Rajendram V. Rajnarayanan

Functional studies of the Ssk1p response regulator protein of Candida albicans as determined by phenotypic analysis of receiver domain point mutants

The Candida albicans response regulator protein Ssk1p regulates oxidant adaptation through the MAPK HOG1 pathway. Deletion mutants lacking SSK1 are oxidant sensitive in vitro and are killed more than wild‐type (WT) cells by human neutrophils. Furthermore, the mutants are avirulent in an invasive murine model, and unable to adhere to human esophageal cells. Transcriptional profiling has indicated that approximately 25% of all changes occur in genes encoding cell wall and stress adaptation functions. In this study, we have investigated the role of amino acid residues in the Ssk1p receiver (or regulatory) domain by constructing point mutants at positions D556 (putative site of protein phosphorylation) and D513 (putative role in divalent metal binding, phosphorylation and conformational switching). For each point mutant, their sensitivity to a variety of oxidant stress conditions was assessed and correlated with in vitro phosphorylation of each Ssk1p receiver domain, phosphorylation of the Hog1p MAP kinase, and translocation to the nucleus. We show that a D556N mutant is sensitive to 5 mM H2O2 or t‐butyl hydroperoxide, similar to a gene knock‐out ssk1 mutant, even though Hog1p is phosphorylated in the D556N mutant. To resolve this apparent paradox, we also demonstrate that Hog1p translocation to the nucleus in the D556N mutant is significantly reduced compared with WT cells (CAF2‐1). In a second point mutant, D513 was changed to a lysine residue (D513K). This mutant had WT levels of resistance to peroxide, but in comparison to WT cells and the D556N mutant, morphogenesis (yeast to hyphae transition) was inhibited in 10% serum or in M‐199 medium at 37°C. In the D513K point mutant, constitutive phosphorylation of Hog1p was observed, suggesting that a non‐conservative change (D513K) traps Ssk1p in an active conformation and therefore constitutive Hog1p phosphorylation. The inhibition of morphogenesis in D513K is related to a downregulation of the transcription factors of morphogenesis, EFG1 and CPH1. Another non‐conserved point mutant (D556R) was also constructed and phenotypically was like the D513K mutant. The receiver domains of the D556N and the D513K mutants could not be appreciably phosphorylated in vitro indicating that constitutive activation of Hog1p occurs in vivo due to the inability of Ssk1p to be phosphorylated at least in the D513K mutant. We speculate that the non‐conservative changes described above in Ssk1p response regulator may cause conformational changes in the Ssk1p that account for phenotype differences compared with the D556N mutant that are also Hog‐independent.

Integrative sequence and tissue expression profiling of chicken and mammalian aquaporins

BackgroundProteins that selectively transport water across the membranes of cells are recognized as important in the normal functioning of the body systems of vertebrates. There are 13 known mammalian aquaporins (AQP0 to AQP12), some of which have been shown to have unexpected cellular roles beyond transmembrane water transport. The availability of non-mammalian vertebrate animal models has the potential to provide insight into the emergence of diverse function in the aquaporins. The domesticated chicken (Gallus gallus) is the premier avian model for biological research; however, only a limited number of studies have compared chicken and mammalian aquaporins. The identification of aquaporins that share functional motifs or are expressed in the same tissues in human and chicken could allow the further functional analyses of homologous aquaporins in both species. We hypothesize that integrative analyses of protein sequences and body site expression of human, mouse, rat and chicken aquaporins has the potential to yield novel biological hypotheses about the unexpected cellular roles of aquaporins beyond transmembrane water transport.ResultsA total of 76 aquaporin transcript models derived from 47 aquaporin genes were obtained for human, mouse, rat and chicken. Eleven body sites (brain, connective tissue, head, heart, liver, muscle, ovary, pancreas, small intestine, spleen and testis) were identified in which there is suggested expression of at least one mammalian and one chicken aquaporin. This study demonstrates that modern on-line analysis tools, a novel matrix integration technique, and the availability of the chicken genome for comparative genomics and expression analysis enables hypothesis generation in several important areas including: (i) alternative transcription and speciation effects on the conservation of functional motifs in vertebrate aquaporins; (ii) the emergence of basolateral targeting in mammalian species; (iii) the potential of the cysteine-rich AQP11 as a possible target in the pathophysiology of neurodegenerative disorders such as autism that involve Purkinje cells; and (iv) possible impairment of function of pancreas-expressed AQP12 during pancreatotropic necrosis in avian influenza virus infection.ConclusionThe investigation of aquaporin function in chicken and mammalian species has the potential to accelerate the discovery of novel knowledge of aquaporins in both avian and mammalian species.

