Alper Uzun

Structure SNP (StSNP): a web server for mapping and modeling nsSNPs on protein structures with linkage to metabolic pathways

SNPs located within the open reading frame of a gene that result in an alteration in the amino acid sequence of the encoded protein [nonsynonymous SNPs (nsSNPs)] might directly or indirectly affect functionality of the protein, alone or in the interactions in a multi-protein complex, by increasing/decreasing the activity of the metabolic pathway. Understanding the functional consequences of such changes and drawing conclusions about the molecular basis of diseases, involves integrating information from multiple heterogeneous sources including sequence, structure data and pathway relations between proteins. The data from NCBIs SNP database (dbSNP), gene and protein databases from Entrez, protein structures from the PDB and pathway information from KEGG have all been cross referenced into the StSNP web server, in an effort to provide combined integrated, reports about nsSNPs. StSNP provides ‘on the fly’ comparative modeling of nsSNPs with links to metabolic pathway information, along with real-time visual comparative analysis of the modeled structures using the Friend software application. The use of metabolic pathways in StSNP allows a researcher to examine possible disease-related pathways associated with a particular nsSNP(s), and link the diseases with the current available molecular structure data. The server is publicly available at http://glinka.bio.neu.edu/StSNP/.

Dynamics of microRNAs in bull spermatozoa

BackgroundMicroRNAs are small non-coding RNAs that regulate gene expression and thus play important roles in mammalian development. However, the comprehensive lists of microRNAs, as well as, molecular mechanisms by which microRNAs regulate gene expression during gamete and embryo development are poorly defined. The objectives of this study were to determine microRNAs in bull sperm and predict their functions.MethodsTo accomplish our objectives we isolated miRNAs from sperm of high and low fertility bulls, conducted microRNA microarray experiments and validated expression of a panel of microRNAs using real time RT-PCR. Bioinformatic approaches were carried out to identify regulated targets.ResultsWe demonstrated that an abundance of microRNAs were present in bovine spermatozoa, however, only seven were differentially expressed; hsa-aga-3155, -8197, -6727, -11796, -14189, -6125, -13659. The abundance of miRNAs in the spermatozoa and the differential expression in sperm from high vs. low fertility bulls suggests that the miRNAs possibly play important functions in the regulating mechanisms of bovine spermatozoa.ConclusionIdentification of specific microRNAs expressed in spermatozoa of bulls with different fertility phenotypes will help better understand mammalian gametogenesis and early development.

Pathway-based genetic analysis of preterm birth.

Preterm birth in the United States is now 12%. Multiple genes, gene networks, and variants have been associated with this disease. Using a custom database for preterm birth (dbPTB) with a refined set of genes extensively curated from literature and biological databases, we analyzed GWAS of preterm birth for complete genotype data on nearly 2000 preterm and term mothers. We used both the curated genes and a genome-wide approach to carry out a pathway-based analysis. There were 19 significant pathways, which withstood FDR correction for multiple testing that were identified using both the curated genes and the genome-wide approach. The analysis based on the curated genes was more significant than genome-wide in 15 out of 19 pathways. This approach demonstrates the use of a validated set of genes, in the analysis of otherwise unsuccessful GWAS data, to identify gene-gene interactions in a way that enhances statistical power and discovery.

dbPTB: a database for preterm birth

Genome-wide association studies (GWAS) query the entire genome in a hypothesis-free, unbiased manner. Since they have the potential for identifying novel genetic variants, they have become a very popular approach to the investigation of complex diseases. Nonetheless, since the success of the GWAS approach varies widely, the identification of genetic variants for complex diseases remains a difficult problem. We developed a novel bioinformatics approach to identify the nominal genetic variants associated with complex diseases. To test the feasibility of our approach, we developed a web-based aggregation tool to organize the genes, genetic variations and pathways involved in preterm birth. We used semantic data mining to extract all published articles related to preterm birth. All articles were reviewed by a team of curators. Genes identified from public databases and archives of expression arrays were aggregated with genes curated from the literature. Pathway analysis was used to impute genes from pathways identified in the curations. The curated articles and collected genetic information form a unique resource for investigators interested in preterm birth. The Database for Preterm Birth exemplifies an approach that is generalizable to other disorders for which there is evidence of significant genetic contributions. Database URL: http://ptbdb.cs.brown.edu/dbPTBv1.php

Different roles of two gamma-tubulin isotypes in the cytoskeleton of the Antarctic ciliate Euplotes focardii: remodelling of interaction surfaces may enhance microtubule nucleation at low temperature.

