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Sample records for protein synthase iii

  1. Evolutionary Implications and Physicochemical Analyses of Selected Proteins of Type III Polyketide Synthase Family

    PubMed Central

    Mallika, V.; Sivakumar, K.C.; Soniya, E.V.

    2011-01-01

    Type III polyketide synthases have a substantial role in the biosynthesis of various polyketides in plants and microorganisms. Comparative proteomic analysis of type III polyketide synthases showed evolutionarily and structurally related positions in a compilation of amino acid sequences from different families. Bacterial and fungal type III polyketide synthase proteins showed <50% similarity but in higher plants, it exhibited >80% among chalcone synthases and >70% in the case of non-chalcone synthases. In a consensus phylogenetic tree based on 1000 replicates; bacterial, fungal and plant proteins were clustered in separate groups. Proteins from bryophytes and pteridophytes grouped immediately near to the fungal cluster, demonstrated how evolutionary lineage has occurred among type III polyketide synthase proteins. Upon physicochemical analysis, it was observed that the proteins localized in the cytoplasm and were hydrophobic in nature. Molecular structural analysis revealed comparatively stable structure comprising of alpha helices and random coils as major structural components. It was found that there was a decline in the structural stability with active site mutation as prophesied by the in silico mutation studies. PMID:21697991

  2. Pullulanase and Starch Synthase III Are Associated with Formation of Vitreous Endosperm in Quality Protein Maize.

    PubMed

    Wu, Hao; Clay, Kasi; Thompson, Stephanie S; Hennen-Bierwagen, Tracie A; Andrews, Bethany J; Zechmann, Bernd; Gibbon, Bryan C

    2015-01-01

    The opaque-2 (o2) mutation of maize increases lysine content, but the low seed density and soft texture of this type of mutant are undesirable. Lines with modifiers of the soft kernel phenotype (mo2) called "Quality Protein Maize" (QPM) have high lysine and kernel phenotypes similar to normal maize. Prior research indicated that the formation of vitreous endosperm in QPM might involve changes in starch granule structure. In this study, we focused on analysis of two starch biosynthetic enzymes that may influence kernel vitreousness. Analysis of recombinant inbred lines derived from a cross of W64Ao2 and K0326Y revealed that pullulanase activity had significant positive correlation with kernel vitreousness. We also found that decreased Starch Synthase III abundance may decrease the pullulanase activity and average glucan chain length given the same Zpu1 genotype. Therefore, Starch Synthase III could indirectly influence the kernel vitreousness by affecting pullulanase activity and coordinating with pullulanase to alter the glucan chain length distribution of amylopectin, resulting in different starch structural properties. The glucan chain length distribution had strong positive correlation with the polydispersity index of glucan chains, which was positively associated with the kernel vitreousness based on nonlinear regression analysis. Therefore, we propose that pullulanase and Starch Synthase III are two important factors responsible for the formation of the vitreous phenotype of QPM endosperms. PMID:26115014

  3. Pullulanase and Starch Synthase III Are Associated with Formation of Vitreous Endosperm in Quality Protein Maize

    PubMed Central

    Wu, Hao; Clay, Kasi; Thompson, Stephanie S.; Hennen-Bierwagen, Tracie A.; Andrews, Bethany J.; Zechmann, Bernd; Gibbon, Bryan C.

    2015-01-01

    The opaque-2 (o2) mutation of maize increases lysine content, but the low seed density and soft texture of this type of mutant are undesirable. Lines with modifiers of the soft kernel phenotype (mo2) called “Quality Protein Maize” (QPM) have high lysine and kernel phenotypes similar to normal maize. Prior research indicated that the formation of vitreous endosperm in QPM might involve changes in starch granule structure. In this study, we focused on analysis of two starch biosynthetic enzymes that may influence kernel vitreousness. Analysis of recombinant inbred lines derived from a cross of W64Ao2 and K0326Y revealed that pullulanase activity had significant positive correlation with kernel vitreousness. We also found that decreased Starch Synthase III abundance may decrease the pullulanase activity and average glucan chain length given the same Zpu1 genotype. Therefore, Starch Synthase III could indirectly influence the kernel vitreousness by affecting pullulanase activity and coordinating with pullulanase to alter the glucan chain length distribution of amylopectin, resulting in different starch structural properties. The glucan chain length distribution had strong positive correlation with the polydispersity index of glucan chains, which was positively associated with the kernel vitreousness based on nonlinear regression analysis. Therefore, we propose that pullulanase and Starch Synthase III are two important factors responsible for the formation of the vitreous phenotype of QPM endosperms. PMID:26115014

  4. 3-Ketoacyl-acyl carrier protein synthase III from spinach (Spinacia oleracea) is not similar to other condensing enzymes of fatty acid synthase.

    PubMed Central

    Tai, H; Jaworski, J G

    1993-01-01

    A cDNA clone encoding spinach (Spinacia oleracea) 3-ketoacyl-acyl carrier protein synthase III (KAS III), which catalyzes the initial condensing reaction in fatty acid biosynthesis, was isolated. Based on the amino acid sequence of tryptic digests of purified spinach KAS III, degenerate polymerase chain reaction (PCR) primers were designed and used to amplify a 612-bp fragment from first-strand cDNA of spinach leaf RNA. A root cDNA library was probed with the PCR fragment, and a 1920-bp clone was isolated. Its deduced amino acid sequence matched the sequences of the tryptic digests obtained from the purified KAS III. Northern analysis confirmed that it was expressed in both leaf and root. The clone contained a 1218-bp open reading frame coding for 405 amino acids. The identity of the clone was confirmed by expression in Escherichia coli BL 21 as a glutathione S-transferase fusion protein. The deduced amino acid sequence was 48 and 45% identical with the putative KAS III of Porphyra umbilicalis and KAS III of E. coli, respectively. It also had a strong local homology to the plant chalcone synthases but had little homology with other KAS isoforms from plants, bacteria, or animals. PMID:8290632

  5. Cellulose synthase interacting protein

    PubMed Central

    Somerville, Chris

    2010-01-01

    Cellulose is the most abundant biopolymer on earth. The great abundance of cellulose places it at the forefront as a primary source of biomass for renewable biofuels. However, the knowledge of how plant cells make cellulose remains very rudimentary. Cellulose microfibrils are synthesized at the plasma membrane by hexameric protein complexes, also known as cellulose synthase complexes. The only known components of cellulose synthase complexes are cellulose synthase (CESA) proteins until the recent identification of a novel component. CSI1, which encodes CESA interacting protein 1 (CSI1) in Arabidopsis. CSI1, as the first non-CESA proteins associated with cellulose synthase complexes, opens up many opportunities. PMID:21150290

