The Auxiliary NADH Dehydrogenase Plays a Crucial Role in Redox Homeostasis of Nicotinamide Cofactors in the Absence of the Periplasmic Oxidation System in Gluconobacter oxydans NBRC3293.

Gluconobacter oxydans has the unique property of a glucose oxidation system in the periplasmic space, where glucose is oxidized incompletely to ketogluconic acids in a nicotinamide cofactor-independent manner. Elimination of the gdhM gene for membrane-bound glucose dehydrogenase, the first enzyme for the periplasmic glucose oxidation system, induces a metabolic change whereby glucose is oxidized in the cytoplasm to acetic acid. G. oxydans strain NBRC3293 possesses two molecular species of type II NADH dehydrogenase (NDH), the primary and auxiliary NDHs that oxidize NAD(P)H by reducing ubiquinone in the cell membrane. The substrate specificities of the two NDHs are different from each other: primary NDH (p-NDH) oxidizes NADH specifically but auxiliary NDH (a-NDH) oxidizes both NADH and NADPH. We constructed G. oxydans NBRC3293 derivatives defective in the ndhA gene for a-NDH, in the gdhM gene, and in both. Our ΔgdhM derivative yielded higher cell biomass on glucose, as reported previously, but grew at a lower rate than the wild-type strain. The ΔndhA derivative showed growth behavior on glucose similar to that of the wild type. The ΔgdhM ΔndhA double mutant showed greatly delayed growth on glucose, but its cell biomass was similar to that of the ΔgdhM strain. The double mutant accumulated intracellular levels of NAD(P)H and thus shifted the redox balance to reduction. Accumulated NAD(P)H levels might repress growth on glucose by limiting oxidative metabolisms in the cytoplasm. We suggest that a-NDH plays a crucial role in redox homeostasis of nicotinamide cofactors in the absence of the periplasmic oxidation system in G. oxydansIMPORTANCE Nicotinamide cofactors NAD+ and NADP+ mediate redox reactions in metabolism. Gluconobacter oxydans, a member of the acetic acid bacteria, oxidizes glucose incompletely in the periplasmic space-outside the cell. This incomplete oxidation of glucose is independent of nicotinamide cofactors. However, if the periplasmic oxidation of glucose is abolished, the cells oxidize glucose in the cytoplasm by reducing nicotinamide cofactors. Reduced forms of nicotinamide cofactors are reoxidized by NADH dehydrogenase (NDH) on the cell membrane. We found that two kinds of NDH in G. oxydans have different substrate specificities: the primary enzyme is NADH specific, and the auxiliary one oxidizes both NADH and NADPH. Inactivation of the latter enzyme in G. oxydans cells in which we had induced cytoplasmic glucose oxidation resulted in elevated intracellular levels of NAD(P)H, limiting cell growth on glucose. We suggest that the auxiliary enzyme is important if G. oxydans grows independently of the periplasmic oxidation system.

Coagulation factor XI induces Ca2+ response and accelerates cell migration in vascular smooth muscle cells via proteinase-activated receptor 1.

Activated coagulation factor XI (FXIa) is a serine proteinase that plays a key role in the intrinsic coagulation pathway. The analysis of FXI-knockout mice has indicated the contribution of FXI to the pathogenesis of atherosclerosis. However, the underlying mechanism remains unknown. We hypothesized that FXIa exerts vascular smooth muscle effects via proteinase-activated receptor 1 (PAR1). Fura-2 fluorometry revealed that FXIa elicited intracellular Ca2+ signal in rat embryo aorta smooth muscle A7r5 cells. The influx of extracellular Ca2+ played a greater role in generating Ca2+ signal than the Ca2+ release from intracellular stores. The FXIa-induced Ca2+ signal was abolished by the pretreatment with atopaxar, an antagonist of PAR1, or 4-amidinophenylmethanesulfonyl fluoride (p-APMSF), an inhibitor of proteinase, while it was also lost in embryonic fibroblasts derived from PAR1-/- mice. FXIa cleaved the recombinant protein containing the extracellular region of PAR1 at the same site (R45/S46) as that of thrombin, a canonical PAR1 agonist. The FXIa-induced Ca2+ influx was inhibited by diltiazem, an L-type Ca2+ channel blocker, and by siRNA targeted to CaV1.2. The FXIa-induced Ca2+ influx was also inhibited by GF109203X and rottlerin, inhibitors of protein kinase C. In a wound healing assay, FXIa increased the rate of cell migration by 2.46-fold of control, which was partly inhibited by atopaxar or diltiazem. In conclusion, FXIa mainly elicits the Ca2+ signal via the PAR1/CaV1.2-mediated Ca2+ influx and accelerates the migration in vascular smooth muscle cells. The present study provides the first evidence that FXIa exerts a direct cellular effect on vascular smooth muscle.