“Teaching old drugs to kill new bugs”: structure-based discovery of anti-SARS drugs

Abstract Severe acute respiratory syndrome (SARS) main protease or 3C-like protease (3CLpro) is essential for the propagation of the coronaviral life cycle and is regarded as one of the main targets for structure-based anti-SARS drug design. It is an attractive approach to find new uses for old drugs as they have already been through extensive clinical testing and could easily be accelerated for clinical approval. Briefly, we performed virtual screening of a database of small molecules against SARS 3CLpro, analyzed inhibitor–protease complexes, and identified several covalent and non-covalent inhibitors. Several old drugs that bind to SARS 3CLpro active site were selected and in silico derivatized to generate covalent irreversible inhibitors with enhanced affinity. Furthermore, we show that pharmacophores derived from clusters of compounds resulting out of virtual screening could be useful probes for future structure–activity relationship studies (SARs) and fine-tune the lead molecules identified.

Developmental Regulation of Genes encoding Universal stress proteins in Schistosoma mansoni

The draft nuclear genome sequence of the snail-transmitted, dimorphic, parasitic, platyhelminth Schistosoma mansoni revealed eight genes encoding proteins that contain the Universal Stress Protein (USP) domain. Schistosoma mansoni is a causative agent of human schistosomiasis, a severe and debilitating Neglected Tropical Disease (NTD) of poverty, which is endemic in at least 76 countries. The availability of the genome sequences of Schistosoma species presents opportunities for bioinformatics and genomics analyses of associated gene families that could be targets for understanding schistosomiasis ecology, intervention, prevention and control. Proteins with the USP domain are known to provide bacteria, archaea, fungi, protists and plants with the ability to respond to diverse environmental stresses. In this research investigation, the functional annotations of the USP genes and predicted nucleotide and protein sequences were initially verified. Subsequently, sequence clusters and distinctive features of the sequences were determined. A total of twelve ligand binding sites were predicted based on alignment to the ATP-binding universal stress protein from Methanocaldococcus jannaschii. In addition, six USP sequences showed the presence of ATP-binding motif residues indicating that they may be regulated by ATP. Public domain gene expression data and RT-PCR assays confirmed that all the S. mansoni USP genes were transcribed in at least one of the developmental life cycle stages of the helminth. Six of these genes were up-regulated in the miracidium, a free-swimming stage that is critical for transmission to the snail intermediate host. It is possible that during the intra-snail stages, S. mansoni gene transcripts for universal stress proteins are low abundant and are induced to perform specialized functions triggered by environmental stressors such as oxidative stress due to hydrogen peroxide that is present in the snail hemocytes. This report serves to catalyze the formation of a network of researchers to understand the function and regulation of the universal stress proteins encoded in genomes of schistosomes and their snail intermediate hosts.

Candidate Single Nucleotide Polymorphism Markers for Arsenic Responsiveness of Protein Targets