  6. Lumazine Synthase Protein Nanoparticle-Gd(III)-DOTA Conjugate as a T1 contrast agent for high-field MRI

    PubMed Central

    Song, YoungKyu; Kang, Young Ji; Jung, Hoesu; Kim, Hansol; Kang, Sebyung; Cho, HyungJoon

    2015-01-01

    With the applications of magnetic resonance imaging (MRI) at higher magnetic fields increasing, there is demand for MRI contrast agents with improved relaxivity at higher magnetic fields. Macromolecule-based contrast agents, such as protein-based ones, are known to yield significantly higher r1 relaxivity at low fields, but tend to lose this merit when used as T1 contrast agents (r1/r2 = 0.5 ~ 1), with their r1 decreasing and r2 increasing as magnetic field strength increases. Here, we developed and characterized an in vivo applicable magnetic resonance (MR) positive contrast agent by conjugating Gd(III)-chelating agent complexes to lumazine synthase isolated from Aquifex aeolicus (AaLS). The r1 relaxivity of Gd(III)-DOTA-AaLS-R108C was 16.49 mM−1s−1 and its r1/r2 ratio was 0.52 at the magnetic field strength of 7 T. The results of 3D MR angiography demonstrated the feasibility of vasculature imaging within 2 h of intravenous injection of the agent and a significant reduction in T1 values were observed in the tumor region 7 h post-injection in the SCC-7 flank tumor model. Our findings suggest that Gd(III)-DOTA-AaLS-R108C could serve as a potential theranostic nanoplatform at high magnetic field strength. PMID:26493381

  7. Lumazine Synthase Protein Nanoparticle-Gd(III)-DOTA Conjugate as a T1 contrast agent for high-field MRI.

    PubMed

    Song, YoungKyu; Kang, Young Ji; Jung, Hoesu; Kim, Hansol; Kang, Sebyung; Cho, HyungJoon

    2015-01-01

    With the applications of magnetic resonance imaging (MRI) at higher magnetic fields increasing, there is demand for MRI contrast agents with improved relaxivity at higher magnetic fields. Macromolecule-based contrast agents, such as protein-based ones, are known to yield significantly higher r1 relaxivity at low fields, but tend to lose this merit when used as T1 contrast agents (r1/r2 = 0.5 ~ 1), with their r1 decreasing and r2 increasing as magnetic field strength increases. Here, we developed and characterized an in vivo applicable magnetic resonance (MR) positive contrast agent by conjugating Gd(III)-chelating agent complexes to lumazine synthase isolated from Aquifex aeolicus (AaLS). The r1 relaxivity of Gd(III)-DOTA-AaLS-R108C was 16.49 mM(-1)s(-1) and its r1/r2 ratio was 0.52 at the magnetic field strength of 7 T. The results of 3D MR angiography demonstrated the feasibility of vasculature imaging within 2 h of intravenous injection of the agent and a significant reduction in T1 values were observed in the tumor region 7 h post-injection in the SCC-7 flank tumor model. Our findings suggest that Gd(III)-DOTA-AaLS-R108C could serve as a potential theranostic nanoplatform at high magnetic field strength. PMID:26493381

  8. Effect of chronic prenatal ethanol exposure on nitric oxide synthase I and III proteins in the hippocampus of the near-term fetal guinea pig.

    PubMed

    Kimura, K A; Chiu, J; Reynolds, J N; Brien, J F

    1999-01-01

    Chronic prenatal ethanol exposure suppresses nitric oxide synthase (NOS) enzymatic activity, in the hippocampus of the near-term fetal guinea pig at gestational day (GD) 62. The objective of this study was to determine if this decrease in NOS activity is the result of decreased NOS I and NOS III protein expression. Pregnant guinea pigs received oral administration of 4 g ethanol/kg maternal body weight/day (n = 8), isocaloric-sucrose/pair feeding (n = 8), or water (n = 8) from GD 2 to GD 61. The NOS I and NOS III protein expression and localization in the hippocampus were determined using Western blot analysis and immunohistochemistry, respectively. The chronic ethanol regimen produced fetal body, brain, and hippocampal growth restriction compared with the isocaloric-sucrose/pair fed and water groups but did not affect the expression or localization of NOS I and NOS III proteins in the hippocampus. The decrease in NOS enzymatic activity induced by chronic prenatal ethanol exposure may be the result of posttranslational modification of NOS I and/or NOS III protein in the hippocampus of the near-term fetal guinea pig. PMID:10386828

  9. Ontogeny of nitric oxide synthase I and III protein expression and enzymatic activity in the guinea pig hippocampus.

    PubMed

    Kimura, K A; Reynolds, J N; Brien, J F

    1999-09-01

    60. NOS enzymatic activity increased throughout prenatal and postnatal life, and attained highest activity in the adult. The developmental profile of NOS III protein expression was similar to that for NOS enzymatic activity. There was differential expression of NOS I protein, which was low in the GD 50 fetus and increased rapidly during fetal development to attain adult level by GD 62. These data suggest that the guinea pig is a reliable animal model in which to investigate the roles of NO in normal hippocampal development and in mediating neuronal injury in this brain region. PMID:10521566

  10. Probing the Mechanism of the Mycobacterium tuberculosis [beta]-Ketoacyl-Acyl Carrier Protein Synthase III mtFabH: Factors Influencing Catalysis and Substrate Specificity

    SciTech Connect

    Brown, Alistair K.; Sridharan, Sudharsan; Kremer, Laurent; Lindenberg, Sandra; Dover, Lynn G.; Sacchettini, James C.; Besra, Gurdyal S.

    2010-11-30

    Mycolic acids are the dominant feature of the Mycobacterium tuberculosis cell wall. These {alpha}-alkyl, {beta}-hydroxy fatty acids are formed by the condensation of two fatty acids, a long meromycolic acid and a shorter C{sub 24}-C{sub 26} fatty acid. The component fatty acids are produced via a combination of type I and II fatty acid synthases (FAS) with FAS-I products being elongated by FAS-II toward meromycolic acids. The {beta}-ketoacyl-acyl carrier protein (ACP) synthase III encoded by mtfabH (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-acyl carrier protein (ACP). The acyl-CoA chain length specificity of mtFabH was assessed in vitro; the enzyme extended longer, physiologically relevant acyl-CoA primers when paired with AcpM, its natural partner, than with Escherichia coli ACP. The ability of the enzyme to use E. coli ACP suggests that a similar mode of binding is likely with both ACPs, yet it is clear that unique factors inherent to AcpM modulate the substrate specificity of mtFabH. Mutation of proposed key mtFabH residues was used to define their catalytic roles. Substitution of supposed acyl-CoA binding residues reduced transacylation, with double substitutions totally abrogating activity. Mutation of Arg{sup 46} revealed its more critical role in malonyl-AcpM decarboxylation than in the acyl-CoA binding role. Interestingly, this effect was suppressed intragenically by Arg{sup 161} {yields} Ala substitution. Our structural studies suggested that His{sup 258}, previously implicated in malonyl-ACP decarboxylation, also acts as an anchor point for a network of water molecules that we propose promotes deprotonation and transacylation of Cys{sup 122}.