Increase in tumor suppressor Arf compensates gene dysregulation in in vitro aged adipocytes.

Deterioration of adipocyte function due to increased oxidative stress predisposes patients to metabolic disorders in advanced age. However, the roles of tumor suppressors in such conditions remain largely unknown. Therefore, we aimed to address their dynamics in aged adipocytes using a long-term culture model. We compared 3T3-L1 adipocytes at 17-19 days (long-term) with those at 8-10 days (short-term) after initiation of adipogenic induction for mimicking 'aged' and 'young' adipocytes, respectively. H2O2 release and dihydroethidium (DHE) staining was increased, while superoxide dismutase (SOD) activity was reduced in long-term cultured adipocytes, which is suggestive of enhanced oxidative stress in this group. Moreover, qRT-PCR revealed increased mRNAs of Nox4 (a subunit of NADPH oxidase complex), Ccl2 (a proinflammatory chemokine) and Il6 [a marker of senescence-associated secretory phenotype (SASP)] along with decreased levels of Pparγ, Adipoq and Slc2a4 (genes related to glucose metabolism). These alterations were associated with increased expression of the tumor suppressors alternate-reading-frame protein p19Arf (Arf) and p16Ink4a. However, silencing of Arf reduced mRNAs of Adipoq and Slc2a4 and enhanced release of Il6. The effect was opposite in Arf overexpressing adipocytes, which showed reduced superoxide production as assessed with DHE staining and SOD activity. Western blots showed that Arf negatively regulates the phosphorylation of Akt. Luciferase assay in Hela cells additionally suggested that Arf negatively regulates Il6 transcriptional activity through a PI3 K/Akt mediated pathway. These findings strongly suggest that the enhanced Arf expression in oxidative stress plays compensatory protective roles against aging-related dysregulation of gene expression in adipocytes.

The structure of the yeast NADH dehydrogenase (Ndi1) reveals overlapping binding sites for water- and lipid-soluble substrates.

Bioenergy is efficiently produced in the mitochondria by the respiratory system consisting of complexes I-V. In various organisms, complex I can be replaced by the alternative NADH-quinone oxidoreductase (NDH-2), which catalyzes the transfer of an electron from NADH via FAD to quinone, without proton pumping. The Ndi1 protein from Saccharomyces cerevisiae is a monotopic membrane protein, directed to the matrix. A number of studies have investigated the potential use of Ndi1 as a therapeutic agent against complex I disorders, and the NDH-2 enzymes have emerged as potential therapeutic targets for treatments against the causative agents of malaria and tuberculosis. Here we present the crystal structures of Ndi1 in its substrate-free, NAD(+)- and ubiquinone- (UQ2) complexed states. The structures reveal that Ndi1 is a peripheral membrane protein forming an intimate dimer, in which packing of the monomeric units within the dimer creates an amphiphilic membrane-anchor domain structure. Crucially, the structures of the Ndi1-NAD(+) and Ndi1-UQ2 complexes show overlapping binding sites for the NAD(+) and quinone substrates.

Therapeutic Effect of Proteinase-Activated Receptor-1 Antagonist on Colitis-Associated Carcinogenesis.

