Novel peptide inhibitor of human tumor necrosis factor-α has antiarthritic activity.

The inhibition of tumor necrosis factor (TNF)-α trimer formation renders it inactive for binding to its receptors, thus mitigating the vicious cycle of inflammation. We designed a peptide (PIYLGGVFQ) that simulates a sequence strand of human TNFα monomer using a series of in silico methods, such as active site finding (Acsite), protein-protein interaction (PPI), docking studies (GOLD and Flex-X) followed by molecular dynamics (MD) simulation studies. The MD studies confirmed the intermolecular interaction of the peptide with the TNFα. Fluorescence-activated cell sorting and fluorescence microscopy revealed that the peptide effectively inhibited the binding of TNF to the cell surface receptors. The cell culture assays showed that the peptide significantly inhibited the TNFα-mediated cell death. In addition, the nuclear translocation of the nuclear factor kappa B (NFκB) was significantly suppressed in the peptide-treated A549 cells, as observed in immunofluorescence and gel mobility-shift assays. Furthermore, the peptide protected against joint damage in the collagen-induced arthritis (CIA) mouse model, as revealed in the micro focal-CT scans. In conclusion, this TNFα antagonist would be helpful for the prevention and repair of inflammatory bone destruction and subsequent loss in the mouse model of CIA as well as human rheumatoid arthritis (RA) patients. This calls upon further clinical investigation to utilize its potential effect as an antiarthritic drug.

Key essential oil components delocalize Candida albicans Kar3p and impact microtubule structure.

BACKGROUND: Treatment of Candida albicans associated infections is often ineffective in the light of resistance, with an urgent need to discover novel antimicrobials. Fungicides require high specificity and can contribute to antifungal resistance, so inhibition of fungal virulence factors is a good strategy for developing new antifungals. OBJECTIVES: Examine the impact of four plant-derived essential oil components (1,8-cineole, α-pinene, eugenol, and citral) on C. albicans microtubules, kinesin motor protein Kar3 and morphology. METHODS: Microdilution assays were used to determine minimal inhibitory concentrations, microbiological assays assessed germ tube, hyphal and biofilm formation, confocal microscopy probed morphological changes and localization of tubulin and Kar3p, and computational modelling was used to examine the theoretical binding of essential oil components to tubulin and Kar3p. RESULTS: We show for the first time that essential oil components delocalize the Kar3p, ablate microtubules, and induce psuedohyphal formation with reduced biofilm formation. Single and double deletion mutants of kar3 were resistant to 1,8-cineole, sensitive to α-pinene and eugenol, but unimpacted by citral. Strains with homozygous and heterozygous Kar3p disruption had a gene-dosage effect for all essential oil components, resulting in enhanced resistance or susceptibility patterns that were identical to that of cik1 mutants. The link between microtubule (αß-tubulin) and Kar3p defects was further supported by computational modeling, showing preferential binding to αß-tubulin and Kar3p adjacent to their Mg2+-binding sites. CONCLUSION: This study highlights how essential oil components interfere with the localization of the kinesin motor protein complex Kar3/Cik1 and disrupt microtubules, leading to their destabilization which results in hyphal and biofilm defects.

Coarse grained modelling highlights the binding differences in the two different allosteric sites of the Human Kinesin EG5 and its implications in inhibitor design.

Kinesins involved in mitotic cell division have gained prominence as promising chemotherapy targets. One such kinesin, EG5, a motor protein responsible for cell division, is a validated chemotherapy target with several compounds at various stages of clinical trials. EG5 has an active site and two different allosteric sites that are known to have ligand specificity. Upon ligand binding, EG5's motor domain will no longer undergo nucleotide-dependent conformational changes required to complete the catalytic cycle. However, there is a lack of in-depth knowledge on the mechanism of inhibitor binding to the two different allosteric sites. To understand the EG5's inhibition mechanism and interactions at allosteric sites and other functionally important regions, we generated two coarse-grained models, Gaussian Network Model (GNM) and Anisotropic Network Model (ANM), to identify the dynamics and its correlation to EG5's function. The first three slowest modes of GNM showed marked differences between the various models of EG5. In the first mode, when the inhibitor is bound at allosteric site 1, there is a presence of a hinge region around residue 166, which is not found when the inhibitor is bound at allosteric site 2 or allosteric sites 1 and 2. The third slowest mode showed a distinctive positively correlated region when the inhibitor is bound at allosteric site 2. These differences indicated that the mechanism of binding at allosteric site 1 and allosteric site 2 are unique. Further, it was observed that the simultaneous ligand binding at allosteric sites 1 and 2 shares structural dynamics and interactions that were found while ligand binds at allosteric sites 1 and 2 independently, leading to a new mechanism. Taken together, our observations suggest that there are different mechanisms at play in each inhibitor bound system considered.