  11. Purification, Characterization, and Identification of Novel Inhibitors of the β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) from Staphylococcus aureus

    PubMed Central

    He, Xin; Reynolds, Kevin A.

    2002-01-01

    Staphylococcus aureus is a versatile and dangerous pathogen and one of the major causes of community-acquired and hospital-acquired infections. The rise of multidrug-resistant strains of S. aureus requires the development of new antibiotics with previously unexploited mechanisms of action, such as inhibition of the β-ketoacyl-acyl carrier protein (ACP) synthase III (FabH). This enzyme initiates fatty acid biosynthesis in a bacterial type II fatty acid synthase, catalyzing a decarboxylative condensation between malonyl-ACP and an acyl coenzyme A (CoA) substrate and is essential for viability. We have identified only one fabH in the genome of S. aureus and have shown that it encodes a protein with 57, 40, and 34% amino acid sequence identity with the FabH proteins of Bacillus subtilis (bFabH1), Escherichia coli (ecFabH), and Mycobacterium tuberculosis (mtFabH). Additional genomic sequence analysis revealed that this S. aureus FabH (saFabH) is not mutated in certain methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) strains. saFabH was expressed in E. coli with an N-terminal polyhistidine tag and subsequently purified by metal chelate and size exclusion chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a molecular mass of 37 kDa, while gel filtration demonstrated a mass of 66.7 kDa, suggesting a noncovalent homodimeric structure for saFabH. The apparent Km for malonyl-ACP was 1.76 ± 0.40 μM, and the enzyme was active with acetyl-CoA (kcat, 16.18 min−1; Km, 6.18 ± 0.9 μM), butyryl-CoA (kcat, 42.90 min−1; Km, 2.32 ± 0.12 μM), and isobutyryl-CoA (kcat, 98.0 min−1; Km, 0.32 ± 0.04 μM). saFabH was weakly inhibited by thiolactomycin (50% inhibitory concentration [IC50], >100 μM) yet was efficiently inhibited by two new FabH inhibitors, 5-chloro-4-phenyl-[1,2]-dithiol-3-one (IC50, 1.87 ± 0.10 μM) and 4-phenyl-5-phenylimino-[1,2,4]dithiazolidin-3-one (IC50, 0.775 ± 0.08 μM). PMID

  12. Human uroporphyrinogen III synthase: NMR-based mapping of the active site.

    PubMed

    Cunha, Luis; Kuti, Miklos; Bishop, David F; Mezei, Mihaly; Zeng, Lei; Zhou, Ming-Ming; Desnick, Robert J

    2008-05-01

    Uroporphyrinogen III synthase (URO-synthase) catalyzes the cyclization and D-ring isomerization of hydroxymethylbilane (HMB) to uroporphyrinogen (URO'gen) III, the cyclic tetrapyrrole and physiologic precursor of heme, chlorophyl, and corrin. The deficient activity of human URO-synthase results in the autosomal recessive cutaneous disorder, congenital erythropoietic porphyria. Mapping of the structural determinants that specify catalysis and, potentially, protein-protein interactions is lacking. To map the active site and assess the enzyme's possible interaction in a complex with hydroxymethylbilane-synthase (HMB-synthase) and/or uroporphyrinogen-decarboxylase (URO-decarboxylase) by NMR, an efficient expression and purification procedure was developed for these cytosolic enzymes of heme biosynthesis that enabled preparation of special isotopically-labeled protein samples for NMR characterization. Using an 800 MHz instrument, assignment of the URO-synthase backbone (13)C(alpha) (100%), (1)H(alpha) (99.6%), and nonproline (1)H(N) and (15)N resonances (94%) was achieved as well as 85% of the side-chain (13)C and (1)H resonances. NMR analyses of URO-synthase titrated with competitive inhibitors N(D)-methyl-1-formylbilane (NMF-bilane) or URO'gen III, revealed resonance perturbations of specific residues lining the cleft between the two major domains of URO synthase that mapped the enzyme's active site. In silico docking of the URO-synthase crystal structure with NMF-bilane and URO'gen III was consistent with the perturbation results and provided a 3D model of the enzyme-inhibitor complex. The absence of chemical shift changes in the (15)N spectrum of URO-synthase mixed with the homogeneous HMB-synthase holoenzyme or URO-decarboxylase precluded occurrence of a stable cytosolic enzyme complex. PMID:18004775

  13. Exploiting the Biosynthetic Potential of Type III Polyketide Synthases.

    PubMed

    Lim, Yan Ping; Go, Maybelle K; Yew, Wen Shan

    2016-01-01

    Polyketides are structurally and functionally diverse secondary metabolites that are biosynthesized by polyketide synthases (PKSs) using acyl-CoA precursors. Recent studies in the engineering and structural characterization of PKSs have facilitated the use of target enzymes as biocatalysts to produce novel functionally optimized polyketides. These compounds may serve as potential drug leads. This review summarizes the insights gained from research on type III PKSs, from the discovery of chalcone synthase in plants to novel PKSs in bacteria and fungi. To date, at least 15 families of type III PKSs have been characterized, highlighting the utility of PKSs in the development of natural product libraries for therapeutic development. PMID:27338328

  14. Crystal structure and substrate specificity of the [beta]-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus

    SciTech Connect

    Qiu, Xiayang; Choudhry, Anthony E.; Janson, Cheryl A.; Grooms, Michael; Daines, Robert A.; Lonsdale, John T.; Khandekar, Sanjay S.

    2010-07-20

    {beta}-Ketoacyl-ACP synthase III (FabH), an essential enzyme for bacterial viability, catalyzes the initiation of fatty acid elongation by condensing malonyl-ACP with acetyl-CoA. We have determined the crystal structure of FabH from Staphylococcus aureus, a Gram-positive human pathogen, to 2 {angstrom} resolution. Although the overall structure of S. aureus FabH is similar to that of Escherichia coli FabH, the primer binding pocket in S. aureus FabH is significantly larger than that present in E. coli FabH. The structural differences, which agree with kinetic parameters, provide explanation for the observed varying substrate specificity for E. coli and S. aureus FabH. The rank order of activity of S. aureus FabH with various acyl-CoA primers was as follows: isobutyryl- > hexanoyl- > butyryl- > isovaleryl- >> acetyl-CoA. The availability of crystal structure may aid in designing potent, selective inhibitors of S. aureus FabH.

  15. Sucrose Synthase: Expanding Protein Function

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sucrose synthase (SUS: EC 2.4.1.13), a key enzyme in plant sucrose catabolism, is uniquely able to mobilize sucrose into multiple pathways involved in metabolic, structural, and storage functions. Our research indicates that the biological function of SUS may extend beyond its catalytic activity. Th...