Arsenic is a toxic metalloid that causes skin cancer and binds to cysteine residues—a property that could be used to infer arsenic responsiveness of a target protein. Non-synonymous Single Nucleotide Polymorphisms (nsSNPs) result in amino acid substitutions and may alter arsenic binding with cysteine residues. Thus, the objective of this investigation was to identify and analyze nsSNPs that lead to substitutions to or from cysteine residues as an indication of increased or decreased arsenic responsiveness. We hypothesize that integration of data on molecular impacts of nsSNPs and arsenic-gene relationships will identify nsSNPs that could serve as arsenic responsiveness markers. We have analyzed functional and structural impacts data for 5,811 nsSNPs linked to 1,224 arsenic-annotated genes. In addition to the identified candidate nsSNPs for increased or reduced arsenic responsiveness, we observed i) a nsSNP that results in the breakage of a disulfide bond, as candidate marker for reduced arsenic responsiveness of KLK7, a secreted serine protease participate in normal shedding of the skin; and ii) 6 pairs of vicinal cysteines in KLK7 protein that could be binding sites for arsenic. In summary, our analysis identified non-synonymous SNPs that could be used to evaluate responsiveness of a protein target to arsenic. In particular, an epidermal expressed serine protease with crucial function in normal skin physiology was prioritized on the basis of abundance of vicinal cysteines for further research on arsenic-induced keratinocyte carcinogenesis.

In silico design of peptidic inhibitors targeting estrogen receptor alpha dimer interface.

Human estrogen receptor alpha (ERα), which acts as a biomarker and as a therapeutic target for breast cancers, is activated by agonist ligands and co-activator proteins. Selective estrogen receptor modulators (SERM) act as antagonists in specific tissues and tamoxifen, a SERM, has served as a drug for decades for ERα-positive breast cancers. However, the ligand-selective and tissue-specific response of ERα biological activity and the resistance to tamoxifen treatment in advanced stages of ERα-positive breast cancers underscores the need to find a ligand-independent inhibitor for ERα. Here we present a ligand-independent approach of inhibiting ERα transactivation targeting its dimerization—a key process of ERα biological activity. Using in silico techniques, we first elucidated the hydrogen bond interactions involved in dimerization and identified three interfacial sequence motifs, where sequence I (DKITD) and sequence II (QQQHQRLAQ) of one monomer form hydrogen bonding with sequence II and sequence I of the second monomer, respectively, and sequence III (LSHIRHMSNK) hydrogen bonds with the same from the second monomer. Studying the structural stability and the binding affinity of the peptides derived from these sequence motifs, we found that an extended and ARG mutated version (LQQQHQQLAQ) of sequence II can act as a suitable template for designing peptidic inhibitors. It provides additional structural stability and interacts more strongly with ERα dimer interface groove formed by helices 9 and 10/11 and prevent ERα dimerization. Our result provides a novel therapeutic designing pipeline for ligand-independent inhibition of ERα.

Functional Annotation Analytics of Rhodopseudomonas palustris Genomes

Rhodopseudomonas palustris, a nonsulphur purple photosynthetic bacteria, has been extensively investigated for its metabolic versatility including ability to produce hydrogen gas from sunlight and biomass. The availability of the finished genome sequences of six R. palustris strains (BisA53, BisB18, BisB5, CGA009, HaA2 and TIE-1) combined with online bioinformatics software for integrated analysis presents new opportunities to determine the genomic basis of metabolic versatility and ecological lifestyles of the bacteria species. The purpose of this investigation was to compare the functional annotations available for multiple R. palustris genomes to identify annotations that can be further investigated for strain-specific or uniquely shared phenotypic characteristics. A total of 2,355 protein family Pfam domain annotations were clustered based on presence or absence in the six genomes. The clustering process identified groups of functional annotations including those that could be verified as strain-specific or uniquely shared phenotypes. For example, genes encoding water/glycerol transport were present in the genome sequences of strains CGA009 and BisB5, but absent in strains BisA53, BisB18, HaA2 and TIE-1. Protein structural homology modeling predicted that the two orthologous 240 aa R. palustris aquaporins have water-specific transport function. Based on observations in other microbes, the presence of aquaporin in R. palustris strains may improve freeze tolerance in natural conditions of rapid freezing such as nitrogen fixation at low temperatures where access to liquid water is a limiting factor for nitrogenase activation. In the case of adaptive loss of aquaporin genes, strains may be better adapted to survive in conditions of high-sugar content such as fermentation of biomass for biohydrogen production. Finally, web-based resources were developed to allow for interactive, user-defined selection of the relationship between protein family annotations and the R. palustris genomes.

