Includes bibliographical references.

Includes bibliographical references. / Maize streak virus (MSV), circular single stranded DNA (ssDNA) virus (~2.7kb), is the causative agent of Maize streak disease, and is a devastating pathogen that causes severe crop losses to subsistence farmers in sub-Saharan Africa. MSV is transmitted by the leafhopper Cicadulina mbila, and is the type member of the Mastrevirus genus (family Geminiviridae). MSV shares a unique twinned icosahedral ("geminate") virion architecture (22 x 38 nm) with all other family members. Geminate particles consist of 110 coat protein (CP) subunits that assemble onto a circular single-stranded DNA (ssDNA) genome. Each T= I unit is an incomplete icosahedron assembled from 55 CPs. The structures of MSV and African cassava mosaic virus (ACMV, genus Begomovirus) have been studied by electron cryo- microscopy (cryo-EM) previously. While these investigations revealed some details about the geminate architecture, the interactions of capsid components have not yet been adequately modelled. The two incomplete icosahedral "heads" of the geminate particle are offset from one another and apparently make distinct CP:CP contacts at this region of the virion. Information regarding the nature of quasi- equivalent CP conformers or the sets of amino acid residues that mediate these interactions has not been forthcoming. Since the experimental results of these previous studies are not available in a public database, we were motivated to revisit the structure of MSV in order to obtain a 3D experimental density that might aid pseudo-atomic modelling. The MSV CP:ssDNA interaction has also been shown to be crucial for systemic movement through the host. Hence, quasi-atomic modelling may inform development of antiviral strategies which aim to interfere with virion assembly. MSV virions were isolated from the leaves of maize plants infected by agro-inoculation and visualized in both heavy metal stain and vitreous ice after they had been adsorbed to a thin-layer of continuous carbon to prevent virion aggregation. Virus preparations consisted of distinct CP assemblies consisting of multiples of the incomplete T=I icosahedral unit. Monopartite (icosahedral), bipartite (geminate), tripartite, and higher assemblies were observed suggesting the MSV CP is not only multifunctional but also structurally versatile being able to package ssDNA of variable sizes. Low-dose images were recorded on film, and 3D reconstruction of both monopartite and bipartite capsid species carried out using standard single-particle image processing methodology. The resolution of the bipartite reconstructions was 26 A for the negative-stain dataset, and 23 A for cryo-EM dataset, while the resolution of the monopartite reconstruction was estimated to be ~15 A. Comparative modelling of the MSV CP was undertaken using the pentamer (CPs) of Satellite tobacco necrosis virus (STNV) as a structural template. Correlation-based fitting of icosahedral and geminate atomic models that varied in geometric arrangement of MSV CPs allowed the geometric parameters of the bipartite capsid to be determined. Fitting ofMSV CPs into the EM densities informed our understanding of interfaces which allow the CP to self-associate, and showed that CPs is in fact displaced within the icosahedral geometry of the heads by a 10° rotation about the 5-fold axes of symmetry in comparison to STNV; hence, while quaternary structure of the pentameric capsomer is conserved between these viruses, the quaternary interactions between capsomers of the T=I unit has diverged considerably. This study shows that the offset between the geminate heads of the MSV virion is ~-11°, and that this geometry appears to arise owing to a distinct set of CP:CP interfaces which occur across the equator between two quasi-icosahedral heads and involve regions that would interact to form the CPs: CPs interfaces within each of the heads (across 2-fold and 3-fold symmetry axes). Notably this offset differs from that reported for ACMV, which has a reported offset of 20°. Additionally, the resolution afforded by the icosahedral monopartite reconstruction provided the first structural evidence to suggest that the calcium ion binding site of the STNV CPs (located on the CS axis) is likely to be conserved in MSV. This result suggests that in common with other plant viruses, depletion of calcium ions may be required for genome egress in a newly infected host cell. This study highlights the importance of future high-resolution studies of this unique virion morphology by both X-ray crystallography and cryo-EM.