BACKGROUND & AIMS: Inflammatory bowel disease is associated with carcinogenesis, which limits the prognosis of the patients. The local expression of proteinases and proteinase-activated receptor 1 (PAR1) increases in inflammatory bowel disease. The present study investigated the therapeutic effects of PAR1 antagonism on colitis-associated carcinogenesis. METHODS: A colitis-associated carcinogenesis model was prepared in mice by treatment with azoxymethane (AOM) and dextran sulfate sodium (DSS). PAR1 antagonist E5555 was administered in long- and short-term protocol, starting on the day of AOM injection and 1 week after completing AOM/DSS treatment, respectively. The fecal samples were collected for metagenome analysis of gut microbiota. The intestinal myofibroblasts of the Crohn's disease patients were used to elucidate underlying cellular mechanisms. Caco-2 cells were used to investigate a possible source of PAR1 agonist proteinases. RESULTS: AOM/DSS model showed weight loss, diarrhea, tumor development, inflammation, fibrosis, and increased production of inflammatory cytokines. The ß-diversity, but not α-diversity, of microbiota significantly differed between AOM/DSS and control mice. E5555 alleviated these pathological changes and altered the microbiota ß-diversity in AOM/DSS mice. The thrombin expression was up-regulated in tumor and non-tumor areas, whereas PAR1 mRNA expression was higher in tumor areas compared with non-tumor areas. E5555 inhibited thrombin-triggered elevation of cytosolic Ca2+ concentration and ERK1/2 phosphorylation, as well as IL6-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation in intestinal myofibroblasts. Caco-2 cell-conditioned medium contained immunoreactive thrombin, which cleaved the recombinant protein containing the extracellular domain of PAR1 at the thrombin cleavage site. CONCLUSIONS: PAR1 antagonism is proposed to be a novel therapeutic strategy for treatment of inflammatory bowel disease and its associated carcinogenesis.

Reaction mechanism of single subunit NADH-ubiquinone oxidoreductase (Ndi1) from Saccharomyces cerevisiae: evidence for a ternary complex mechanism.

The flavoprotein rotenone-insensitive internal NADH-ubiquinone (UQ) oxidoreductase (Ndi1) is a member of the respiratory chain in Saccharomyces cerevisiae. We reported previously that bound UQ in Ndi1 plays a key role in preventing the generation of reactive oxygen species. Here, to elucidate this mechanism, we investigated biochemical properties of Ndi1 and its mutants in which highly conserved amino acid residues (presumably involved in NADH and/or UQ binding sites) were replaced. We found that wild-type Ndi1 formed a stable charge transfer (CT) complex (around 740 nm) with NADH, but not with NADPH, under anaerobic conditions. The intensity of the CT absorption band was significantly increased by the presence of bound UQ or externally added n-decylbenzoquinone. Interestingly, however, when Ndi1 was exposed to air, the CT band transiently reached the same maximum level regardless of the presence of UQ. This suggests that Ndi1 forms a ternary complex with NADH and UQ, but the role of UQ in withdrawing an electron can be substitutable with oxygen. Proteinase K digestion analysis showed that NADH (but not NADPH) binding induces conformational changes in Ndi1. The kinetic study of wild-type and mutant Ndi1 indicated that there is no overlap between NADH and UQ binding sites. Moreover, we found that the bound UQ can reversibly dissociate from Ndi1 and is thus replaceable with other quinones in the membrane. Taken together, unlike other NAD(P)H-UQ oxidoreductases, the Ndi1 reaction proceeds through a ternary complex (not a ping-pong) mechanism. The bound UQ keeps oxygen away from the reduced flavin.

Biological and immunological characteristics of hepatitis E virus-like particles based on the crystal structure.

Hepatitis E virus (HEV) is a causative agent of acute hepatitis. The crystal structure of HEV-like particles (HEV-LP) consisting of capsid protein was determined at 3.5-A resolution. The capsid protein exhibited a quite different folding at the protruding and middle domains from the members of the families of Caliciviridae and Tombusviridae, while the shell domain shared the common folding. Tyr-288 at the 5-fold axis plays key roles in the assembly of HEV-LP, and aromatic amino acid residues are well conserved among the structurally related viruses. Mutational analyses indicated that the protruding domain is involved in the binding to the cells susceptive to HEV infection and has some neutralization epitopes. These structural and biological findings are important for understanding the molecular mechanisms of assembly and entry of HEV and also provide clues in the development of preventive and prophylactic measures for hepatitis E.

Endovascular Treatment Using a Small Amount of Contrast Medium in a Patient with a Posterior Communicating Artery Ruptured Dissecting Aneurysm and Chronic Kidney Disease: A Case Report.