Identification of nsSNPs of transcription factor E2F1 predisposing individuals to lung cancer and head and neck cancer.

E2Fs transcription factors family is involved in the G1/S transition and DNA replication and their deregulated expression have been reported in various human cancers. Studies have shown that the genetic variants of E2F1 family members play an important role in head and neck carcinogenesis. In this study, we predicted six highly deleterious nsSNPs (C227F, R252H, V295D, C298Y, R56W, and Y59C) of E2F1 gene through in silico analyses. The latter was based on protein structure, function, and amino acid conservation. Molecular dynamics studies showed a deviation of the structures of the mutant proteins from the global protein parameters. Further, a case-control study that included total 535 samples (305 cancer patients and 230 controls) was conducted to find the association of the predicted SNPs with the susceptibility to lung cancer (LC) and head and neck cancer (HNC). The genotyping was done applying in-house artificial-RFLP method. Statistical analysis showed that the mutant alleles/genotypes of rs3213172 (R252H) were found to increase ∼ 2-5 fold risk of LC and HNC in all the genetic models. These results suggest that the rs3213172C/T polymorphism of the E2F1 gene could be used as an effective biomarker for genetic susceptibility to LC and HNC in our population.

Genome-Wide Mutagenesis of Hepatitis C Virus Reveals Ability of Genome To Overcome Detrimental Mutations.

To gain insight into the impact of mutations on the viability of the hepatitis C virus (HCV) genome, we created a set of full-genome mutant libraries, differing from the parent sequence as well as each other, by using a random mutagenesis approach; the proportion of mutations increased across these libraries with declining template amount or dATP concentration. The replication efficiencies of full-genome mutant libraries ranged between 71 and 329 focus-forming units (FFU) per 105 Huh7.5 cells. Mutant libraries with low proportions of mutations demonstrated low replication capabilities, whereas those with high proportions of mutations had their replication capabilities restored. Hepatoma cells transfected with selected mutant libraries, with low (4 mutations per 10,000 bp copied), moderate (33 mutations), and high (66 mutations) proportions of mutations, and their progeny were subjected to serial passage. Predominant virus variants (mutants) from these mutant libraries (Mutantl, Mutantm, and Mutanth, respectively) were evaluated for changes in growth kinetics and particle-to-FFU unit ratio, virus protein expression, and modulation of host cell protein synthesis. Mutantm and Mutantl variants produced >3.0-log-higher extracellular progeny per ml than the parent, and Mutanth produced progeny at a rate 1.0-log lower. More than 80% of the mutations were in a nonstructural part of the mutant genomes, the majority were nonsynonymous, and a moderate to large proportion were in the conserved regions. Our results suggest that the HCV genome has the ability to overcome lethal/deleterious mutations because of the high reproduction rate but highly selects for random, beneficial mutations.IMPORTANCE Hepatitis C virus (HCV) in vivo displays high genetic heterogeneity, which is partly due to the high reproduction and random substitutions during error-prone genome replication. It is difficult to introduce random substitutions in vitro because of limitations in inducing mutagenesis from the 5' end to the 3' end of the genome. Our study has overcome this limitation. We synthesized full-length genomes with few to several random mutations in the background of an HCV clone that can recapitulate all steps of the life cycle. Our study provides evidence of the capability of the HCV genome to overcome deleterious mutations and remain viable. Mutants that emerged from the libraries had diverse phenotype profiles compared to the parent, and putative adaptive mutations mapped to segments of the conserved nonstructural genome. We demonstrate the potential utility of our system for the study of sequence variation that ensures the survival and adaptation of HCV.

Cinnamate-CoA ligase is involved in biosynthesis of benzoate-derived biphenyl phytoalexin in Malus × domestica 'Golden Delicious' cell cultures.