Includes bibliographical references. / Angiotensin-converting enzyme (ACE), a zinc metalloprotease, is a key regulator of the mammalian renin-angiotensin system (RAS) Primarily, ACF is a dipeptidl peptidase which cleaves angiotensin I to produce angiotensin II, a potent vasoconstrictor. By the same enzymatic mechanism, ACE also inactivates the vasodilator bradykinin. The main overall effect of these actions is an increase in blood pressure. Several ACF inhibitors have been developed as drugs for the treatment of myocardial infarction, hypertension, kidney failure and heart failure.

Includes bibliographical references (leaves 123-136). / Amidases are a group of carbon-nitrogen hydrolysing enzymes that catalyze the conversion of amides to corresponding carboxylic acids and ammonia. These enzymes are of great interest in synthetic industries where they are used for mass production of acidic products. Aliphatic amidase from Geobacillus pallidus RAPcS (RAPcS amidase), which belongs to the nitrilase superfamily of enzymes, has recently been characterised biochemically. It shows both amide hydrolysis and acyl transfer activities, and also exhibits stereo selectivity for some enantiomeric substrates. This enzyme can therefore be exploited in large-scale production of enantio-pure compounds. Structural characterization of this amidase would yield insights into the basis of this substrate selectivity and activity. This would inform future experiments that aims at modifying this enzyme to alter its substrate specificity. This work presents structural characterization of RAPcS amidase. Gel filtration chromatography and electron microscopic analyses provided useful information on the quaternary structure of RAPcS amidase. Crystals were grown, and an X-ray diffraction dataset to 1.9 Å collected using an in-house X-ray source. The space group of this data was determined to be primitive cubic P4₂32, and the structure was solved by molecular replacement using the backbone of the hypothetical protein PH0642 from Pyrococcus horikoshii (PDB ID, Ij31) that had all non-identical side chains substituted with alanines, as a search probe. The molecular replacement rotational and translational searches were performed using PHASER. The model was rebuilt with PHENIX before refinement using REFMAC5. The final model was of high quality with minimal errors. RAPcS amidase is homohexameric in solution and has a four-layer α-β-β-α structural fold that highly resembles nitrilase superfamily enzymes. It has an extended C-terminal tail that is essential for strengthening the interacting dimer interfaces by participating in domain swapping. The active site pocket has Glu, Lys, Cys catalytic triad that is conserved in the nitrilase superfamily. The substrate binding pocket is small in size, explaining the specificity of this enzyme for short aliphatic amides. These findings have made steps towards understanding the catalytic mechanism, and the basis for substrate specificity in this enzyme. It has also provided useful information on the overall structure, as well as the structure of the active site, not only for RAPcS amidase but also for related enzymes, which will form the basis for designing future structural characterization work in the nitrilase-related amidases.

The bacterial type six secretion system (T6SS) is an injectisome responsible for the translocation of effector molecules directly into host cells or competing bacteria. The system is widely distributed among proteobacteria and is found in both clinically relevant strains as well as environmental stains and represents an important system for the study of both microbial ecology and virulence. The apparatus itself is believed to have arisen from a combination of genes from bacteria and bacteriophage due to seqeuence and structural identity between T6SS components and structural bacteriophage proteins. The current model of the T6SS apparatus consists essentially of an inverted phage body that is attached to the donor cell membrane complex. The phage-like structure can contract and force a sharp needle point complex along with effector proteins into the target cell. The phage derived components have received a considerable amount of attention and the mode of assembly is relatively well understood. However, little detailed information on the assembly and function of the membrane embedded complex is available. The first major goal of this thesis was to structurally characterize the proteins of the membrane embedded complex of the type six secretion system. The structures of IglE and TssL from Francisella sp. were solved and represent a platform for further characterization of the T6SS assembly and function. The periplasmic domain of a TssL homologue from P. aeruginosa was also solved and this structure represents a subset of evolved TssL proteins that bind peptidoglycan through an unknown mechanism. Biochemical and structural analysis probed this system but came short of a definitive model for peptidoglycan binding. However, the data collected from this study will further the field of peptidoglycan binding modules and help to characterise differences among T6SSs. The translocated proteins of the T6SS are often bactericidal and attack the peptidoglycan, lipid bilayer or DNA of the target cell. However, one secreted substrate, Tse2 from Pseudomonas aeruginosa is targeted to other neighbouring cells of the same species. This toxin shares no sequence identity with any known protein but has been shown to be toxic to not only bacteria but also yeast and mammalian cells. The structure of the complex between Tse2 and its immunity protein was solved and led to two interrelated discoveries. The first was that the molecular details behind the immunity protein inhibiting Tse2 where it binds directly to the active site. The second was that based on structural identity with ADP-ribosylating toxins, the active site of Tse2 was identified. These results carry the study of this protein forward significantly although the precise function of Tse2 remains unknown. This structure is the first co-structure of a cytotoxic T6SS substrate and has significant implications for the cell in terms of handling the toxin for delivery rather than self intoxication. / Graduate

