"The class IX myosin is a member of the myosin superfamily and found in variety of tissues. Myosin IX is quite unique among the myosin superfamily in that the tail region contains a GTPase activating protein (GAP) domain for the small GTP-binding protein, Rho. Recently it was reported that myosin IX shows processive movement that travels on an actin filament for a long distance. This was an intriguing discovery, because myosin IX is a “single-headed” myosin unlike other processive myosins which have “double-headed” structure. It has been thought that “processive” motors walk on their track with their two heads, thus traveling for a long distance. Therefore, it is reasonable to expect that the processive movement of single headed myosin IX is based on the unique feature of myosin IX motor function. In this study, I investigated the mechanism of processive movement of single-headed myosins by analyzing the mechanism of ATPase cycle of myosin IX that is closely correlated with the cross-bridge cycle (the mechanical cycle of actomyosin). In the first part, I performed the transient enzyme kinetic analysis of myosin IX using the motor domain construct to avoid the complexity raised by the presence of the tail domain. It was revealed that the kinetical characteristics of myosin IX ATPase is quite different from other processive myosins. It was particularly notable that the affinity of the weak actin binding state of Myosin IX was extremely high comparing with known myosins. It is thought that the high affinity for actin throughout the ATPase cycle is a major component to explain the processive movement of myosin IX. In the second part of this study, I cloned full length human myosin IX construct to further investigate the regulation of motor activity of myosin IX. It was revealed that the basal ATPase activity but not the actin dependent ATPase activity of myosin IX is inhibited by its tail region. Furthermore full-length myosin IX is regulated by calcium, presumably due to the calcium binding to the CaM light chain. These result suggest that the tail domain serves as a regulatory component of myosin IX."

Peptide hormones are critical modulators of physiology and development in humans and have been well characterized for their effects on humans and other mammals. The question of the origin of the many families of peptide hormones in mammals is pressing, as it gives us a window into the evolution of important systems in all extant animals and their common ancestors. The focus of this thesis was to examine the origin of a select group of peptide hormone families including the secretin superfamily, reproductive neuropeptides, insulin and the insulin-like peptides, and stanniocalcin. The evolution of the secretin superfamily was found to have originated with the vertebrates, and new information from the genomes of basal vertebrates like the lamprey Petromyon marinus and elephant shark Callorhinchus milii allows us to better piece together the gene duplications that produced the current hormone family in humans and fish. The reproductive hormones, including gonadotropin-releasing hormone (GnRH), vasopressin/oxytocin, and kisspeptin were examined, with a focus on the evolution of their G protein-coupled receptors. GnRH was found to have originated in the early bilaterians, and its receptors clearly belong to a superfamily also containing receptors of the related neuropeptides adipokinetic hormone and corazonin, which have only been found in protostome invertebrates. Vasopressin/oxytocin receptors share a common ancestor with the GnRH receptors, although their peptides are not structurally related, and evolved at a similar time. Kisspeptin evolved later, within the vertebrates, however its receptors are closely related to an orphan receptor in protostome invertebrates, GPR54, with an unknown ligand. Insulin family members from the tunicate Ciona intestinalis and the amphioxus Branchiostoma floridae were identified, isolated and characterized to determine the nature of the insulin superfamily at the origin of the chordates, and it appears this family was well-developed already. Finally, the calcium-regulator stanniocalcin was identified, isolated and characterized in C. intestinalis and compared with the vertebrate and amphioxus stanniocalcins. A group of stanniocalcins were also discovered in a wide range of both protostomes and unicellular eukaryotes, indicating this ancient group of neurohormones appeared early in eukaryotic evolution. / Graduate

No description available.