Objective: We report a rare case of a patient with a ruptured posterior communicating artery (P-com A) dissecting aneurysm and chronic kidney disease (CKD) treated by endovascular embolization using a small amount of contrast medium. Case Presentation: An 88-year-old female patient had sudden onset of headache and vomit due to subarachnoid hemorrhage. MRI revealed a ruptured dissecting aneurysm of the right P-com A. The patient had CKD of severity grade 4. Endovascular treatment was performed using only 10 mL of diluted contrast medium with injection through a microcatheter. The postoperative course was uneventful, and no deterioration of renal function occurred. Conclusion: With minimal amount of contrast medium, endovascular treatment could be safely and effectively performed for patients with P-com A dissecting aneurysms and severe CKD.

Lactulose Modulates the Structure of Gut Microbiota and Alleviates Colitis-Associated Tumorigenesis.

Lactulose, a galactose-fructose disaccharide, is made from the milk sugar lactose by heating or isomerization processes. Lactulose is proposed to modulate gut microbiota and thus expected to be beneficial in treating inflammatory bowel disease. In the present study, we investigated the therapeutic effect of lactulose on gastrointestinal inflammation and inflammation-related tumorigenesis in a mouse model of colorectal cancer as well as its effect on gut microbiota composition. Azoxymethane (AOM)/dextran sulfate sodium (DSS) model was used in this study. Lactulose treatment was performed by feeding 2% lactulose for 14 weeks. Stool samples collected at 4 time points were used for metagenomic analysis of the microbiota. Pathological analysis was performed 21 weeks after AOM injection. AOM/DSS increased the macrophage counts, inflammatory cytokine expression, colorectal tumorigenesis, and imbalance in gut microbiota composition, as evidenced by increased pathogen abundance (e.g., Escherichia and Clostridium). Lactulose significantly inhibited the inflammatory events, and ameliorated inflammation and tumorigenesis. The composition of the intestinal microbiota was also restored upon lactulose treatment, and lactulose reduced pathogen abundance and increased the abundance of Muribaculum and Lachnospiraceae. Meanwhile, the pathways related to Crohn's disease were downregulated after lactulose treatment. Our findings suggest that lactulose restores the structure and composition of the intestinal microbiota, mitigates inflammation, and suppresses inflammatory tumorigenesis.

Idiopathic dissecting cerebral aneurysm of the distal anterior cerebral artery in an infant successfully treated with aneurysmectomy: illustrative case.

BACKGROUND: Idiopathic dissecting cerebral aneurysms (IDCAs) are male dominant but are extremely rare in children. Many IDCAs in children are located in the posterior cerebral artery and the supraclinoid internal cervical artery. No cases of IDCA of the distal anterior cerebral artery (ACA) have been reported. OBSERVATIONS: A previously healthy 7-month-old boy experienced afebrile seizures and presented at the authors' hospital 1 week after the first seizure. He was not feeling well but had no neurological deficits. The authors diagnosed a ruptured aneurysm of the right distal ACA based on imaging results. He underwent emergency craniotomy to prevent re-rupture of the aneurysm. Using intraoperative indocyanine green videoangiography, the authors confirmed peripheral blood flow and then performed aneurysmectomy. Pathological examination of the aneurysm revealed a thickened intima, fragmentation of the internal elastic lamina, and a hematoma in the aneurysmal wall. The authors ultimately diagnosed IDCA because no cause was indicated, including a history of trauma. The boy recovered after surgery and was subsequently discharged with no complications. LESSONS: The authors reported, for the first time, IDCA of the distal ACA in an infant. The trapping technique is often used for giant fusiform aneurysms in infants. Indocyanine green videoangiography is useful for evaluating peripheral blood flow during trapping in this case.

Characterization of the ubiquinone binding site in the alternative NADH-quinone oxidoreductase of Saccharomyces cerevisiae by photoaffinity labeling.

