WWOX is a tumor suppressor and the down-regulation of WWOX has been demonstrated in prostate, lung, breast, gastric cancers. However, the role of WWOX in renal cell carcinoma (RCC) remains unknown. It has been demonstrated that WWOX addressed in mitochondria, golgi apparatus, rough ER, lysosome, plasma membrane and nuclear. The Subcellular localization of WWOX has been controversial. There are two parts in this study: (I) The expression of WWOX in RCC and the probability of WWOX to be a diagnostic and a prognostic marker. (II) The regulation of WWOX by phosphorylation. For the study of WWOX expression in RCC, we prepared polyclonal WWOX antibody and characterized the specificity of the antibody. We applied this specific antibody to 33 NT paring RCC tissue specimen for immunoblotting study and 138 cases of paraffin-embedded specimens for IHC, respectively. Our results demonstrated that hWOX1 was specifically down-regulated in clear cell type RCC (p=0.018). The percentage of down-regulation in patient specimen is 60.7 % and 90.7 % in immunoblotting and IHC study, respectively. And in clear cell and clear-granular combined type RCC, down-regulation of WWOX was significantly correlated with the survival rate of patients (p=0.0482). Therefore, WWOX could be used as a diagnostic and a prognostic marker in clear cell type RCC. Besides, we performed bioinformatics to predict the phosphorylation site of WWOX and investigated the effect of phosphorylation on WWOX subcellular localization. Our results demonstrated that hWOX1 was phosphorylated by PKC at Thr49 and Thr102 and the phosphorylation regulated the subcellular localization of WWOX.

Epidemiological evidence has correlated consumption of sorghum with reduced incidences of gastrointestinal (GI) tract cancer, especially esophageal cancer. There is little evidence on how phenols of sorghum may affect chemoprevention. Seventeen sorghum varieties were screened for phenolic profiles and antioxidant capacity. The ability of crude sorghum extracts to induce NAD(P)H:quinone oxidoreductase (QR, a phase II protective enzyme), and inhibit proliferation of colon (HT-29) and esophageal (OE33) carcinoma cells using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) and PicoGreen assays, were determined in vitro. 3- Deoxyanthocyanidins, apigeninidin, luteolinidin and their methoxylated derivatives were also investigated for antioxidant capacity, QR inducing and antiproliferative potential. Tannin sorghum generally showed higher antioxidant capacity than non-tannin sorghum varieties. Sorghum varieties containing extractable condensed tannins did not show any significant QR inducing potential; on the other hand, non-tannin sorghums increased QR activity by 1.5-2.7 times; black sorghum (Tx430) was most potent (doubled QR activity at 25 mg/mL, 2.7-fold increase at 100 mg/mL). All sorghum extracts showed relatively strong antiproliferation activity with IC50s (the concentration needed to inhibit cancer cell growth by 50%) of 49.7-883 mg/mL. Tannin-containing sorghums had stronger cancer cell proliferation inhibitory potential (IC50s 49.7-131 mg/mL) than non-tannin sorghums (IC50s 141-883 mg/mL). Total phenol content and antioxidant capacity of crude sorghum extracts positively correlated with their antiproliferative potential (r2 0.71-0.97). Among tested 3-deoxyanthocyanidins, methoxylation on A-ring improved QR inducing potency. 5,7-Dimethoxyluteolinidin had the greatest QR inducing potency (4.3- fold at 100 mM). Methoxylation also improved their antiproliferation potential; the IC50s trend was di-methoxylated (8.3-105 mM) > mono-methoxylated (40.1-192 mM) > apigeninidin and luteolinidin (81.5-284 mM). This study provides information for how phenolic compositions of sorghum and 3-deoxyanthocyanidin structure affect their capacity to induce QR activity and inhibit GI tract cancer cell proliferation. The information is useful to promote the utilization of sorghum in functional foods, beverages, dietary supplements, and other health-related industries. Further study will focus on, fractioned and isolated sorghum phenols, the effect of food processing on their chemopreventive potential, as well as cellular mechanisms involved.