  16. Rat hepatic uroporphyrinogen III co-synthase. Purification and evidence for a bound folate coenzyme participating in the biosynthesis of uroporphyrinogen III.

    PubMed Central

    Kohashi, M; Clement, R P; Tse, J; Piper, W N

    1984-01-01

    Rat hepatic uroporphyrinogen III co-synthase was isolated and purified 73-fold with a 13% yield by (NH4)2SO4 fractionation and sequential chromatography on DEAE-Sephacel, Sephadex G-100 (superfine grade) and folate-AH-Sepharose 4B. The purified co-synthase has an Mr of approx. 42 000, and is resolved into two bands, each possessing co-synthase activity, by polyacrylamide-gel electrophoresis. A factor was dissociated from the purified co-synthase. Results of both microbiological and competitive protein-binding assays suggest that it is a pteroylpolyglutamate. The isolated pteroylpolyglutamate factor was co-eluted with authentic N5-methyltetrahydropteroylheptaglutamate on DEAE-Sephacel. Uroporphyrinogen III is formed by cosynthase-free preparations of uroporphyrinogen I synthase in the presence of tetrahydropteroylglutamate. Tetrahydropeteroylheptaglutamate is also able to direct the formation of equivalent amounts of uroporphyrinogen III at a concentration approximately one-hundredth that of tetrahydropteroylmonoglutamate. These results suggest that a reduced pteroylpolyglutamate factor is associated with rat hepatic uroporphyrinogen III co-synthase, and that this may function as a coenzyme for the biosynthesis of uroporphyrinogen III. Images Fig. 5. PMID:6466301

  17. Protein preparation, crystallization and preliminary X-ray analysis of Polygonum cuspidatum bifunctional chalcone synthase/benzalacetone synthase.

    PubMed

    Lu, Heshu; Yang, Mingfeng; Liu, Chunmei; Lu, Ping; Cang, Huaixing; Ma, Lanqing

    2013-08-01

    The chalcone synthase (CHS) superfamily of type III polyketide synthases (PKSs) generate the backbones of a variety of plant secondary metabolites. An active bifunctional chalcone synthase/benzalacetone synthase (CHS/BAS) from Polygonum cuspidatum was overexpressed in Escherichia coli as a C-terminally polyhistidine-tagged fusion protein, purified to homogeneity and crystallized using polyethylene glycol 4000 as a precipitant. The production of well shaped crystals of the complex between PcPKS1 and benzalacetone was dependent on the presence of sorbitol and barium chloride as additives. The crystals belonged to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a = 80.23, b = 81.01, c = 122.89 Å, and diffracted X-rays to at least 2.0 Å resolution. PMID:23908031

  18. Type III polyketide synthase repertoire in Zingiberaceae: computational insights into the sequence, structure and evolution.

    PubMed

    Mallika, Vijayanathan; Aiswarya, Girija; Gincy, Paily Thottathil; Remakanthan, Appukuttan; Soniya, Eppurathu Vasudevan

    2016-07-01

    Zingiberaceae or 'ginger family' is the largest family in the order 'Zingiberales' with more than 1300 species in 52 genera, which are mostly distributed throughout Asia, tropical Africa and the native regions of America with their maximum diversity in Southeast Asia. Many of the members are important spice, medicinal or ornamental plants including ginger, turmeric, cardamom and kaempferia. These plants are distinguished for the highly valuable metabolic products, which are synthesised through phenylpropanoid pathway, where type III polyketide synthase is the key enzyme. In our present study, we used sequence, structural and evolutionary approaches to scrutinise the type III polyketide synthase (PKS) repertoire encoded in the Zingiberaceae family. Highly conserved amino acid residues in the sequence alignment and phylogram suggested strong relationships between the type III PKS members of Zingiberaceae. Sequence and structural level investigation of type III PKSs showed a small number of variations in the substrate binding pocket, leading to functional divergence among these PKS members. Molecular evolutionary studies indicate that type III PKSs within Zingiberaceae evolved under strong purifying selection pressure, and positive selections were rarely detected in the family. Structural modelling and protein-small molecule interaction studies on Zingiber officinale PKS 'a representative from Zingiberaceae' suggested that the protein is comparatively stable without much disorder and exhibited wide substrate acceptance. PMID:27138283

  19. A type III polyketide synthase from Wachendorfia thyrsiflora and its role in diarylheptanoid and phenylphenalenone biosynthesis.

    PubMed

    Brand, S; Hölscher, D; Schierhorn, A; Svatos, A; Schröder, J; Schneider, B

    2006-07-01

    Chalcone synthase (CHS) related type III plant polyketide synthases (PKSs) are likely to be involved in the biosynthesis of diarylheptanoids (e.g. curcumin and polycyclic phenylphenalenones), but no such activity has been reported. Root cultures from Wachendorfia thyrsiflora (Haemodoraceae) are a suitable source to search for such enzymes because they synthesize large amounts of phenylphenalenones, but no other products that are known to require CHSs or related enzymes (e.g. flavonoids or stilbenes). A homology-based RT-PCR strategy led to the identification of cDNAs for a type III PKS sharing only approximately 60% identity with typical CHSs. It was named WtPKS1 (W. thyrsiflora polyketide synthase 1). The purified recombinant protein accepted a large variety of aromatic and aliphatic starter CoA esters, including phenylpropionyl- and side-chain unsaturated phenylpropanoid-CoAs. The simplest model for the initial reaction in diarylheptanoid biosynthesis predicts a phenylpropanoid-CoA as starter and a single condensation reaction to a diketide. Benzalacetones, the expected release products, were observed only with unsaturated phenylpropanoid-CoAs, and the best results were obtained with 4-coumaroyl-CoA (80% of the products). With all other substrates, WtPKS1 performed two condensation reactions and released pyrones. We propose that WtPKS1 catalyses the first step in diarylheptanoid biosynthesis and that the observed pyrones are derailment products in the absence of downstream processing proteins. PMID:16496097

  20. A Homologue of the 3-Oxoacyl-(Acyl Carrier Protein) Synthase III Gene Located in the Glycosylation Island of Pseudomonas syringae pv. tabaci Regulates Virulence Factors via N-Acyl Homoserine Lactone and Fatty Acid Synthesis▿

    PubMed Central

    Taguchi, Fumiko; Ogawa, Yujiro; Takeuchi, Kasumi; Suzuki, Tomoko; Toyoda, Kazuhiro; Shiraishi, Tomonori; Ichinose, Yuki