The tumour necrosis factor (TNF) superfamily is a group of cytokines that orchestrate a variety of functions, both in the development of the architecture of immune organs and of the immune response. The mammalian TNF superfamily consists of 19 ligands and 29 receptors, whereas in the chicken only 10 ligands and 15 receptors are present. Chickens do not develop lymph nodes, possibly due to the absence of the lymphotoxin genes (TNF superfamily members) in their genome. New members of the chicken TNF superfamily have recently been identified in the genome, namely chicken receptor activator of NF-κB ligand (chRANKL), its signalling receptor, chRANK, and its decoy receptor, osteoprotegerin (chOPG). In mammals, RANKL and RANK are transmembrane proteins expressed on the surface of Th1 cells and mature dendritic cells (DC), respectively. OPG is expressed as a soluble protein from osteoblasts and DC, regulating the interaction between RANKL and RANK. To investigate the bioactivity of this triad of molecules, the extracellular soluble domains of chRANKL and chRANK and full-length chOPG were identified and cDNAs cloned. ChRANKL, chRANK and chOPG mRNA are ubiquitously expressed across non-lymphoid and lymphoid tissues and immune cells in the chicken. Similar to mammals, chRANK and chOPG mRNA expression levels are upregulated in mature bone marrow-derived DC (BMDC). ChRANKL transcription is regulated by Ca2+-mobilisation and is further enhanced by the activation of the protein kinase C pathway, as seen in mammals. The biological activities of chRANKL, chRANK and chOPG were investigated by the production of recombinant soluble fusion proteins. The extracellular, TNF-homology, domain of chRANKL (schRANKL) was sub-cloned into a modified pCI-neo vector expressing an in-frame isoleucine zipper to encourage trimer formation. FLAG-tagged schRANKL produced in COS-7 cells predominantly forms homotrimers and chOPG is expressed as homodimers, both signatures of their mammalian TNF superfamily orthologues. SchRANKL enhances the mRNA expression levels of pro-inflammatory cytokines in mature BMDC and BM-derived macrophages (BMDM). Pre-incubation with soluble chRANK-Fc or chOPG-Fc blocked the schRANKL-mediated increase in pro-inflammatory cytokine mRNA expression levels in BMDC. Expression of surface markers on BMDC and BMDM were not affected by schRANKL treatment. SchRANKL enhances the survival rates of BMDC and BMDM and can drive osteoclast differentiation from monocyte/macrophage progenitor cells. The chRANKL signalling receptor, chRANK, does not contain an intracellular catalytic domain but requires the binding of intracellular TNF receptor-associated factors (TRAF) to initiate signalling. TRAFs are a family of seven proteins (TRAF1-7) grouped due to their highly conserved RING domains, zinc finger domains, TRAF-N and TRAF-C domains. ChRANK possesses four of the five TRAF peptide-binding motifs found in mammalian RANK. The "missing" chRANK TRAF peptide-binding motif is TRAF6-specific, a vital protein for RANKL-mediated osteoclastogenesis. All seven members of the mammalian TRAF family are present in the chicken genome. To investigate the conservation of RANK-specific TRAF signalling proteins, chicken TRAF2 (chTRAF2), chTRAF5, chTRAF6 and a newly found member, chTRAF7, were identified and their cDNAs cloned. ChTRAF5, chTRAF6 and chTRAF7 had mRNA expression patterns, in non-lymphoid and lymphoid tissues and in a number of immune cells, similar to their orthologues in mammals. Interestingly, chTRAF2 has two variants, the full-length chTRAF2 and a novel isoform (chTRAF2S) lacking exon 4. ChTRAF2S lacks a portion of zinger finger one, all of zinc finger two and a portion of zinc finger three, producing a protein with a hybrid of zinc fingers 1 and 3 and intact zinc fingers 4 and 5. RT-PCR analyses indicated differential expression of both of the chTRAF2 isoforms in a number of non-lymphoid and lymphoid tissues, splenocyte subsets and in a kinetic study of ConA-stimulated splenocytes. ChTRAF2S is biologically active compared to chTRAF2, inducing higher levels of NF-κB activation. Co-transfections indicate that chTRAF2 may regulate chTRAF2S bioactivity as no synergistic effect was identified when cells were transfected with both isoforms. Knowledge gained from this study will help work to further dissect the interactions between chRANKL-expressing T cells and chRANK-expressing DC to drive Th1 immune responses and to understand how the chicken mounts an effective immune response while expressing a minimal essential repertoire of the TNF superfamily.