Despite the importance of molecular recognition in nearly all aspects of protein function, the determinants of specificity for enzyme-substrate and protein-protein interactions are poorly understood. The majority of these complexes involving bacterial luciferase from V. harveyi have yet to be fully characterized. Luciferase catalyzes the reaction of molecular oxygen, FMNH2 and a long-chain aliphatic aldehyde yielding FMN, the corresponding carboxylic acid and blue-green light. In vivo, luciferase is thought to obtain FMNH2 following transfer from a transiently associated oxidoreductase. To identify the oxidoreductase responsible for providing FMNH2 in E. coli, bioluminescence was compared using single gene deletion strains deficient in either a homolog to the endogenous V. harveyi oxidoreductase (Frp) or an oxidoreductase distantly related to luxG from V. fischeri (Fre). Fre is responsible for reducing flavin in vivo but does not physically interact with luciferase. The association between luciferase and the flavin product is also described. Luciferase was crystallized and subjected to soaking with high concentrations of FMN. A model was obtained for luciferase bound to FMN. Using molecular dynamics, models for the enzyme:aldehyde, enzyme:FMNH2, and luciferase bound to several reaction intermediates are presented. Finally, a conserved loop region adjacent to the active center was investigated for the ability to facilitate protein:protein interaction between luciferase and the endogenous Frp oxidoreductase. Following alanine mutagenesis of the charged residues throughout this loop, it appears that the residues targeted by this study are not components of a docking platform but facilitate a lid-gating mechanism of paramount importance for catalytic function.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21137号 / 農博第2263号 / 新制||農||1057(附属図書館) / 学位論文||H30||N5111(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 三芳 秀人, 教授 宮川 恒, 教授 加納 健司 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

No description available.

Oxidative decarboxylation of pyruvate is a fundamental reaction of living organisms in general, leading to energy conservation. In some anaerobic or microaerophilic eukaryotic or prokaryotic organisms pyruvate decarboxylation is carried out by a single enzyme, pyruvate:ferredoxin oxidoreductase (PFO). PFO contains Fe-S clusters and thiamin pyrophosphate cofactor (TPP). In the reaction catalyzed by PFO, from pyruvate and Co-A arise acetyl-CoA, CO2, and two electrons are released. Those electrons are accepted by low molecular carrier proteins. Most frequently these proteins are ferredoxins or flavodoxins such as in nitrogen fixating bacteria. PFO can perform a reversible reaction. Trichomonads are mostly parasitic or endosymbiotic organisms with mitochondria-like organelles, hydrogenosomes. These organelles possess PFO which is one of the key enzymes in the metabolism of Trichomonas vaginalis hydrogenosomes. PFO of T. vaginalis, a sexually transmitted pathogen of man, plays also a role in a term of medical importance. PFO is, by a universally accepted concept, one of the key proteins acting in the activation of antimicrobial drugs against trichomoniasis 5-nitroimidazoles, including metronidazole. In the genome of T. vaginalis seven PFO genes were identified. They were named PFO A, B1, B2, C, D, E and...

Extreme thermophiles and hyperthermophiles are microorganisms capable of growing optimally at 65-79??C and 80??C plus, respectively. Many of the enzymes isolated from them are thermostable, which makes them a potential resource for research and industrial applications. An increasing number of hyper/thermophiles is shown to be able to produce ethanol as an end-metabolite. Despite characterization of many alcohol dehydrogenases (ADHs) with a potential role in the production of ethanol, to date there has been no significant progress in identifying the enzymes responsible for the production of acetaldehyde, which is an intermediate in production of ethanol from pyruvate.<br> Pyruvate decarboxylase (PDC encoded by pdc) is a thiamine pyrophosphate (TPP)-containing enzyme responsible for conversion of pyruvate to acetaldehyde in many mesophilic organisms. However, no pdc/PDC homolog has yet been found in fully sequenced genomes of hyper/thermophiles. The only PDC activity reported in hyperthermophiles is a bifunctional, TPP- and CoA-dependent pyruvate ferredoxin oxidoreductase (POR)/PDC enzyme from the hyperthermophilic archaeon Pyrococcus furiosus.<br> The bifunctional and TPP-containing POR/PDC enzyme was isolated and characterized from the ethanol-producing hyperthermophilic archaeon Thermococcus guaymasensis (Topt=88??C), as well as the bacteria Thermotoga hypogea (Topt=70??C) and Thermotoga maritima (Topt=80??C). The T. guaymasensis enzyme was purified anaerobically to homogeneity as judged by SDS-PAGE analysis. POR and PDC activities were co-eluted from each of the chromatographic columns, and the ratio of POR to PDC activities remained constant throughout the purification steps. All of the enzyme activities were CoA- and TPP-dependent and highly sensitive toward exposure to air. The apparent kinetic parameters were determined for the main substrates, including pyruvate and CoA for each activity. Since the genome sequence of T. guaymasensis and T. hypogea were not available, sequences of the genes encoding POR were determined via primer walking and inverse PCR.<br> A novel enzyme capable of catalyzing the production of acetaldehyde from pyruvate in hyperthermophiles was also characterized. The enzyme contained TPP and flavin and was expressed as recombinant histidine-tagged protein in the mesophilic host Escherichia coli. The new enzyme was a bifunctional enzyme catalyzing another reaction as the major reaction besides catalyzing the non-oxidative decarboxylation of pyruvate to acetaldehyde.<br> Another enzyme known to be involved in catalysis of acetaldehyde production from pyruvate is CoA-acetylating acetaldehyde dehydrogenase (AcDH encoded by mhpF and adhE). Pyruvate is oxidized into acetyl-CoA by either POR or pyruvate formate lyase (PFL), and AcDH catalyzes the reduction of acetyl-CoA to acetaldehyde. AcDH is present in some mesophilic (such as clostridia) and thermophilic bacteria (e.g. Geobacillus and Thermoanaerobacter). However, no AcDH gene or protein homologs could be found in the released genomes of hyperthermophiles. Moreover, no such activity was detectable from the cell-free extracts of different hyperthermophiles used in this study.<br> In conclusion, no commonly-known PDCs was found in hyperthermophiles, but two types of acetaldehyde-producing enzymes were present in various bacterial and archaeal hyperthermophiles. Although the deduced amino acid sequences from different hyperthermophiles are quite similar, the levels of POR and PDC activities appeared to vary significantly between the archaeal and bacterial enzymes, which most likely reflects the different physiological implications of each activity.

