor Nω-hydroxy-nor-l-arginine nor-NOHA (IC50 = 1.7 ± 0.2 µM). Enzyme kinetic studies showed that CAPA was a competitive inhibitor of arginase with Ki = 5.5 ± 1 µM. Whereas the activity of nor-NOHA was retained (IC50 = 5.7 ± 0.6 µM) using a human recombinant arginase I (h-ARG I), CAPA showed poorer activity (IC50 = 60.3 ± 7.8 µM). However, our study revealed that the cinnamoyl moiety and catechol function were important for inhibitory activity. Docking results on h-ARG I demonstrated that the caffeoyl moiety could penetrate into the active-site pocket of the enzyme, and the catechol function might interact with the cofactor Mn2+ and several crucial amino acid residues involved in the hydrolysis mechanism of arginase. The results of this study suggest that 3,4-dihydroxycinnamides are worth being considered as potential mammalian arginase inhibitors, and could be useful for further research on the development of new arginase inhibitors.

Critical regions and residues for self-interaction of grapevine leafroll-associated virus 2 protein p24

The 24-kDa protein (p24) encoded by grapevine leafroll-associated virus 2 (GLRaV-2) is an RNA-silencing suppressor. In this work, a yeast two-hybrid system (YTHS) and bimolecular fluorescence complementation analyses showed that GLRaV-2 p24 can interact with itself, and that this interaction occurs in the cytoplasm of Nicotiana benthamiana cells. To identify the functional region(s) and crucial amino acid residues required for p24 self-interaction, various truncated and substitution mutants were generated. YTHS assay showed that in both homologous pairing and pairing with the wild-type p24, the functional regions mapped to aa 10–180 or 1–170 which contain, respectively, all seven α-helices or the first six α-helices and the N-terminal end (aa 1–9) of the protein. When only the full-length p24 was an interaction partner, the functional region of aa 1–170 could be further mapped to aa 1–140 which contains four α-helices plus most of the fifth α-helix. Further analysis with substitution mutants demonstrated that hydrophobic residues I35/F38/V85/V89/W149 and V162/L169/L170, which may, respectively, mediate the inter-domain interaction of the same p24 monomer and the tail-to-tail association between two p24 counterparts, are crucial for homotypic p24–p24 interaction. In addition, substitution of two basic residues—R2 or R86—of p24, which may play important functional roles in RNA binding, did not seem to affect self-interaction of the mutants in yeast but had obvious effects in plant cells. Taken together, our results demonstrate the functional regions and crucial amino acids for p24 self-interaction.

Insights into the structure activity relationship of mPGES-1 inhibitors: Hints for better inhibitor design

Microsomal prostaglandin E synthase-1 (mPGES-1) is a membrane protein which plays crucial role in arachidonic acid metabolism, in the catalysis of PGH2 to PGE2. It is a potential drug target involved in variety of human cancers and inflammatory disorders. In the present study we made an attempt to identify crucial amino acid residues involved in the effective binding of its inhibitors at the active site. Molecular docking and Structure Activity Relationship (SAR) studies were performed. In the present study 127 inhibitors having significant variability in parent scaffold were considered. The results clearly indicated that in the GSH and PGH2 binding site Arg70, Arg73, Asn74, Glu77, His113, Tyr117, Arg126, Ser127, Tyr130, Thr131 and Ala138 consistently form crucial interactions with inhibitors of different classes/scaffolds. These findings are consistent with that of existing reports on the active site residues pivotal at mPGES-1 active site. Further analysis suggested that out of all important amino acid residues identified; Arg73, Asn74, His113, Tyr117, Arg126, Ser127, Tyr130, Thr131 and Ala138 play a crucial role in hydrogen and Ͽ-Ͽ interactions. The identified amino acid residues can act as target sites for the design and development of drug candidates against mPGES-1.