γ‐Tubulin belongs to the tubulin superfamily and plays an essential role in the nucleation of cellular microtubules. In the present study, we report the characterization of γ‐tubulin from the psychrophilic Antarctic ciliate Euplotes focardii. In this organism, γ‐tubulin is encoded by two genes, γ‐T1 and γ‐T2, that produce distinct isotypes. Comparison of the γ‐T1 and γ‐T2 primary sequences to a Euplotesγ‐tubulin consensus, derived from mesophilic (i.e. temperate) congeneric species, revealed the presence of numerous unique amino acid substitutions, particularly in γ‐T2. Structural models of γ‐T1 and γ‐T2, obtained using the 3D structure of human γ‐tubulin as a template, suggest that these substitutions are responsible for conformational and/or polarity differences located: (a) in the regions involved in longitudinal ‘plus end’ contacts; (b) in the T3 loop that participates in binding GTP; and (c) in the M loop that forms lateral interactions. Relative to γ‐T1, the γ‐T2 gene is amplified by approximately 18‐fold in the macronuclear genome and is very strongly transcribed. Using confocal immunofluorescence microscopy, we found that the γ‐tubulins of E. focardii associate throughout the cell cycle with basal bodies of the non‐motile dorsal cilia and of all of the cirri of the ventral surface (i.e. adoral membranelles, paraoral membrane, and frontoventral transverse, caudal and marginal cirri). By contrast, only γ‐T2 interacts with the centrosomes of the spindle during micronuclear mitosis. We also established that the γ‐T1 isotype associates only with basal bodies. Our results suggest that γ‐T1 and γ‐T2 perform different functions in the organization of the microtubule cytoskeleton of this protist and are consistent with the hypothesis that γ‐T1 and γ‐T2 have evolved sequence‐based structural alterations that facilitate template nucleation of microtubules by the γ‐tubulin ring complex at cold temperatures.

Active site prediction for comparative model structures with thematics.

THEMATICS (Theoretical Microscopic Titration Curves) is a simple, reliable computational predictor of the active sites of enzymes from structure. Our method, based on well-established Finite Difference Poisson-Boltzmann techniques, identifies the ionisable residues with anomalous predicted titration behavior. A cluster of two or more such perturbed residues is a very reliable predictor of the active site. The protein does not have to bear any resemblance in sequence or structure to any previously characterized protein, but the method does require the three-dimensional structure. We now present evidence that THEMATICS can also locate the active site in structures built by comparative modeling from similar structures. Results are given for a total of 21 sets of proteins, including 21 templates and 83 comparative model structures. Detailed results are presented for three sets of orthologous proteins (Triosephosphate isomerase, 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase, and Aspartate aminotransferase) and for one set of human homologues of Aldose reductase with different functions. THEMATICS correctly locates the active site in the model structures. This suggests that the method can be applicable to a much larger set of proteins for which an experimentally determined structure is unavailable. With a few exceptions, the predicted active sites in the comparative model structures are similar to that of the corresponding template structure.

Bioinformatic Approach to the Genetics of Preeclampsia

OBJECTIVE: To identify candidate genes and genetic variants for preeclampsia using a bioinformatic approach to extract and organize genes and variants from the published literature. METHODS: Semantic data-mining and natural language processing were used to identify articles from the published literature meeting criteria for potential association with preeclampsia. Articles were manually reviewed by trained curators. Cluster analysis was used to aggregate the extracted genes into gene sets associated with preeclampsia or severe preeclampsia, early or late preeclampsia, maternal or fetal tissue sources, and concurrent conditions (ie, fetal growth restriction, gestational hypertension, or hemolysis, elevated liver enzymes, and low platelet count [HELLP]). Gene ontology was used to organize this large group of genes into ontology groups. RESULTS: From more than 22 million records in PubMed, with 28,000 articles on preeclampsia, our data-mining tool identified 2,300 articles with potential genetic associations with preeclampsia-related phenotypes. After curation, 729 articles were “accepted” that contained “statistically significant” associations with 535 genes. We saw distinct segregation of these genes by severity and timing of preeclampsia, by maternal or fetal source, and with associated conditions (eg, gestational hypertension, fetal growth restriction, or HELLP syndrome). CONCLUSION: The gene sets and ontology groups identified through our systematic literature curation indicate that preeclampsia represents several distinct phenotypes with distinct and overlapping maternal and fetal genetic contributions. LEVEL OF EVIDENCE: III

Efficacy of a Non-Hypercalcemic Vitamin-D2 Derived Anti-Cancer Agent (MT19c) and Inhibition of Fatty Acid Synthesis in an Ovarian Cancer Xenograft Model

