Hox proteins are evolutionarily conserved transcription factors that control important developmental pathways in morphogenesis of the embryo. The Hoxa2 gene is expressed in the developing central nervous system in rhombomeres 2 to 7 and affects cellular differentiation. Few target genes of Hoxa2 protein have been identified so far and its mechanisms of regulating gene expression remain elusive. Previous work in our laboratory isolated Hoxa2 protein binding sequences from the E18 mouse spinal cord and hindbrain tissues using chromatin immunoprecipitation (ChIP). All isolated DNA fragments contain conserved GATG motifs. Sequence analysis revealed that one fragment belongs to the high temperature requirement factor A 3 (HtrA3) gene and another fragment belongs to the Dual specificity tyrosine kinase 4 (Dyrk4) gene. In this study, direct binding of Hoxa2 protein to the HtrA3 and Dyrk4 fragments was confirmed by electrophoretic mobility shift assays (EMSA). Site-directed mutagenesis and EMSA studies revealed that Hoxa2 protein binds to the multiple GATG motifs within these fragments. HtrA3 fragment also repressed luciferase gene expression in transient transfection and luciferase assays. Mutation of the DNA fragment showed that the repressive activity was affected by the GATG motifs, suggesting Hoxa2 protein regulated gene expression by binding to the GATG motif in the cis-regulatory element. In contrast to the inhibitory activity of Hoxa2 protein, a Hoxa2-VP16 fusion protein (Hoxa2 fused with an activation domain of a virion protein from herpes simplex virus) transactivates the luciferase expression by binding to GATG sites. RT-PCR and immunohistochemistry analysis revealed an upregulation of HtrA3 expression in Hoxa2-/- mice. This observation correlates with the inhibitory role of Hoxa2 protein acting upon the HtrA3 fragment in luciferase assays. Our data suggest that HtrA3 is a direct in vivo downstream target of Hoxa2 protein and support the activity regulation model in which Hox proteins selectively regulate target genes through occupation of multiple monomer binding sites.

Hox proteins are evolutionarily conserved transcription factors that control important developmental pathways in morphogenesis of the embryo. The Hoxa2 gene is expressed in the developing central nervous system in rhombomeres 2 to 7 and affects cellular differentiation. Few target genes of Hoxa2 protein have been identified so far and its mechanisms of regulating gene expression remain elusive. Previous work in our laboratory isolated Hoxa2 protein binding sequences from the E18 mouse spinal cord and hindbrain tissues using chromatin immunoprecipitation (ChIP). All isolated DNA fragments contain conserved GATG motifs. Sequence analysis revealed that one fragment belongs to the high temperature requirement factor A 3 (HtrA3) gene and another fragment belongs to the Dual specificity tyrosine kinase 4 (Dyrk4) gene. In this study, direct binding of Hoxa2 protein to the HtrA3 and Dyrk4 fragments was confirmed by electrophoretic mobility shift assays (EMSA). Site-directed mutagenesis and EMSA studies revealed that Hoxa2 protein binds to the multiple GATG motifs within these fragments. HtrA3 fragment also repressed luciferase gene expression in transient transfection and luciferase assays. Mutation of the DNA fragment showed that the repressive activity was affected by the GATG motifs, suggesting Hoxa2 protein regulated gene expression by binding to the GATG motif in the cis-regulatory element. In contrast to the inhibitory activity of Hoxa2 protein, a Hoxa2-VP16 fusion protein (Hoxa2 fused with an activation domain of a virion protein from herpes simplex virus) transactivates the luciferase expression by binding to GATG sites. RT-PCR and immunohistochemistry analysis revealed an upregulation of HtrA3 expression in Hoxa2-/- mice. This observation correlates with the inhibitory role of Hoxa2 protein acting upon the HtrA3 fragment in luciferase assays. Our data suggest that HtrA3 is a direct in vivo downstream target of Hoxa2 protein and support the activity regulation model in which Hox proteins selectively regulate target genes through occupation of multiple monomer binding sites.

Influenza A is one of the most prevalent and significant viral infections worldwide, resulting in annual epidemics and occasional pandemics. Upon infection, antiviral drugs targeting the neuraminidase protein and M2 protein are the only treatment options available. However, the emergence of antiviral drug resistance is concerning, therefore the aim of this work was to identify inhibitor molecules that may bind to highly conserved regions of selected internal influenza A proteins. Sequences of the non-structural protein 1 (NS1), nuclear export protein (NEP) and polymerase basic protein 2 (PB2) from all hosts and subtypes were aligned and the degree of amino acid conservation was calculated based on Valdar's scoring method. Missing parts of the experimental structures were predicted using the I-TASSER server and ligand binding hot spots were identified with computational solvent mapping. Selected binding sites in conserved regions were subjected to virtual screening against two compound libraries using AutoDock Vina and AutoDock 4. Two out of twelve top hit compounds predicted to target the NS1 protein showed capability of reducing influenza A H1N1 replication in plaque reduction assays at concentrations below 100 μM, although the target protein and mechanism of action could not be confirmed. For the NEP, conservation analysis was based on 3000 sequences and binding hot spots were located in common areas amongst three structures. Docking results revealed predicted binding affinities of up to -8.95 kcal/mol, and conserved amino acid residues interacting with top compounds include Arg42, Asp43, Lys39, Ile80, Gln101, Leu105, and Val109. For the PB2 protein, conservation analysis was based on ~12,000 sequences and fifteen potential binding hot spots were identified. Docking results revealed predicted binding affinities of up to -10.3 kcal/mol, with top molecules interacting with the highly conserved residues Gln138, Gly222, Ile539, Asn540, Gly541, Tyr531 and Thr530. The findings from this research could provide starting points for in vitro experiments, as well as the development of antiviral drugs that function to inhibit influenza A replication without leading to resistance.