The Glyoxalase system is composed of two metalloenzymes, Glyoxalase I and Glyoxalase II, that catalyze the conversion of toxic, metabolically produced alpha-ketoaldehydes, such as methyglyoxal, in the presence of a thiol cofactor, such as glutathione, into their corresponding nontoxic 2-hydroxycarboxylic acids, leading to detoxification of these cellular metabolites. Previous studies on the first enzyme in the Glyoxalase system, Glyoxalase I (GlxI), in yeast, protozoa, animals, human, plants and Gram-negative bacteria suggest two metal activation classes, zinc-activation or non-zinc-activation (but exhibiting selective nickel/cobalt-activation). This thesis provides the key discoveries of the Glyoxalase system from Gram-positive microorganisms using the major thiol cofactor/cosubstrate that produced within that particular organisms as well as the relatedness of the proteins in the same beta-alpha-beta-beta-beta protein superfamily.

The amidohydrolase superfamily is a functionally diverse set of enzymes that catalyzes predominantly hydrolysis reactions involving sugars, nucleic acids, amino acids, and organophosphate esters. A more divergent member of this superfamily, URI (uronate isomerase) from Escherichia coli, catalyzes the isomerization of D-glucuronate to D-fructuronate and D-galacturonate to D-tagaturonate. In Bacillus halodurans, two distinct operons were identified for the metabolism of D-glucuronate and D-galacturonate based on kinetics and genomic context. The canonical uronate isomerase is encoded by the gene Bh0705. A second URI in this organism, Bh0493, is the outlier of the group in terms of sequence similarity. Kinetic evidences indicate that Bh0705 is relatively specific for the isomerization of D-glucuronate, while Bh0493 is specific for the Dgalacturonate pathway. Bell-shaped pH-rate profiles were observed for the wild type URI from Escherichia coli. Primary isotope effects with [2-2H]-D-glucuronate and solvent viscosity studies are consistent with product release as the rate limiting step. X-ray structure of Bh0493 was determined in the presence of D-glucuronate. A chemical mechanism is proposed that utilizes a proton transfer from C-2 of D-glucuronate to C-1 that is initiated by the combined actions of Asp-355 and the C-5 hydroxyl of the substrate that is bound to the metal ion. The formation of the cis-enediol intermediate is further facilitated by the shuttling of the proton between the C-2 and C-1 oxygens by the conserved Tyr-50 and/or Arg-357. Another divergent member of the AHS is the enzyme renal dipeptidase. Structural studies of the enzyme from Streptomyces coelicolor (Sco3058) demonstrate that the active site consists of a binuclear metal center. Bell-shaped pH-rate profiles are observed for both Zn2+ and Cd2+ enzymes. A chemical mechanism for renal dipeptidase is proposed based on structural analysis of the enzyme-inhibitor complex. The reaction is initiated by the polarization of the amide bond by the B-metal. Asp-320 activates the bridging hydroxide for nucleophilic attack at the peptide carbon center, forming a tetrahedral intermediate that is stabilized by the metal center and His-150. The protonated Asp-320 donates the proton to the a-amino group of the leaving group, causing the collapse of the tetrahedral intermediate and cleavage of the carbon-nitrogen bond.

The Glyoxalase system is composed of two metalloenzymes, Glyoxalase I and Glyoxalase II, that catalyze the conversion of toxic, metabolically produced alpha-ketoaldehydes, such as methyglyoxal, in the presence of a thiol cofactor, such as glutathione, into their corresponding nontoxic 2-hydroxycarboxylic acids, leading to detoxification of these cellular metabolites. Previous studies on the first enzyme in the Glyoxalase system, Glyoxalase I (GlxI), in yeast, protozoa, animals, human, plants and Gram-negative bacteria suggest two metal activation classes, zinc-activation or non-zinc-activation (but exhibiting selective nickel/cobalt-activation). This thesis provides the key discoveries of the Glyoxalase system from Gram-positive microorganisms using the major thiol cofactor/cosubstrate that produced within that particular organisms as well as the relatedness of the proteins in the same beta-alpha-beta-beta-beta protein superfamily.