No description available.

Interferon (IFN) regulatory factor family member 4 (IRF4) is a transcription factor that serves specific roles in transcriptional regulation of IFN responsive genes and is indispensable in B- & T-cell differentiation. IRF4 like the other members of the family has two major domains- the N-terminal DNA binding domain (DBD) essential for its recognition and binding to the Interferon Stimulated Response Element DNA sequence and a C-terminal Interferon activation domain (IAD) thought to maintain IRF4 in an auto-inhibited inactive state and is also critical in its activation. A putative unstructured linker connects the DBD and IAD. Activation in most members of the IRF family requires phosphorylation to induce homo and hetero-dimerization. In contrast, IRF4 functions primarily through ternary complex formation involving different proteins including PU.1 and MyD88. The IRF4IAD has a C-terminal auto-inhibitory region (AIR) that has been proposed to physically impede the DBD from interacting with DNA in the absence of its binding partner. To understand the activation mechanism in molecular detail we determined the crystal structure of the IAD of IRF4 and also performed small-angle X-ray scattering (SAXS) studies. Our data reveals that the surface electrostatics of IAD and presence of additional loops confers exclusivity to IRF4 in the IRF family. SAXS studies suggest that the AIR is structured and makes interactions with the putative linker. We also performed analytical ultracentrifugation studies, fluorescence anisotropy binding experiments and SAXS studies on full-length IRF4 as well as on constructs where the first 20 residues, exclusive to IRF4 or the AIR were removed. We observe that the first 20 residues are critical in decreasing the binding affinity of full-length IRF4 to DNA. In addition, the putative linker of IRF4 connecting the N- and C-termini appears to be a folded domain and interacts with AIR. Also, overall full-length IRF4 appears as an elongated molecule and the N- and the C-terminal domains are arranged on either ends of full-length IRF4. Moreover, there are no signs of huge conformational changes in the protein during the activation process. Taken together, based on our data we propose that there is no auto-inhibited state for IRF4. Furthermore, it is the binding affinity of full-length IRF4 that is increased in the presence of its binding partner most likely through modest conformational changes.

In summary, the main points to support the view that dextran synthesis is unessential to the dental caries process are presented below: 1 . Dextran-fed, microbe-bearing rats do not experience dental caries. 2 . Lactobacillus casei 4646 has no extracellular dextransucrase enzyme or other extracellular enzymes which release glucose or fructose from sucrose . 3. L. casei 4646 does not synthesize extracellular polysaccharide of the dextran class when grown on sucrose-containing medium. 4. L. casei 4646 does not exhibit in vitro, adherent growth on stainless-steel wire when grown on sucrose-containing medium . 5. L. casei 4646 does not cause dental plaque formation on the molars of sucrose-fed gnotobiotic rats (Rosen, Lenney, and O'Malley, 1968). 6. L. casei 4646 induces dental caries of the pit and fissure class when introduced as a monoinfecting agent in sucrose-fed gnotobiotic rats (Rosen, Lenney, and O'Malley, 1968). These observations advocated rejection of the hypothesis that extracellular dextran is an absolute requirement in the dental caries process. Therefore, dextranase will be an effective anticaries agent only against organisms such as Streptococcus E-49 (Fitzgerald, Keyes, Stoudt, and Spinell, 1968) which depend on the synthesis of dextran from sucrose for tooth colonization. This position predicts that dextranase treatment of sucrose-fed gnotobiotic rats infected with Lactobacillus casei 4646 would have no effect on the rats' caries experience.