The immunologic relatedness of various cofactor-binding sites of enzymes requiring different nucleotide cofactors was examined. Chicken antibodies specific for NADPH- or CoA-binding domains were raised using an NADPH- or CoA-requiring enzyme as an immunogen. Antibodies specific for either NADPH- or CoA-binding domains were isolated by immunoaffinity chromatography of the respective antisera using unrelated NADPH- or CoA-requiring enzymes as affinity ligands. The reactivities of the NADPH- and CoA-binding-site-specific antibodies with a variety of enzymes that required different cofactors was shown on Western blots of SDS-PAGE of the enzymes. Variable cross-reactivities were observed among all nucleotide-cofactor requiring enzymes with each specific cofactor-domain-antibody population. Numerous proteins not physiologically associated with nucleotide cofactors, including acyl carrier protein, were completely unreactive. Proteins that bound phosphoryl compounds either as substrates or cofactors showed varying degrees of reactivity with each population of specific antibodies. These included aldolase, ribulose-1,5-bisphosphate carboxylase/oxygenase, ribonuclease A, carbonic anhydrase and triosephosphate isomerase. The immunologic cross-reactivity suggested that these proteins share a common structural feature, probably a primary structure epitope, since the proteins had been subjected to denaturing polyacrylamide gel electrophoresis. A candidate for this common structural feature is a glycine-rich sequence comprising a phosphate binding loop.

No description available.

An increasing number of proteins with unknown function have their three-dimensional structure solved at high resolution. This situation, largely due to structural genomics initiatives, has been stimulating the development of automated structure-based function prediction methods. Knowledge of residues important for function – and more particularly – for binding can help automated prediction of function in different ways. The properties of a binding site such as its shape or amino acid composition can provide clues on the ligand that may bind to it. Also, having information on functionally important regions in similar proteins can refine the process of annotation transfer between homologues. Experimental results indicate that functional residues often have an unfavourable contribution to the stability of the folded state of a protein. This observation is the underlying principle of several computational methods for predicting the location of functional sites in protein structures. These methods search protein structures for destabilising residues, with the assumption that these are likely to be important for function. We have developed a method to detect clusters of destabilising residues which are in close spatial proximity within a protein structure. Individual residue contributions to protein stability are evaluated using detailed atomic models and an energy function based on fundamental physico-chemical principles. Our overall aim in this work was to evaluate the overlap between these clusters of destabilising residues and known binding sites in proteins. Unfortunately, reliable benchmark datasets of known binding sites in proteins are sorely lacking. Therefore, we have undertaken a comprehensive approach to define binding sites unambiguously from structural data. We have rigorously identified seven issues which should be considered when constructing datasets of binding sites to validate prediction methods, and we present the construction of two new datasets in which these problems are handled. In this regard, our work constitute a major improvement over previous studies in the field. Our first dataset consists of 70 proteins with binding sites for diverse types of ligands (e.g. nucleic acids, metal ions) and was constructed using all available data, including literature curation. The second dataset contains 192 proteins with binding sites for small ligands and polysaccharides, does not require literature curation, and can therefore be automatically updated. We have used our dataset of 70 proteins to evaluate the overlap between destabilising regions and binding sites (the second dataset of 192 proteins was not used for that evaluation as it constitutes a later improvement). The overlap is on average limited but significantly larger than random. The extent of the overlap varies with the type of bound ligand. Significant overlap is obtained for most polysaccharide- and small ligand-binding sites, whereas no overlap is observed for nucleic acid-binding sites. These differences are rationalised in terms of the geometry and energetics of the binding sites. Although destabilising regions, as detected in this work, can in general not be used to predict all types of binding sites in protein structures, they can provide useful information, particularly on the location of binding sites for polysaccharides and small ligands. In addition, our datasets of binding sites in proteins should help other researchers to derive and validate new function prediction methods. We also hope that the criteria which we use to define binding sites may be useful in setting future standards in other analyses.