Apple (Malus sp.) and other genera belonging to the sub-tribe Malinae of the Rosaceae family produce unique benzoic acid-derived biphenyl phytoalexins. Cell cultures of Malus domestica cv. 'Golden Delicious' accumulate two biphenyl phytoalexins, aucuparin and noraucuparin, in response to the addition of a Venturia inaequalis elicitor (VIE). In this study, we isolated and expressed a cinnamate-CoA ligase (CNL)-encoding sequence from VIE-treated cell cultures of cv. 'Golden Delicious' (M. domestica CNL; MdCNL). MdCNL catalyses the conversion of cinnamic acid into cinnamoyl-CoA, which is subsequently converted to biphenyls. MdCNL failed to accept benzoic acid as a substrate. When scab-resistant (cv. 'Shireen') and moderately scab-susceptible (cv. 'Golden Delicious') apple cultivars were challenged with the V. inaequalis scab fungus, an increase in MdCNL transcript levels was observed in internodal regions. The increase in MdCNL transcript levels could conceivably correlate with the pattern of accumulation of biphenyls. The C-terminal signal in the MdCNL protein directed its N-terminal reporter fusion to peroxisomes in Nicotiana benthamiana leaves. Thus, this report records the cloning and characterisation of a cinnamoyl-CoA-forming enzyme from apple via a series of in vivo and in vitro studies. Defining the key step of phytoalexin formation in apple provides a biotechnological tool for engineering elite cultivars with improved resistance.

Structure and function prediction of arsenate reductase from Deinococcus indicus DR1.

Arsenic prevalence in the environment impelled many organisms to develop resistance over the course of evolution. Tolerance to arsenic, either as the pentavalent [As(V)] form or the trivalent form [As(III)], by bacteria has been well studied in prokaryotes, and the mechanism of action is well defined. However, in the rod-shaped arsenic tolerant Deinococcus indicus DR1, the key enzyme, arsenate reductase (ArsC) has not been well studied. ArsC of D. indicus belongs to the Grx-linked prokaryotic arsenate reductase family. While it shares homology with the well-studied ArsC of Escherichia coli having a catalytic cysteine (Cys 12) and arginine triad (Arg 60, 94, and 107), the active site of D.indicus ArsC contains four residues Glu 9, Asp 53, Arg 86, and Glu 100, and with complete absence of structurally equivalent residue for crucial Cys 12. Here, we report that the mechanism of action of ArsC of D. indicus is different as a result of convergent evolution and most likely able to detoxify As(V) using a mix of positively- and negatively-charged residues in its active site, unlike the residues of E. coli. This suggests toward the possibility of an alternative mechanism of As (V) degradation in bacteria.

Crystal Structures of SgcE6 and SgcC, the Two-Component Monooxygenase That Catalyzes Hydroxylation of a Carrier Protein-Tethered Substrate during the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027 in Streptomyces globisporus.

C-1027 is a chromoprotein enediyne antitumor antibiotic produced by Streptomyces globisporus. In the last step of biosynthesis of the (S)-3-chloro-5-hydroxy-ß-tyrosine moiety of the C-1027 enediyne chromophore, SgcE6 and SgcC compose a two-component monooxygenase that hydroxylates the C-5 position of (S)-3-chloro-ß-tyrosine. This two-component monooxygenase is remarkable for two reasons. (i) SgcE6 specifically reacts with FAD and NADH, and (ii) SgcC is active with only the peptidyl carrier protein (PCP)-tethered substrate. To address the molecular details of substrate specificity, we determined the crystal structures of SgcE6 and SgcC at 1.66 and 2.63 Å resolution, respectively. SgcE6 shares a similar ß-barrel fold with the class I HpaC-like flavin reductases. A flexible loop near the active site of SgcE6 plays a role in FAD binding, likely by providing sufficient space to accommodate the AMP moiety of FAD, when compared to that of FMN-utilizing homologues. SgcC shows structural similarity to a few other known FADH2-dependent monooxygenases and sheds light on some biochemically but not structurally characterized homologues. The crystal structures reported here provide insights into substrate specificity, and comparison with homologues provides a catalytic mechanism of the two-component, FADH2-dependent monooxygenase (SgcE6 and SgcC) that catalyzes the hydroxylation of a PCP-tethered substrate.

Crystal structure of SgcJ, an NTF2-like superfamily protein involved in biosynthesis of the nine-membered enediyne antitumor antibiotic C-1027.

Comparative analysis of the enediyne biosynthetic gene clusters revealed sets of conserved genes serving as outstanding candidates for the enediyne core. Here we report the crystal structures of SgcJ and its homologue NCS-Orf16, together with gene inactivation and site-directed mutagenesis studies, to gain insight into enediyne core biosynthesis. Gene inactivation in vivo establishes that SgcJ is required for C-1027 production in Streptomyces globisporus. SgcJ and NCS-Orf16 share a common structure with the nuclear transport factor 2-like superfamily of proteins, featuring a putative substrate binding or catalytic active site. Site-directed mutagenesis of the conserved residues lining this site allowed us to propose that SgcJ and its homologues may play a catalytic role in transforming the linear polyene intermediate, along with other enediyne polyketide synthase-associated enzymes, into an enzyme-sequestered enediyne core intermediate. These findings will help formulate hypotheses and design experiments to ascertain the function of SgcJ and its homologues in nine-membered enediyne core biosynthesis.