    2006-01-01

    Pseudomonas syringae pv. tabaci 6605 possesses a genetic region involved in flagellin glycosylation. This region is composed of three open reading frames: orf1, orf2, and orf3. Our previous study revealed that orf1 and orf2 encode glycosyltransferases; on the other hand, orf3 has no role in posttranslational modification of flagellin. Although the function of Orf3 remained unclear, an orf3 deletion mutant (Δorf3 mutant) had reduced virulence on tobacco plants. Orf3 shows significant homology to a 3-oxoacyl-(acyl carrier protein) synthase III in the fatty acid elongation cycle. The Δorf3 mutant had a significantly reduced ability to form acyl homoserine lactones (AHLs), which are quorum-sensing molecules, suggesting that Orf3 is required for AHL synthesis. In comparison with the wild-type strain, swarming motility, biosurfactant production, and tolerance to H2O2 and antibiotics were enhanced in the Δorf3 mutant. A scanning electron micrograph of inoculated bacteria on the tobacco leaf surface revealed that there is little extracellular polymeric substance matrix surrounding the cells in the Δorf3 mutant. The phenotypes of the Δorf3 mutant and an AHL synthesis (ΔpsyI) mutant were similar, although the mutant-specific characteristics were more extreme in the Δorf3 mutant. The swarming motility of the Δorf3 mutant was greater than that of the ΔpsyI mutant. This was attributed to the synergistic effects of the overproduction of biosurfactants and/or alternative fatty acid metabolism in the Δorf3 mutant. Furthermore, the amounts of iron and biosurfactant seem to be involved in biofilm development under quorum-sensing regulation in P. syringae pv. tabaci 6605. PMID:17028280

  1. Divinyl ether synthase gene and protein, and uses thereof

    DOEpatents

    Howe, Gregg A.; Itoh, Aya

    2011-09-13

    The present invention relates to divinyl ether synthase genes, proteins, and methods of their use. The present invention encompasses both native and recombinant wild-type forms of the synthase, as well as mutants and variant forms, some of which possess altered characteristics relative to the wild-type synthase. The present invention also relates to methods of using divinyl ether synthase genes and proteins, including in their expression in transgenic organisms and in the production of divinyl ether fatty acids, and to methods of suing divinyl ether fatty acids, including in the protection of plants from pathogens.

  2. Divinyl ether synthase gene, and protein and uses thereof

    DOEpatents

    Howe, Gregg A.; Itoh, Aya

    2006-12-26

    The present invention relates to divinyl ether synthase genes, proteins, and methods of their use. The present invention encompasses both native and recombinant wild-type forms of the synthase, as well as mutants and variant forms, some of which possess altered characteristics relative to the wild-type synthase. The present invention also relates to methods of using divinyl ether synthase genes and proteins, including in their expression in transgenic organisms and in the production of divinyl ether fatty acids, and to methods of suing divinyl ether fatty acids, including in the protection of plants from pathogens.

  3. Biochemical and Structural Characterization of Germicidin Synthase: Analysis of a Type III Polyketide Synthase That Employs Acyl-ACP as a Starter Unit Donor

    SciTech Connect

    Chemler, Joseph A.; Buchholz, Tonia J.; Geders, Todd W.; Akey, David L.; Rath, Christopher M.; Chlipala, George E.; Smith, Janet L.; Sherman, David H.

    2012-08-10

    Germicidin synthase (Gcs) from Streptomyces coelicolor is a type III polyketide synthase (PKS) with broad substrate flexibility for acyl groups linked through a thioester bond to either coenzyme A (CoA) or acyl carrier protein (ACP). Germicidin synthesis was reconstituted in vitro by coupling Gcs with fatty acid biosynthesis. Since Gcs has broad substrate flexibility, we directly compared the kinetic properties of Gcs with both acyl-ACP and acyl-CoA. The catalytic efficiency of Gcs for acyl-ACP was 10-fold higher than for acyl-CoA, suggesting a strong preference toward carrier protein starter unit transfer. The 2.9 {angstrom} germicidin synthase crystal structure revealed canonical type III PKS architecture along with an unusual helical bundle of unknown function that appears to extend the dimerization interface. A pair of arginine residues adjacent to the active site affect catalytic activity but not ACP binding. This investigation provides new and surprising information about the interactions between type III PKSs and ACPs that will facilitate the construction of engineered systems for production of novel polyketides.

  4. [Cloning, expression and functional identification of a type III polyketide synthase gene from Huperzia serrata].

    PubMed

    Ye, Jin-cui; Zhang, Ping; Sun, Jie-yin; Guo, Chao-tan; Chen, Guo-shen; Abe, Ikuro; Noguchi, Hiroshi

    2011-10-01

    A cDNA encoding novel type III polyketide synthase (PKS) was cloned and sequenced from young leaves of Chinese club moss Huperzia serrata (Thunb.) Trev. by RT-PCR using degenerated primers based on the conserved sequences of known CHSs, and named as H. serrata PKS2. The terminal sequences of cDNA were obtained by the 3'- and 5'-RACE method. The full-length cDNA of H. serrata PKS2 contained a 1212 bp open reading frame encoding a 46.4 kDa protein with 404 amino acids. The deduced amino acid sequence of H. serrata PKS2 showed 50%-66% identities to those of other chalcone synthase super family enzymes of plant origin. The recombinant H. serrata PKS2 was functionally expressed in Escherichia coli with an additional hexahistidine tag at the N-terminus and showed unusually versatile catalytic potency to produce various aromatic tetraketides, including chalcones, benzophenones, phloroglucinols, and acridones. In particular, the enzyme accepted bulky starter substrates N-methylanthraniloyl-CoA, and carried out three condensations with malonyl-CoA to produce 1, 3-dihydroxy-N-methylacridone. Interestingly, H. serrata PKS2 lacks most of the consensus active site sequences with acridone synthase from Ruta graveolens (Rutaceae). PMID:22242464

  5. Identification of a cryptic type III polyketide synthase (1,3,6,8-tetrahydroxynaphthalene synthase) from Streptomyces peucetius ATCC 27952.