The biosynthetic pathway for the production of the dipeptide antibiotic bacilysin has been the subject of intense research over the past three decades. These studies revealed the role of multiple enzymes in the biosynthesis of this antibiotic. The identification of different enzymes was initially guided by genetic studies on different strains of Bacillus. The functional role of some these enzymes have been validated in vitro in the recent past. Despite this, the in vitro synthesis of bacilysin still remains elusive. The focus of this study was on two oxidoreductases - BacC and BacG. In the course of these studies, several variations to conventional oxidoreductase mechanisms were observed. These studies also provided us an opportunity to examine an oxidoreductase, BacC, at atomic resolution. This thesis describes these structural studies alongside efforts to achieve the biosynthesis of bacilysin in vitro. Chapter 1 provides an introduction to the broad goals of this thesis. First, the diversity of naturally occurring antibiotics is described. This is followed by a description of nonribosomal peptides and their preferred route for antibiotic synthesis. A summary of previous work in this area is provided to place this study in perspective. Earlier studies performed in this laboratory and others provided a framework for understanding the role of BacC and BacG. These studies have been described with an emphasis on the pivotal role of oxidoreductases in this process. In this context, known features of oxidoreductases, classification of the enzyme family, known reaction mechanisms, preferred substrates and cofactors of the enzyme have been summarized in this chapter. Chapter 2 describes the structural and biochemical characterization of B.subtilis BacG. The crystal structures of BacG determined in the apo form and ligand bound states could capture different conformational states of this enzyme. These structures revealed a basis to understand the ping-pong reaction mechanism. The catalytic residues Tyr-Ser-Lys-Asn involved in the proton relay were examined by mutational analysis. These biochemical studies could corroborate our observations derived from structural analysis. Put together, these studies suggest synchronized conformational changes in BacG that can rationalize cofactor specificity and catalytic action on di hydroxyphenyl pyruvate to form tetra hydroxyphenyl pyruvate en route to anticapsin biosynthesis. Bacillus subtilis BacC could be structurally characterized at 1.19Å resolution. The atomic resolution structure formed the basis for the analysis reported in this chapter. The structure revealed aspects of non-covalent interactions that could be unambiguously determined due to the high resolution diffraction data. The atomic resolution structure also enabled us to conduct charge density analysis on this protein. Atomic displacement parameters were used as a tool to explore paths of non-covalent interactions. A commercially available substrate, 3-Quinuclidinone, was used to characterize enzymatic activity. We note that this enzyme follows a rapid equilibrium random mechanism. Furthermore, the kinetic profiles were conclusive to draw inferences on allosteric interactions. A comparison between the NADH-complex and the apo enzyme structure suggests aspects of nuanced atomic displacement that governs the intra structural signal transduction. Taken together, this study provided a template to analyze the role of non-covalent interactions in regulating enzymatic activity. Chapter 4 is based on a survey of oxidoreductases that have been previously described in literature. During this study, we collated the extensive structural and biochemical data in this family of enzymes. However, we noted that the data remains disperse thereby limiting efforts to understand the reaction mechanism from a structural perspective. Here we collate information of known sequences, structures, cofactors, ligand preferences, reaction mechanisms and their influence on higher order association and catalytic activity in this class of enzymes. Chapter 5 summarizes the findings on two oxidoreductases (BacC, BacG). These studies on two closely related oxidoreductases BacC and BacG performing different roles in the same biosynthetic pathway revealed aspects biosynthesis that are often poorly recognized in protein engineering. The role of the reaction mechanism and their influence on the cofactor specificity could be inferred from the studies on these two enzymes. These studies also suggest the feasibility of evaluating aspects of enzyme activity and regulation provided the wealth of a priori information that is currently available. Put together, these studies provide a data-set for protein engineering efforts on oxidoreductases with general inferences for other enzymes in the short-chain dehydrogenases/ reductases (SDR) family. Appendix 1 provides a schematic representation of our efforts to biosynthetically obtain bacilysin in vitro. The identification, mass spectrometry of the products and substrates en route to bacilysin biosynthesis are compiled in this section. Appendix 2 describes the preliminary characterization of B.subtilis BacF. This part of the work describes the cloning, expression and purification of BacF and attempts to obtain suitable diffracting crystals for structural analysis.