The Ndi1 enzyme found in the mitochondrial membrane of Saccharomyces cerevisiae is an NDH-2-type alternative NADH-quinone oxidoreductase. As Ndi1 is expected to be a possible remedy for complex I defects of mammalian mitochondria, a detailed biochemical characterization of the enzyme is needed. To identify the ubiquinone (UQ) binding site in Ndi1, we conducted photoaffinity labeling using a photoreactive biotinylated UQ mimic (compound 2) synthesized following a concept of the least possible modification of the substituents on the quinone ring. Cleavage with CNBr of Ndi1 cross-linked by 2 revealed the UQ ring of 2 to be specifically cross-linked to the Phe281-Met410 region (130 amino acids). Digestion of the CNBr fragment with V8 protease and lysylendopeptidase (Lys-C) gave approximately 8 and approximately 4 kDa peptides, respectively. The approximately 8 kDa V8 digest was identified as the Thr329-Glu399 region (71 amino acids) by an N-terminal sequence analysis. Although the approximately 4 kDa Lys-C digest could not be identified by N-terminal sequence analysis, the band was thought to cover the Gly374-Lys405 region (32 amino acids). Taken together, the binding site of the Q ring of 2 must be located in a common region of the V8 protease, and Lys-C digests Gly374-Glu399 (26 amino acids). Superimposition of the Ndi1 sequence onto a three-dimensional structural model of NDH-2 from Escherichia coli suggested that the C-terminal portion of this region is close to the isoalloxazine ring of FAD.

Transforming growth factor-beta1 downregulates caveolin-1 expression and enhances sphingosine 1-phosphate signaling in cultured vascular endothelial cells.

In vascular endothelial cells, specialized microdomains of plasma membrane termed caveolae modulate various receptor signal transduction pathways regulated by caveolin-1, a resident protein of caveolae. We examined whether transforming growth factor-beta1 (TGF-beta1), a multifunctional cytokine, alters expression levels of caveolin-1 and influences heterologous receptor signaling. Treatment of cultured bovine aortic endothelial cells (BAEC) with TGF-beta1 induces marked decreases in caveolin-1 expression in a time- and dose-dependent fashion at both levels of protein and mRNA. A pharmacological inhibitor of activin receptor-like kinase 5 (ALK-5) counteracts caveolin-1 downregulation by TGF-beta1, indicating the involvement of ALK-5 receptor subtype for TGF-beta1. Sphingosine 1-phosphate (S1P) is a serum-borne angiogenic lipid growth factor that exerts a wide variety of biological actions. S1P modulates G protein-coupled S1P receptors, activating downstream molecules kinases AMP-activated protein kinase (AMPK), and Akt as well as a small G protein Rac1, ultimately to promote migration. Because S1P receptor signaling is associated with caveolae/caveolin-1, we examined whether pretreatment with TGF-beta1 enhances effects of S1P on BAEC. Whereas S1P alone evokes robust BAEC responses to S1P, pretreatment with TGF-beta1 leads to even higher magnitudes of S1P-elicited signaling responses and cell migration. Conversely, genetic knockdown of caveolin-1 using small interfering RNA mimics TGF-beta1-induced promotion of BAEC responses to S1P. Collectively, these data demonstrate that TGF-beta1 downregulates caveolin-1 of cultured endothelial cells, involving ALK-5 receptor subtype. Because downregulation of caveolin-1 by TGF-beta1 promotes subsequent heterologous receptor signaling by S1P, these results may also identify novel point of cross-talk between cytokines and sphingolipids within endothelial signal transduction machineries.

Crystallization and preliminary X-ray diffraction analysis of recombinant hepatitis E virus-like particle.

Hepatitis E virus (HEV) accounts for the majority of enterically transmitted hepatitis infections worldwide. Currently, there is no specific treatment for or vaccine against HEV. The major structural protein is derived from open reading frame (ORF) 2 of the viral genome. A potential oral vaccine is provided by the virus-like particles formed by a protein construct of partial ORF3 protein (residue 70-123) fused to the N-terminus of the ORF2 protein (residues 112-608). Single crystals obtained by the hanging-drop vapour-diffusion method at 293 K diffract X-rays to 8.3 A resolution. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 337, b = 343, c = 346 A, alpha = beta = gamma = 90 degrees , and contain one particle per asymmetric unit.

Ubiquinone binding site of yeast NADH dehydrogenase revealed by structures binding novel competitive- and mixed-type inhibitors.