    PubMed

    Ghimire, Gopal Prasad; Oh, Tae-Jin; Liou, Kwangkyoung; Sohng, Jae Kyung

    2008-10-31

    We identified a 1,134-bp putative type III polyketide synthase from the sequence analysis of Streptomyces peucetius ATCC 27952, named Sp-RppA, which is characterized as 1,3,6,8-tetrahydroxynaphthalene synthase and shares 33% identity with SCO1206 from S. coelicolor A3(2) and 32% identity with RppA from S. griseus. The 1,3,6,8-tetrahydroxynaphthalene synthase is known to catalyze the sequential decarboxylative condensation, intramolecular cyclization, and aromatization of an oligoketide derived from five units of malonyl-CoA to give 1,3,6,8-tetrahydroxynaphthalene, which spontaneously oxidizes to form 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin). In this study, we report the in vivo expression and in vitro synthesis of flaviolin from purified gene product (Sp-RppA). PMID:18612244

  6. The cellulose synthase companion proteins act non-redundantly with CELLULOSE SYNTHASE INTERACTING1/POM2 and CELLULOSE SYNTHASE 6

    PubMed Central

    Endler, Anne; Schneider, Rene; Kesten, Christopher; Lampugnani, Edwin R.; Persson, Staffan

    2016-01-01

    ABSTRACT Cellulose is a cell wall constituent that is essential for plant growth and development, and an important raw material for a range of industrial applications. Cellulose is synthesized at the plasma membrane by massive cellulose synthase (CesA) complexes that track along cortical microtubules in elongating cells of Arabidopsis through the activity of the protein CELLULOSE SYNTHASE INTERACTING1 (CSI1). In a recent study we identified another family of proteins that also are associated with the CesA complex and microtubules, and that we named COMPANIONS OF CELLULOSE SYNTHASE (CC). The CC proteins protect the cellulose synthesising capacity of Arabidopsis seedlings during exposure to adverse environmental conditions by enhancing microtubule dynamics. In this paper we provide cell biology and genetic evidence that the CSI1 and the CC proteins fulfil distinct functions during cellulose synthesis. We also show that the CC proteins are necessary to aid cellulose synthesis when components of the CesA complex are impaired. These data indicate that the CC proteins have a broad role in aiding cellulose synthesis during environmental changes and when core complex components are non-functional. PMID:26829351

  7. Direct transfer of starter substrates from type I fatty acid synthase to type III polyketide synthases in phenolic lipid synthesis.

    PubMed

    Miyanaga, Akimasa; Funa, Nobutaka; Awakawa, Takayoshi; Horinouchi, Sueharu

    2008-01-22

    Alkylresorcinols and alkylpyrones, which have a polar aromatic ring and a hydrophobic alkyl chain, are phenolic lipids found in plants, fungi, and bacteria. In the Gram-negative bacterium Azotobacter vinelandii, phenolic lipids in the membrane of dormant cysts are essential for encystment. The aromatic moieties of the phenolic lipids in A. vinelandii are synthesized by two type III polyketide synthases (PKSs), ArsB and ArsC, which are encoded by the ars operon. However, details of the synthesis of hydrophobic acyl chains, which might serve as starter substrates for the type III polyketide synthases (PKSs), were unknown. Here, we show that two type I fatty acid synthases (FASs), ArsA and ArsD, which are members of the ars operon, are responsible for the biosynthesis of C(22)-C(26) fatty acids from malonyl-CoA. In vivo and in vitro reconstitution of phenolic lipid synthesis systems with the Ars enzymes suggested that the C(22)-C(26) fatty acids produced by ArsA and ArsD remained attached to the ACP domain of ArsA and were transferred hand-to-hand to the active-site cysteine residues of ArsB and ArsC. The type III PKSs then used the fatty acids as starter substrates and carried out two or three extensions with malonyl-CoA to yield the phenolic lipids. The phenolic lipids in A. vinelandii were thus found to be synthesized solely from malonyl-CoA by the four members of the ars operon. This is the first demonstration that a type I FAS interacts directly with a type III PKS through substrate transfer. PMID:18199837

  8. A type III ACC synthase, ACS7, is involved in root gravitropism in Arabidopsis thaliana

    PubMed Central

    Chang, Ing-Feng

    2013-01-01

    Ethylene is an important plant hormone that regulates developmental processes in plants. The ethylene biosynthesis pathway is a highly regulated process at both the transcriptional and post-translational level. The transcriptional regulation of these ethylene biosynthesis genes is well known. However, post-translational modifications of the key ethylene biosynthesis enzyme 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) are little understood. In vitro kinase assays were conducted on the type III ACS, AtACS7, fusion protein and peptides to determine whether the AtACS7 protein can be phosphorylated by calcium-dependent protein kinase (CDPK). AtACS7 was phosphorylated at Ser216, Thr296, and Ser299 by AtCDPK16 in vitro. To investigate further the function of the ACS7 gene in Arabidopsis, an acs7-1 loss-of-function mutant was isolated. The acs7-1 mutant exhibited less sensitivity to the inhibition of root gravitropism by treatment with the calcium chelator ethylene glycol tetraacetic acid (EGTA). Seedlings were treated with gradient concentrations of ACC. The results showed that a certain concentration of ethylene enhanced the gravity response. Moreover, the acs7-1 mutant was less sensitive to inhibition of the gravity response by treatment with the auxin polar transport inhibitor 1-naphthylphthalamic acid, but exogenous ACC application recovered root gravitropism. Altogether, the results indicate that AtACS7 is involved in root gravitropism in a calcium-dependent manner in Arabidopsis. PMID:23943848

  9. Biosynthesis of Dictyostelium discoideum differentiation-inducing factor by a hybrid type I fatty acid-type III polyketide synthase.

    PubMed

    Austin, Michael B; Saito, Tamao; Bowman, Marianne E; Haydock, Stephen; Kato, Atsushi; Moore, Bradley S; Kay, Robert R; Noel, Joseph P

    2006-09-01

    Differentiation-inducing factors (DIFs) are well known to modulate formation of distinct communal cell types from identical Dictyostelium discoideum amoebas, but DIF biosynthesis remains obscure. We report complimentary in vivo and in vitro experiments identifying one of two approximately 3,000-residue D. discoideum proteins, termed 'steely', as responsible for biosynthesis of the DIF acylphloroglucinol scaffold. Steely proteins possess six catalytic domains homologous to metazoan type I fatty acid synthases (FASs) but feature an iterative type III polyketide synthase (PKS) in place of the expected FAS C-terminal thioesterase used to off load fatty acid products. This new domain arrangement likely facilitates covalent transfer of steely N-terminal acyl products directly to the C-terminal type III PKS active sites, which catalyze both iterative polyketide extension and cyclization. The crystal structure of a steely C-terminal domain confirms conservation of the homodimeric type III PKS fold. These findings suggest new bioengineering strategies for expanding the scope of fatty acid and polyketide biosynthesis. PMID:16906151

  10. Biosynthesis of Dictyostelium discoideum differentiation-inducing factor by a hybrid type I fatty acid–type III polyketide synthase

    PubMed Central

    Austin, Michael B; Saito, Tamao; Bowman, Marianne E; Haydock, Stephen; Kato, Atsushi; Moore, Bradley S; Kay, Robert R; Noel, Joseph P