Sphingolipids and ceramides are essential components of cellular membranes and important signalling molecules. Because of a growing appreciation for their diverse biological roles, understanding of the biosynthesis and regulation of sphingolipids has recently become a key goal in drug discovery. Serine palmitoyltransferase (SPT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyses the condensation between L-serine and a long-chain acyl thioester such as palmitoyl-CoA (C16-CoA). This first step in sphingolipid biosynthesis is conserved in all organisms studied to date, from microbes to man. The fungal natural product myriocin is a potent inhibitor of SPT; however, the molecular details of inhibition are not fully understood. Myriocin contains a long alkyl chain and a polar head group thus it displays features of both SPT substrates. Therefore, the prevailing hypothesis is that inhibition of SPT occurs because myriocin acts as a mimic of a key transition state of the catalytic mechanism. Through a combination of UV-vis spectroscopy, mass spectrometry, x-ray crystallography and enzyme inhibition assays it has been possible to study the interaction between S. paucimobilis SPT and myriocin. I have shown that myriocin initially forms an inhibitory PLP:myriocin aldimine complex in the active site that displays a Ki of 967 nM. Interestingly, this complex is susceptible to unexpected, slow enzymatic degradation. The mechanism for myriocin breakdown has been elucidated as a retro-aldol type reaction, which results in cleavage of the C2-C3 bond producing a C18 aldehyde. This aldehyde is then capable of covalently modifying the active site lysine265, forming a second (suicide) inhibitory complex and rendering the enzyme catalytically inactive. Substitution of the active site lysine produced SPT K265A, an inactive enzyme that did not catalyse the breakdown of the PLP:myriocin complex. However, the determination of the crystal structure of the SPT K265A:PLP-myriocin complex revealed that the myriocin had undergone decarboxylation. Nevertheless, this SPT:PLP:decarboxymyriocin structure revealed details about myriocin’s mechanism of inhibition for the first time. The novel mechanism of myriocin degradation has implications on the structure activity relationship (SAR) and design of drugs targeted towards SPT, the role of feedback regulation by long chain aldehydes and further expands the range of reactions catalysed by this important enzyme. As well as inhibition studies the structure of bacterial SPT was also examined by preparing an N-terminally truncated S. paucimobilis SPT. This version, shortened by 21 amino acids, was ~5-fold slower than the wild-type enzyme and suggests that the N-terminus may play a role in catalysis. Additional work has been undertaken to study an unusual membrane-bound viral SPT, composed of two naturally fused open reading frames (SPT2-SPT1) with the proposed SPT2 domain at the N-terminus and the SPT1 domain at the C-terminus. To study soluble mimics of this interesting fusion I prepared a bacterial S. paucimobilis SPT fused wild-type and mutant construct and isolated a fused SPT2-SPT1 with what appears to be single PLPbinding site.

<p>The genomes of several organisms have been sequenced and the need for methods to analyse the data is growing. In this project a method is described that tries to identify co-regulated genes. The method identifies transcription factor binding sites, documented in TRANSFAC, in the non-coding regions of genes. The algorithm counts the number of common binding sites and the number of unique binding sites for each pair of genes and decides if the genes are co-regulated. The result of the method is compared with the correlation between the gene expression patterns of the genes. The method is tested on 21 gene pairs from the genome of Saccharomyces cerevisiae. The algorithm first identified binding sites from all organisms. The accuracy of the program was very low in this case. When the algorithm was modified to only identify binding sites found in plants the accuracy was much improved, from 52% to 76% correct predictions.</p>

Multiple lines of evidence suggest that elevated plasma lipoprotein(a) (Lp(a)) concentrations are a significant risk factor for the development of a number of vascular diseases including coronary heart disease and stroke. Lp(a) consists of a low-density lipoprotein (LDL)-like moiety and an unique glycoprotein, apolipoprotein(a) (apo(a)), that is covalently attached to the apolipoproteinB-100 (apoB-100) component of LDL by a single disulfide bond. Many studies have suggested a role for Lp(a) in the process of endothelial dysfunction. Indeed, Lp(a) has been shown to increase both the expression of adhesion molecules on endothelial cells (EC), as well as monocyte and leukocyte chemotactic activity in these cells. We have previously demonstrated that Lp(a), through its apo(a) moiety, increases actomyosin-driven EC contraction which, as a consequence, increases EC permeability. In this thesis, we have demonstrated a role for the strong lysine-binding site in the kringle IV type 10 domain of apo(a) in increasing EC permeability, which occurs through a Rho/Rho kinase-dependent pathway. We have further validated these findings using mouse mesenteric arteries in a pressure myograph system. We also have dissected another major signaling pathway initiated by apo(a) that involves in a disruption of adherens junctions in EC. In this pathway, apo(a)/Lp(a) activates the PI3K/Akt/GSK3β-dependent pathway to facilitate nuclear translocation of beta-catenin. In the nucleus beta-catenin induced the expression of cyclooxygenase-2 (COX-2) and the secretion of prostaglandin E2 (PGE2) from the EC. Finally, we have presented data to suggest a novel inflammatory role for apo(a) in which it induces the activation of nuclear factor-kappaB through promotion of the dissociation of IkappaB from the inactive cytoplasmic complex; this allows the nuclear translocation of NFkappaB with attendant effects on the transcription of pro-inflammatory genes. Taken together, our findings may facilitate the development of new drug targets for mitigating the harmful effects of Lp(a) on vascular EC which corresponds to an early step in the process of atherogenesis. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2009-09-22 19:24:04.594

No description available.