Yeast Ndi1 is a monotopic alternative NADH dehydrogenase. Its crystal structure in complex with the electron acceptor, ubiquinone, has been determined. However, there has been controversy regarding the ubiquinone binding site. To address these points, we identified the first competitive inhibitor of Ndi1, stigmatellin, along with new mixed-type inhibitors, AC0-12 and myxothiazol, and thereby determined the crystal structures of Ndi1 in complexes with the inhibitors. Two separate binding sites of stigmatellin, STG-1 and STG-2, were observed. The electron density at STG-1, located at the vicinity of the FAD cofactor, further demonstrated two binding modes: STG-1a and STG-1b. AC0-12 and myxothiazol are also located at the vicinity of FAD. The comparison of the binding modes among stigmatellin at STG-1, AC0-12, and myxothiazol revealed a unique position for the aliphatic tail of stigmatellin at STG-1a. Mutations of amino acid residues that interact with this aliphatic tail at STG-1a reduced the affinity of Ndi1 for ubiquinone. In conclusion, the position of the aliphatic tail of stigmatellin at STG-1a provides a structural basis for its competitive inhibition of Ndi1. The inherent binding site of ubiquinone is suggested to overlap with STG-1a that is distinct from the binding site for NADH.

Possibility of transkingdom gene therapy for complex I diseases.

Defects of complex I are involved in many human mitochondrial diseases, and therefore we have proposed to use the NDI1 gene encoding a single subunit NADH dehydrogenase of Saccharomyces cerevisiae for repair of respiratory activity. The yeast NDI1 gene was successfully introduced into mammalian cell lines. The expressed NDI1 protein was correctly targeted to the matrix side of the inner mitochondrial membranes, was fully functional and restored the NADH oxidase activity to the complex I-deficient cells. The NDI1-transduced cells were more resistant to complex I inhibitors and diminished production of reactive oxygen species induced by rotenone. It was further shown that the NDI1 protein can be functionally expressed in tissues such as skeletal muscles and the brain of rodents, which scarcely induced an inflammatory response. The use of NDI1 as a potential molecular therapy for complex I-deficient diseases is briefly discussed, including the proposed animal model.

Can a single subunit yeast NADH dehydrogenase (Ndi1) remedy diseases caused by respiratory complex I defects?

The proton-translocating NADH-quinone oxidoreductase (complex I) is one of five enzyme complexes in the oxidative phosphorylation system in mammalian mitochondria. Complex I is composed of 46 different subunits, 7 of which are encoded by mitochondrial DNA. Defects of complex I are involved in many human mitochondrial diseases; therefore, the authors proposed to use the NDI1 gene encoding a single subunit NADH dehydrogenase of Saccharomyces cerevisiae for repair of respiratory activity. The yeast NDI1 gene was successfully introduced into 10 mammalian cell lines (two of which were complex I-deficient mutants). The expressed Ndi1 protein was correctly targeted to the matrix side of the inner mitochondrial membranes, was fully functional, and restored the NADH oxidase activity to the complex I-deficient cells. The NDI1-transduced cells were more resistant to complex I inhibitors and diminished production of reactive oxygen species. It was further shown that the Ndi1 protein can be functionally expressed in tissues such as skeletal muscles and brain of rodents. The Ndi1 expression scarcely induced an inflammatory response as assessed by hematoxylin and eosin (H&E) staining. The Ndi1 protein expressed in the substantia nigra (SN) elicited protective effects against neurodegeneration caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment. The Ndi1 protein has a great potential as a molecular remedy for complex I deficiencies.

Crystallization and preliminary crystallographic study of carnosinase CN2 from mice.

Mammalian tissues contain several histidine-containing dipeptides, of which L-carnosine is the best characterized and is found in various tissues including the brain and skeletal muscles. However, the mechanism for its biosynthesis and degradation have not yet been fully elucidated. Crystallographic study of carnosinase CN2 from mouse has been undertaken in order to understand its enzymatic mechanism from a structural viewpoint. CN2 was crystallized by the hanging-drop vapour-diffusion technique using PEG 3350 as a precipitant. Crystals were obtained in complex with either Mn(2+) or Zn(2+). Both crystals of CN2 belong to the monoclinic space group P2(1) and have almost identical unit-cell parameters (a = 54.41, b = 199.77, c = 55.49 A, beta = 118.52 degrees for the Zn(2+) complex crystals). Diffraction data were collected to 1.7 and 2.3 A for Zn(2+) and Mn(2+) complex crystals, respectively, using synchrotron radiation. Structure determination is ongoing using the multiple-wavelength anomalous diffraction (MAD) method.