    2010-01-01

    Differentiation-inducing factors (DIFs) are well known to modulate formation of distinct communal cell types from identical Dictyostelium discoideum amoebas, but DIF biosynthesis remains obscure. We report complimentary in vivo and in vitro experiments identifying one of two ~3,000-residue D. discoideum proteins, termed ‘steely’, as responsible for biosynthesis of the DIF acylphloroglucinol scaffold. Steely proteins possess six catalytic domains homologous to metazoan type I fatty acid synthases (FASs) but feature an iterative type III polyketide synthase (PKS) in place of the expected FAS C-terminal thioesterase used to off load fatty acid products. This new domain arrangement likely facilitates covalent transfer of steely N-terminal acyl products directly to the C-terminal type III PKS active sites, which catalyze both iterative polyketide extension and cyclization. The crystal structure of a steely C-terminal domain confirms conservation of the homodimeric type III PKS fold. These findings suggest new bioengineering strategies for expanding the scope of fatty acid and polyketide biosynthesis. PMID:16906151

  11. Crystallization and preliminary crystallographic analysis of an octaketide-producing plant type III polyketide synthase

    SciTech Connect

    Morita, Hiroyuki; Kondo, Shin; Kato, Ryohei; Wanibuchi, Kiyofumi; Noguchi, Hiroshi; Sugio, Shigetoshi; Abe, Ikuro; Kohno, Toshiyuki

    2007-11-01

    Octaketide synthase from A. arborescens has been overexpressed in E. coli, purified and crystallized. Diffraction data have been collected to 2.6 Å. Octaketide synthase (OKS) from Aloe arborescens is a plant-specific type III polyketide synthase that produces SEK4 and SEK4b from eight molecules of malonyl-CoA. Recombinant OKS expressed in Escherichia coli was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to space group I422, with unit-cell parameters a = b = 110.2, c = 281.4 Å, α = β = γ = 90.0°. Diffraction data were collected to 2.6 Å resolution using synchrotron radiation at BL24XU of SPring-8.

  12. Identification of sucrose synthase as an actin-binding protein

    NASA Technical Reports Server (NTRS)

    Winter, H.; Huber, J. L.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.

  13. Expression, purification and crystallization of a fungal type III polyketide synthase that produces the csypyrones

    PubMed Central

    Yang, Dengfeng; Mori, Takahiro; Matsui, Takashi; Hashimoto, Makoto; Morita, Hiroyuki; Fujii, Isao; Abe, Ikuro

    2014-01-01

    CsyB from Aspergillus oryzae is a novel type III polyketide synthase that catalyzes the formation of csypyrone B1 [4-(3-acetyl-4-hydroxy-2-oxo-2H-pyran-6-yl)butyric acid] from fatty acyl-CoA, malonyl-CoA and acetoacetyl-CoA. Recombinant CsyB expressed in Escherichia coli was crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space P21, with unit-cell parameters a = 70.0, b = 104.8, c = 73.5 Å, β = 114.4°. PMID:24915080

  14. Protein-water dynamics in antifreeze protein III activity

    NASA Astrophysics Data System (ADS)

    Xu, Yao; Bäumer, Alexander; Meister, Konrad; Bischak, Connor G.; DeVries, Arthur L.; Leitner, David M.; Havenith, Martina

    2016-03-01

    We combine Terahertz absorption spectroscopy (THz) and molecular dynamics (MD) simulations to investigate the underlying molecular mechanism for the antifreeze activity of one class of antifreeze protein, antifreeze protein type III (AFP-III) with a focus on the collective water hydrogen bond dynamics near the protein. After summarizing our previous work on AFPs, we present a new investigation of the effects of cosolutes on protein antifreeze activity by adding sodium citrate to the protein solution of AFP-III. Our results reveal that for AFP-III, unlike some other AFPs, the addition of the osmolyte sodium citrate does not affect the hydrogen bond dynamics at the protein surface significantly, as indicated by concentration dependent THz measurements. The present data, in combination with our previous THz measurements and molecular simulations, confirm that while long-range solvent perturbation is a necessary condition for the antifreeze activity of AFP-III, the local binding affinity determines the size of the hysteresis.

  15. Molecular cloning, modeling, and site-directed mutagenesis of type III polyketide synthase from Sargassum binderi (Phaeophyta).

    PubMed

    Baharum, Hariyanti; Morita, Hiroyuki; Tomitsuka, Akifumi; Lee, Fong-Chin; Ng, Kim-Yong; Rahim, Raha Abdul; Abe, Ikuro; Ho, Chai-Ling

    2011-10-01

    Type III polyketide synthases (PKSs) produce an array of metabolites with diverse functions. In this study, we have cloned the complete reading frame encoding type III PKS (SbPKS) from a brown seaweed, Sargassum binderi, and characterized the activity of its recombinant protein biochemically. The deduced amino acid sequence of SbPKS is 414 residues in length, sharing a higher sequence similarity with bacterial PKSs (38% identity) than with plant PKSs. The Cys-His-Asn catalytic triad of PKS is conserved in SbPKS with differences in some of the residues lining the active and CoA binding sites. The wild-type SbPKS displayed broad starter substrate specificity to aliphatic long-chain acyl-CoAs (C(6)-C(14)) to produce tri- and tetraketide pyrones. Mutations at H(331) and N(364) caused complete loss of its activity, thus suggesting that these two residues are the catalytic residues for SbPKS as in other type III PKSs. Furthermore, H227G, H227G/L366V substitutions resulted in increased tetraketide-forming activity, while wild-type SbPKS produces triketide α-pyrone as a major product. On the other hand, mutant H227G/L366V/F93A/V95A demonstrated a dramatic decrease of tetraketide pyrone formation. These observations suggest that His(227) and Leu(366) play an important role for the polyketide elongation reaction in SbPKS. The conformational changes in protein structure especially the cavity of the active site may have more significant effect to the activity of SbPKS compared with changes in individual residues. PMID:21181422

  16. Class III viral membrane fusion proteins

    PubMed Central

    Backovic, Marija

    2010-01-01

    SUMMARY Accumulating structural studies of viral fusion glycoproteins have revealed unanticipated structural relationships between unrelated virus families and allowed the grouping of these membrane fusogens into three distinct classes. Here we review the newly identified group of class III viral fusion proteins, whose members include fusion proteins from rhabdoviruses, herpesviruses and baculoviruses. While clearly related in structure, the class III viral fusion proteins exhibit distinct structural features in their architectures as well as in their membrane-interacting fusion loops, which are likely related to their virus-specific differences in cellular entry. Further study of the similarities and differences in the class III viral fusion glycoproteins may provide greater insights into protein:membrane interactions that are key to promoting efficient bilayer fusion during virus entry. PMID:19356922

  17. Distinct Structural Elements Dictate the Specificity of the Type III Pentaketide Synthase from Neurospora crassa

    SciTech Connect

    Rubin-Pitel, Sheryl B.; Zhang, Houjin; Vu, Trang; Brunzelle, Joseph S.; Zhao, Huimin; Nair, Satish K.