Les thiol-disulfure oxydoréductases sont des protéines qui jouent un rôle majeur dans la cellule. Elles sont impliquées dans l'activité de nombreuses protéines cytoplasmiques, ainsi que dans la maturation et la stabilité des protéines extracytoplasmiques. Les particularités structurales conservées chez les TDORs, comme le repliement thiorédoxine et le motif CxxC, les propriétés physico-chimiques, leur environnement physiologique et leurs substrats sont autant de facteurs qui influencent la capacité de ces protéines à catalyser préférentiellement la réduction, l'oxydation, ou l'isomérisation des ponts disulfures in vivo.Je me suis intéressée aux TDORs atypiques cytoplasmiques pouvant présenter une activité thiol-oxydase ou disulfure-isomérase dans le cytoplasme des bactéries. J'ai caractérisées deux thiorédoxines atypiques, l'une provenant de l'organisme anaérobie Desulfovibrio vulgaris Hildenborough, Dtrx, et l'autre provenant de la bactérie pathogène Pseudomonas aeruginosa PAO1, PsTrx. Dtrx, possédant une séquence consensus thiol-oxydase CPHC, présente des propriétés in vitro en accord avec cette séquence. Nous avons proposé un mécanisme qui peut être appliqué de façon réversible dans le sens de la réduction et de l'oxydation des cystéines des substrats.PsTrx contient une séquence consensus CGHC dans son site actif, qui est généralement conservée chez PDI, protéine eucaryote. Les propriétés physico-chimiques, et la structure tridimensionnelle déterminées pour PsTrx par RMN, présentent des caractéristiques identiques à celles observées pour le domaine catalytique de PDI. / Thiol/disulfide oxidoreductases catalyze important redox reactions in the cell. They are implicated in the reduction of disulfide bonds in cytoplasm, and disulfide bond formation during folding of secreted proteins. All of the members of this family share the thioredoxin fold and an active site with two conserved cysteine residues that specify the biological activity of the protein in the reduction, oxidation or isomerisation of disulfide bond in vivo.In this work, I have studied atypical cytoplasmic TDORs catalyzing the oxidation or isomerisation of disulfide bond in bacteria. I have characterized two atypical thioredoxin proteins, one from the anaerobe Desulfovibrio vulgaris Hildenborough, Dtrx, and one from the pathogenic Pseudomonas aeruginosa PAO1, PsTrx.Dtrx, with the CPHC active site, presents important activities in the thiol-oxidation of proteins. We proposed a reversible mechanism for the disulfide-reduction or thiol-oxidation of substrate proteins.PsTrx presents the CGHC active site shown in the eukaryote PDI protein. Physico-chemical properties and tridimensional structure solved by NMR are the same that those of the catalytical domain of PDI.This work presents the properties of the two atypical thioredoxins, Dtrx and PsTrx. These proteins have similar functional and structural characteristics in vitro, but probably different redox functions in vivo.