β-arrestin2, previously recognized as a facilitator for G-protein associated 7 TMR desensitization/ internalization, has now been appreciated as an independent signal transducer that regulates multiple cellular responses including inflammation. Cecal ligation and puncture procedure (CLP) induced septic shock is an acute inflammatory response characterized by uncontrolled systemic inflammation. Myocardial ischemia/reperfusion is a chronic sterilize inflammation that requires the reaction of macrophages, fibroblasts and cardiac stem cells for regeneration and remodeling of the infarcted myocardium. Restrained chronic stress is an immune suppression model in which the inactivation of macrophages may be involved. Here we showed β-arrestin2 overexpression inhibited CLP-induced heart dysfunction in septic shock, stabilized the cardiovascular system, and eventually promoted survival. Inhibition of the activation of p38 that downstream of the IL-6 pathway may be a key regulatory target for β-arrestin2. To rescue cardiomyocytes from ischemia and reperfusion injury, Sca-1+ CSC from Wide-type or β-arrestin2 Knockout mice were delivered to the risked area before reperfusion; β-arrestin2 was shown to be a required factor and a promoter for the differentiation of the cardiac stem cells. A β-arrestin2/miR-155/GSK3β pathway was identified in this study. TLR-9 is an important part of the innate immune system which has been shown to be regulated by β-arrestin2 in various inflammatory models. Here we found, the immune suppression induced by restrained stress is mediated by Toll-like receptor 9 (TLR-9). TLR-9 facilitated the elevation of IL-1β, IL-10 and IL-17 levels in serum and decrease of the levels of plasma IFN-γ. Furthermore, macrophage apoptosis was alleviated in TLR-9 deficiency mice. In summary, β-arrestin2 and associated proteins like TLR-9 are important regulators of the immune response in a variety of disease conditions. Therapeutic strategies should be generated to balance the inflammation and anti-inflammation response by modulating β-arrestin2 expression and functions.

The 1839 bp cDNA for rat pulmonary carboxylesterase was cloned by reverse transcription polymerase chain reaction (RT PCR) from total rat lung RNA using specific primers derived from the 5' and 3' untranslated regions of rat hepatic cholesteryl ester hydrolase (CEH). The unique cDNA was sequenced and found to have 99% homology with hepatic CEH. This homology extends to the predicted amino acid sequences which show only six amino acid residue differences in the coding region: three conserved and three nonconserved changes. However, the catalytic activitites of the two proteins are dramatically different. While CEH hydrolyzes cholesterol oleate, the pulmonary carboxylesterase has no activity towards this substrate. The active recombinant lung carboxylesterase was purified using a baculovirus expression system. The substrate specifities were determined using p-nitrophenyl acetate, p-nitrophenyl caprylate and cholesterol oleate. Also, the KM, Vmax and pH optima were determined for each substrate. Comparison of the substrate specificities of the recombinant pulmonary carboxylesterase with the recombinant CEH further establish the critical role of the six amino acid residues in determining the differences in the catalytic activities of these two proteins. Cumulative mutations were made in the lung carboxylesterase sequence to those of the hepatic CEH sequence, in order to determine the role(s) of these six amino acid residues in conferring cholesterol oleate hydrolytic activity. These studies showed that GIn186 is vital for activity towards hydrophilic substrates, while the region around amino acid residue 500 consisting of Ser491, Lys492, Asn506 and Asn504 may be responsible for the absence of catalytic activity towards hydrophobic substrates.