Autism from a Biometric Perspective

Purpose: The aim of this pilot study was to test autistic children, siblings and their parents using a biometric device based on the gas discharge visualization (GDV) technique in order to assess their psycho-emotional and physiological functional state based on the activity of the autonomic nervous system. Hypothesis: We hypothesize that the biometric assessment based on GDV will enable us: (1) to evaluate some specific features associated with autism spectrum disorder (ASD) as well as to compare autistic children to their siblings and to controls; (2) to analyze the differences in individual values of parents of autistic children versus parents of normal children. Results: Out of total of 48 acupuncture points present on ten fingertips of both hands and associated to organs/organ systems, autistic children differed significantly from controls (p < 0.05) in 36 (images without filter) and 12 (images with filter), siblings differed significantly from controls (p < 0.05) in 12 (images without filter) and seven (images with filter), autistic children differed significantly (p < 0.05) from siblings in eight (images without filter) and one (images with filter), fathers of autistic children differed significantly (p < 0.05) from controls in 14 (images without filter) and three (images with filter) and mothers of autistic children differed significantly (p < 0.05) from controls in five (images without filter) and nine (images with filter) acupuncture points. Conclusions: All compared groups have shown significant difference on both psycho-emotional (images without filter) and physiological (images with filter) levels. However, the differences between autistic children and controls expressed on psycho-emotional level were the most significant as compared to the other groups. Therefore, the activity of the sympathetic autonomic nervous system is significantly altered in children with autism. The biometric method based on GDV is a promising step in autism research that may lead towards creating a disease profile and identify unique signature/biomarker for autism. Further work should involve more participants in order to augment our findings.

Regulation of Phenobarbital-Mediated Induction of CYP102 (Cytochrome P450BM-3) in Bacillus megaterium by Phytochemicals from Soy and Green Tea

Cytochrome P450 102 (CYP102 or Cytochrome P450(BM)(-)(3)) is induced in Bacillus megaterium by barbiturates, perioxisome proliferators, estrogen, and nonsteroidal antiinflammatory drugs. We have previously demonstrated that a CYP102 construct (BMC 143) coupled with a luciferase reporter gene can be used to identify the inducers of CYP102. We now describe the effect of added phytochemicals on the induction of CYP102 by phenobarbital (PB) in B. megaterium. The isoflavones genistein, biochanin A, coumestrol, and equol, the green tea flavanoid epicatechin, and the fungal toxin zearalenone inhibit the induction of CYP102 by PB in a dose-dependent manner. However, the isoflavone daidzein, the phytoalexin glyceollin, and catechin, an epimer of epicatechin, failed to exhibit a similar inhibitory effect on PB-mediated CYP102 induction.

Carbamate Insecticides Target Human Melatonin Receptors

Carbaryl (1-naphthyl methylcarbamate) and carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) are among the most toxic insecticides, implicated in a variety of diseases including diabetes and cancer among others. Using an integrated pharmacoinformatics based screening approach, we have identified these insecticides to be structural mimics of the neurohormone melatonin and were able to bind to the putative melatonin binding sites in MT1 and MT2 melatonin receptors in silico. Carbaryl and carbofuran then were tested for competition with 2-[125I]-iodomelatonin (300 pM) binding to hMT1 or hMT2 receptors stably expressed in CHO cells. Carbaryl and carbofuran showed higher affinity for competition with 2-[125I]-iodomelatonin binding to the hMT2 compared to the hMT1 melatonin receptor (33 and 35-fold difference, respectively) as predicted by the molecular modeling. In the presence of GTP (100 μM), which decouples the G-protein linked receptors to modulate signaling, the apparent efficacy of carbaryl and carbofuran for 2-[125I]-iodomelatonin binding for the hMT1 melatonin receptor was not affected but significantly decreased for the hMT2 melatonin receptor compatible with receptor antagonist/inverse agonist and agonist efficacy, respectively. Altogether, our data points to a potentially new mechanism through which carbamate insecticides carbaryl and carbofuran could impact human health by altering the homeostatic balance of key regulatory processes by directly binding to melatonin receptors.