Structure-guided functional characterization of enediyne self-sacrifice resistance proteins, CalU16 and CalU19.

Calicheamicin γ1I (1) is an enediyne antitumor compound produced by Micromonospora echinospora spp. calichensis, and its biosynthetic gene cluster has been previously reported. Despite extensive analysis and biochemical study, several genes in the biosynthetic gene cluster of 1 remain functionally unassigned. Using a structural genomics approach and biochemical characterization, two proteins encoded by genes from the 1 biosynthetic gene cluster assigned as "unknowns", CalU16 and CalU19, were characterized. Structure analysis revealed that they possess the STeroidogenic Acute Regulatory protein related lipid Transfer (START) domain known mainly to bind and transport lipids and previously identified as the structural signature of the enediyne self-resistance protein CalC. Subsequent study revealed calU16 and calU19 to confer resistance to 1, and reminiscent of the prototype CalC, both CalU16 and CalU19 were cleaved by 1 in vitro. Through site-directed mutagenesis and mass spectrometry, we identified the site of cleavage in each protein and characterized their function in conferring resistance against 1. This report emphasizes the importance of structural genomics as a powerful tool for the functional annotation of unknown proteins.

Crystal structure of SsfS6, the putative C-glycosyltransferase involved in SF2575 biosynthesis.

The molecule known as SF2575 from Streptomyces sp. is a tetracycline polyketide natural product that displays antitumor activity against murine leukemia P388 in vivo. In the SF2575 biosynthetic pathway, SsfS6 has been implicated as the crucial C-glycosyltransferase (C-GT) that forms the C-C glycosidic bond between the sugar and the SF2575 tetracycline-like scaffold. Here, we report the crystal structure of SsfS6 in the free form and in complex with TDP, both at 2.4 Å resolution. The structures reveal SsfS6 to adopt a GT-B fold wherein the TDP and docked putative aglycon are consistent with the overall C-glycosylation reaction. As one of only a few existing structures for C-glycosyltransferases, the structures described herein may serve as a guide to better understand and engineer C-glycosylation.

Predictive inference on cytoplasmic and mitochondrial thioredoxin peroxidases in the highly radioresistant Lepidopteran insect Spodoptera frugiperda.

Lepidopteran insects show remarkable resistance to radiation and chemical stress than insects of other orders. Despite this, the antioxidant machinery of insects of this order is poorly understood. Recently we demonstrated the significance of cytoplasmic NOS and a stronger mitochondrial antioxidant enzyme system in the stress-resistance of Lepidopteran insects. In the present study, we hypothesize two thioredoxin peroxidase orthologues (Sf-TPx1 and Sf-TPx2) in Lepidopteran insect Spodoptera frugiperda and demonstrate their structural/functional features important for cellular antioxidant activity and stress resistance. Results show a higher mitochondrial localization score (WoLFPSORT) of Sf-TPx2 (mitochondria-18.0, cytoplasm-7.0, nucleus-4.0) than its Drosophila orthologue Jafrac2 (secretory-30.0; mitochondria/nucleus/cytoplasm-no signal), which is important for antioxidant activity, and a higher cytoplasmic localization score of Sf-TPx1 (mitochondria-no signal; cytoplasm-22.0; nucleus-3.5) than the Drosophila Jafrac1 (mitochondria-17; nucleus- 11; cytoplasm-no signal). Structural modeling data show certain motifs present in Jafrac1 and Jafrac2 that affect active site conformation and separate cysteine residues at distances not suitable for disulphide bridge formation (5.21Å; 5.73Å). These motifs are absent in Sf-TPx1 and Sf-TPx2, yielding shorter distance (2.01Å; 2.05Å) between the cysteine residues suitable for disulphide bridge formation. Taken together, the disulphide bridge as well as mitochondrial and cytoplasmic localization are crucial for peroxidatic activity of TPx's. Therefore,we hypothesize that the Spodoptera TPx's offer potentially stronger anti-oxidant activity than that of Drosophila orthologues, and may contribute in the high radioresistance of Lepidopteran insects.