Background Numerous vitamin-D analogs exhibited poor response rates, high systemic toxicities and hypercalcemia in human trials to treat cancer. We identified the first non-hypercalcemic anti-cancer vitamin D analog MT19c by altering the A-ring of ergocalciferol. This study describes the therapeutic efficacy and mechanism of action of MT19c in both in vitro and in vivo models. Methodology/Principal Finding Antitumor efficacy of MT19c was evaluated in ovarian cancer cell (SKOV-3) xenografts in nude mice and a syngenic rat ovarian cancer model. Serum calcium levels of MT19c or calcitriol treated animals were measured. In-silico molecular docking simulation and a cell based VDR reporter assay revealed MT19c–VDR interaction. Genomewide mRNA analysis of MT19c treated tumors identified drug targets which were verified by immunoblotting and microscopy. Quantification of cellular malonyl CoA was carried out by HPLC-MS. A binding study with PPAR-Y receptor was performed. MT19c reduced ovarian cancer growth in xenograft and syngeneic animal models without causing hypercalcemia or acute toxicity. MT19c is a weak vitamin-D receptor (VDR) antagonist that disrupted the interaction between VDR and coactivator SRC2-3. Genome-wide mRNA analysis and western blot and microscopy of MT19c treated xenograft tumors showed inhibition of fatty acid synthase (FASN) activity. MT19c reduced cellular levels of malonyl CoA in SKOV-3 cells and inhibited EGFR/phosphoinositol-3kinase (PI-3K) activity independently of PPAR-gamma protein. Significance Antitumor effects of non-hypercalcemic agent MT19c provide a new approach to the design of vitamin-D based anticancer molecules and a rationale for developing MT19c as a therapeutic agent for malignant ovarian tumors by targeting oncogenic de novo lipogenesis.

Functional genomics of HMGN3a and SMARCAL1 in early mammalian embryogenesis.

BackgroundEmbryonic genome activation (EGA) is a critical event for the preimplantation embryo, which is manifested by changes in chromatin structure, transcriptional machinery, expression of embryonic genes, and degradation of maternal transcripts. The objectives of this study were to determine transcript abundance of HMGN3a and SMARCAL1 in mature bovine oocytes and early bovine embryos, to perform comparative functional genomics analysis of these genes across mammals.ResultsNew annotations of both HMGN3a and SMARCAL1 were submitted to the Bovine Genome Annotation Submission Database at BovineGenome.org. Careful analysis of the bovine SMARCAL1 consensus gene set for this protein (GLEAN_20241) showed that the NCBI protein contains sequencing errors, and that the actual bovine protein has a high degree of homology to the human protein. Our results showed that there was a high degree of structural conservation of HMGN3a and SMARCAL1 in the mammalian species studied. HMGN3a transcripts were present at similar levels in bovine matured oocytes and 2–4-cell embryos but at higher levels in 8–16-cell embryos, morulae and blastocysts. On the other hand, transcript levels of SMARCAL1 decreased throughout preimplantation development.ConclusionThe high levels of structural conservation of these proteins highlight the importance of chromatin remodeling in the regulation of gene expression, particularly during early mammalian embryonic development. The greater similarities of human and bovine HMGN3a and SMARCAL1 proteins may suggest the cow as a valuable model to study chromatin remodeling at the onset of mammalian development. Understanding the roles of chromatin remodeling proteins during embryonic development emphasizes the importance of epigenetics and could shed light on the underlying mechanisms of early mammalian development.

An AP endonuclease 1-DNA polymerase β complex: Theoretical prediction of interacting surfaces

Abasic (AP) sites in DNA arise through both endogenous and exogenous mechanisms. Since AP sites can prevent replication and transcription, the cell contains systems for their identification and repair. AP endonuclease (APEX1) cleaves the phosphodiester backbone 5′ to the AP site. The cleavage, a key step in the base excision repair pathway, is followed by nucleotide insertion and removal of the downstream deoxyribose moiety, performed most often by DNA polymerase beta (pol-β). While yeast two-hybrid studies and electrophoretic mobility shift assays provide evidence for interaction of APEX1 and pol-β, the specifics remain obscure. We describe a theoretical study designed to predict detailed interacting surfaces between APEX1 and pol-β based on published co-crystal structures of each enzyme bound to DNA. Several potentially interacting complexes were identified by sliding the protein molecules along DNA: two with pol-β located downstream of APEX1 (3′ to the damaged site) and three with pol-β located upstream of APEX1 (5′ to the damaged site). Molecular dynamics (MD) simulations, ensuring geometrical complementarity of interfaces, enabled us to predict interacting residues and calculate binding energies, which in two cases were sufficient (∼−10.0 kcal/mol) to form a stable complex and in one case a weakly interacting complex. Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-β at the 3′-side of APEX1 are those most likely to occur in vivo. Additional multiple sequence analyses of APEX1 and pol-β in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces. Based on these results, we propose that pol-β in the open or closed conformation interacts and makes a stable interface with APEX1 bound to a cleaved abasic site on the 3′ side. The method described here can be used for analysis in any DNA-metabolizing pathway where weak interactions are the principal mode of cross-talk among participants and co-crystal structures of the individual components are available.