Metal ions play essential roles in biological functions including catalysis, protein stability, DNA-protein interactions and cell signaling. It is estimated that 30% of proteins utilize metals in some fashion. Additionally, methods by which metal ions can be visualized have been utilized to study metal concentrations and localizations in relation to disease. Understanding the roles metals play in biological systems has great potential in medicine and technology. Chapters 1 and 2 of this dissertation analyzes the structure and function of the Mn-dependent enzyme oxalate decarboxylase (OxDc) and Chapter 2 presents a bioinformatic analysis of the cupin superfamily that provides the structural scaffold of the decarboxylase. The X-ray crystal structure of the W132F variant was determined and utilized together with EPR data to develop a computational approach to determining EPR spectra of the enzyme’s two metal-binding centers. Furthermore, a variant in which the catalytic Glu162 was deleted revealed the binding mode of oxalate, the first substrate-bound structure of OxDc. OxDc is a member of the cupin superfamily, which comprises a wide variety of proteins and enzymes with great sequence and functional diversity. A bioinformatics analysis of the superfamily was performed to analyze how sequence variation determines function and metal utilization. Chapters 3 and 4 discuss the expansion of lanthanide-binding tags (LBTs) to in cellulo studies. Lanthanide-binding tags are short sequences of amino acids that have high affinity and selectivity for lanthanide ions. An EGF-LBT construct used to quantify EGF receptors on the surface of A431 and HeLa cells. The results from the LBT quantification are consistent with previous studies of EGFR receptors in these cell types, validating the use of this method for future studies. The potential of using LBTs for X-ray fluorescence microscopy (XFM) was also investigated. LBT-labeled constructs were utilized to investigate if membrane bound as well as cytosolic LBT-containing proteins could be visualized and localized to their cell compartments via XFM; both membrane-localized and cytosolic proteins were successfully visualized. With the high resolution (< 150 Å) obtainable with new synchrotron beamline configurations LBTs could be used to study nanoscale biological structures in their near-native state.

abstract: Here I document the breadth of the CAP (Cysteine-RIch Secretory Proteins (CRISP), Antigen 5 (Ag5), and the Pathogenesis-Related 1 (PR)) protein superfamily and trace some of the major events in the evolution of this family with particular focus on vertebrate CRISP proteins. Specifically, I sought to study the origin of these CAP subfamilies using both amino acid sequence data and gene structure data, more precisely the positions of exon/intron borders within their genes. Counter to current scientific understanding, I find that the wide variety of CAP subfamilies present in mammals, where they were originally discovered and characterized, have distinct homologues in the invertebrate phyla contrary to the common assumption that these are vertebrate protein subfamilies. In addition, I document the fact that primitive eukaryotic CAP genes contained only one exon, likely inherited from prokaryotic SCP-domain containing genes which were, by nature, free of introns. As evolution progressed, an increasing number of introns were inserted into CAP genes, reaching 2 to 5 in the invertebrate world, and 5 to 15 in the vertebrate world. Lastly, phylogenetic relationships between these proteins appear to be traceable not only by amino acid sequence homology but also by preservation of exon number and exon borders within their genes. / Dissertation/Thesis / Masters Thesis Biology 2016

NtdB is an enzyme encoded within the ntd operon in Bacillus subtilis. This operon is reported to contain a complete set of genes necessary for the biosynthesis of 3,3'-neotrehalosadiamine (NTD), a compound composed of two kanosamine subunits linked together by a 1,1'-(α,β)-linkage. Both NTD and kanosamine have reported antibiotic properties. The function of NtdB has been a matter of speculation, but has never been investigated in vitro. Using a phosphate assay and an array of substrates, NtdB was determined to be a phosphatase, specific to kanosamine-6-phosphate (K6P) (kcat = 32 ± 1 s-1, Km = 93 ± 7 µM). Site-directed mutagenesis of amino acid residues in the core and the cap domains of the enzyme identified residues important for the catalytic reaction and substrate specificity. These mutations confirmed the presence of four motifs, characteristic of members of the haloacid dehalogenase (HAD) superfamily, and allowed identification of the substrate binding site of the enzyme. KabB, a homologue of NtdB from Bacillus cereus, showed notably lower activity with K6P than NtdB. This research defines the role of NtdB as a specific K6P phosphatase and challenges the previously reported NTD biosynthesis pathway by demonstrating a novel pathway for the production of the antibiotic kanosamine.