Structure at 1.9 A resolution of a quinohemoprotein alcohol dehydrogenase from Pseudomonas putida HK5.

The type II quinohemoprotein alcohol dehydrogenase of Pseudomonas putida is a periplasmic enzyme that oxidizes substrate alcohols to the aldehyde and transfers electrons first to pyrroloquinoline quinone (PQQ) and then to an internal heme group. The 1.9 A resolution crystal structure reveals that the enzyme contains a large N-terminal eight-stranded beta propeller domain (approximately 60 kDa) similar to methanol dehydrogenase and a small C-terminal c-type cytochrome domain (approximately 10 kDa) similar to the cytochrome subunit of p-cresol methylhydoxylase. The PQQ is bound near the axis of the propeller domain about 14 A from the heme. A molecule of acetone, the product of the oxidation of isopropanol present during crystallization, appears to be bound in the active site cavity.

A key role of PGC-1α transcriptional coactivator in production of VEGF by a novel angiogenic agent COA-Cl in cultured human fibroblasts.

We previously demonstrated a potent angiogenic effect of a newly developed adenosine-like agent namedCOA-Cl.COA-Cl exerted tube forming activity in human umbilical vein endothelial cells in the presence of normal human dermal fibroblasts (NHDF). We therefore explored whether and howCOA-Cl modulates gene expression and protein secretion ofVEGF, a master regulator of angiogenesis, inNHDFRT-PCRandELISArevealed thatCOA-Cl upregulatedVEGF mRNAexpression and protein secretion inNHDFHIF1α(hypoxia-inducible factor 1α), a transcription factor, andPGC-1α(peroxisome proliferator-activated receptor-γcoactivator-1α), a transcriptional coactivator, are known to positively regulate theVEGFgene. Immunoblot andRT-PCRanalyses revealed thatCOA-Cl markedly upregulated the expression ofPGC-1αprotein andmRNACOA-Cl had no effect on the expression ofHIF1αprotein andmRNAin both hypoxia and normoxia. SilencingPGC-1αgene, but notHIF1αgene, by small interferingRNAattenuated the ability ofCOA-Cl to promoteVEGFsecretion. When an N-terminal fragment ofPGC-1αwas cotransfected with its partner transcription factorERRα(estrogen-related receptor-α) inCOS-7 cells,COA-Cl upregulated the expression of the endogenousVEGF mRNA However,COA-Cl had no effect on the expression ofVEGF, whenHIF1αwas transfected.COA-Cl inducesVEGFgene expression and protein secretion in fibroblasts. The transcriptional coactivatorPGC-1α, in concert withERRα, plays a key role in theCOA-Cl-inducedVEGFproduction.COA-Cl-induced activation ofPGC-1α-ERRα-VEGFpathway has a potential as a novel means for therapeutic angiogenesis.

Rhodoquinone reaction site of mitochondrial complex I, in parasitic helminth, Ascaris suum.

The components and organization of the respiratory chain in helminth mitochondria vary remarkably depending upon the stage of the life cycle. Mitochondrial complex I in the parasitic helminth Ascaris suum uses ubiquinone-9 (UQ(9)) and rhodoquinone-9 (RQ(9)) under aerobic and anaerobic conditions, respectively. In this study, we investigated structural features of the quinone reduction site of A. suum complex I using a series of quinazoline-type inhibitors and also by the kinetic analysis of rhodoquinone-2 (RQ(2)) and ubiquinone-2 (UQ(2)) reduction. Structure-activity profiles of the inhibition by quinazolines were comparable, but not completely identical, between NADH-RQ(2) and NADH-UQ(2) oxidoreductase activities. However, the inhibitory mechanism of quinazolines was competitive and partially competitive against RQ(2) and UQ(2), respectively. The pH profiles of both activities differed remarkably; NADH-RQ(2) oxidoreductase activity showed an optimum pH at 7.6, whereas NADH-UQ(2) oxidoreductase activity showed two optima pH at 6.4 and 7.2. Our results indicate that although A. suum complex I uses both RQ(2) and UQ(2) as an electron acceptor, the manner of reaction (or binding) of the two quinones differs.