    2009-01-15

    The fungal type III polyketide synthase 2'-oxoalkylresorcyclic acid synthase (ORAS) primes with a range of acyl-Coenzyme A thioesters (C{sub 4}--C{sub 20}) and extends using malonyl-Coenzyme A to produce pyrones, resorcinols, and resorcylic acids. To gain insight into this unusual substrate specificity and product profile, we have determined the crystal structures of ORAS to 1.75 {angstrom} resolution, the Phe-252{yields}Gly site-directed mutant to 2.1 {angstrom} resolution, and a binary conplex of ORAS with eicosanoic acid to 2.0 {angstrom} resolution. The structures reveal a distinct rearrangement of structural elements near the active site that allows accomodation of long-chain fatty acid esters and a reorientation of the gating mechanism that controls cyclization and polyketide chain length. The roles of these structural elements are further elucidated by characterization of various structure-based site-directed variants. These studies establish an unexpected plasticity to the PKS fold, unanticipated from structural studies of other members of this enzyme family.

  18. Phasin proteins activate Aeromonas caviae polyhydroxyalkanoate (PHA) synthase but not Ralstonia eutropha PHA synthase.

    PubMed

    Ushimaru, Kazunori; Motoda, Yoko; Numata, Keiji; Tsuge, Takeharu

    2014-05-01

    In this study, we performed in vitro and in vivo activity assays of polyhydroxyalkanoate (PHA) synthases (PhaCs) in the presence of phasin proteins (PhaPs), which revealed that PhaPs are activators of PhaC derived from Aeromonas caviae (PhaCAc). In in vitro assays, among the three PhaCs tested, PhaCAc was significantly activated when PhaPs were added at the beginning of polymerization (prepolymerization PhaCAc), whereas the prepolymerization PhaCRe (derived from Ralstonia eutropha) and PhaCDa (Delftia acidovorans) showed reduced activity with PhaPs. The PhaP-activated PhaCAc showed a slight shift of substrate preference toward 3-hydroxyhexanoyl-CoA (C6). PhaPAc also activated PhaCAc when it was added during polymerization (polymer-elongating PhaCAc), while this effect was not observed for PhaCRe. In an in vivo assay using Escherichia coli TOP10 as the host strain, the effect of PhaPAc expression on PHA synthesis by PhaCAc or PhaCRe was examined. As PhaPAc expression increased, PHA production was increased by up to 2.3-fold in the PhaCAc-expressing strain, whereas it was slightly increased in the PhaCRe-expressing strain. Taken together, this study provides evidence that PhaPs function as activators for PhaCAc both in vitro and in vivo but do not activate PhaCRe. This activating effect may be attributed to the new role of PhaPs in the polymerization reaction by PhaCAc. PMID:24584238

  19. Molecular and biochemical characterization of benzalacetone synthase and chalcone synthase genes and their proteins from raspberry (Rubus idaeus L.).

    PubMed

    Zheng, Desen; Hrazdina, Geza

    2008-02-15

    Two new members of the polyketide synthase (PKS) gene family (RiPKS4 and RiPKS5) were cloned from raspberry fruits (Rubus idaeus L., cv Royalty) and expressed in Escherichia coli. Characterization of the recombinant enzyme products indicated that RiPKS4 is a bifunctional polyketide synthase producing both 4-hydroxybenzalacetone and naringenin chalcone. The recombinant RiPKS4 protein, like the native protein from raspberry fruits [W. Borejsza-Wysocki, G. Hrazdina, Plant Physiol. 1996;110: 791-799] accepted p-coumaryl-CoA and ferulyl-CoA as starter substrates and catalyzed the formation of both naringenin chalcone, 4-hydroxy-benzalacetone and 3-methoxy-4-hydroxy-benzalacetone. Although activity of RiPKS4 was higher with ferulyl-CoA than with p-coumaryl-CoA, the corresponding product, 3-methoxy-4-hydroxy phenylbutanone could not be detected in raspberries to date. Sequence analysis of the genes and proteins suggested that this feature of RiPKS4 was created by variation in the C-terminus due to DNA recombination at the 3' region of its coding sequence. RiPKS5 is a typical chalcone synthase (CHS) that uses p-coumaryl-CoA only as starter substrate and produces naringenin chalcone exclusively as the reaction product. PMID:18068110

  20. Functional Promiscuity of Two Divergent Paralogs of Type III Plant Polyketide Synthases.

    PubMed

    Pandith, Shahzad A; Dhar, Niha; Rana, Satiander; Bhat, Wajid Waheed; Kushwaha, Manoj; Gupta, Ajai P; Shah, Manzoor A; Vishwakarma, Ram; Lattoo, Surrinder K

    2016-08-01

    Plants effectively defend themselves against biotic and abiotic stresses by synthesizing diverse secondary metabolites, including health-protective flavonoids. These display incredible chemical diversity and ubiquitous occurrence and confer impeccable biological and agricultural applications. Chalcone synthase (CHS), a type III plant polyketide synthase, is critical for flavonoid biosynthesis. It catalyzes acyl-coenzyme A thioesters to synthesize naringenin chalcone through a polyketidic intermediate. The functional divergence among the evolutionarily generated members of a gene family is pivotal in driving the chemical diversity. Against this backdrop, this study was aimed to functionally characterize members of the CHS gene family from Rheum emodi, an endangered and endemic high-altitude medicinal herb of northwestern Himalayas. Two full-length cDNAs (1,179 bp each), ReCHS1 and ReCHS2, encoding unique paralogs were isolated and characterized. Heterologous expression and purification in Escherichia coli, bottom-up proteomic characterization, high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis, and enzyme kinetic studies using five different substrates confirmed their catalytic potential. Phylogenetic analysis revealed the existence of higher synonymous mutations in the intronless divergents of ReCHS. ReCHS2 displayed significant enzymatic efficiency (Vmax/Km) with different substrates. There were significant spatial and altitudinal variations in messenger RNA transcript levels of ReCHSs correlating positively with metabolite accumulation. Furthermore, the elicitations in the form of methyl jasmonate, salicylic acid, ultraviolet B light, and wounding, chosen on the basis of identified cis-regulatory promoter elements, presented considerable differences in the transcript profiles of ReCHSs. Taken together, our results demonstrate differential propensities of CHS paralogs in terms of the accumulation of flavonoids and

  1. Structural Analysis of Protein-Protein Interactions in Type I Polyketide Synthases

    PubMed Central

    Xu, Wei; Qiao, Kangjian; Tang, Yi

    2013-01-01

    Polyketide synthases (PKSs) are responsible for synthesizing a myriad of natural products with agricultural, medicinal relevance. The PKSs consist of multiple functional domains of which each can catalyze a specified chemical reaction leading to the synthesis of polyketides. Biochemical studies showed that protein-substrate and protein-protein interactions play crucial roles in these complex regio-/stereo- selective biochemical processes. Recent developments on X-ray crystallography and protein NMR techniques have allowed us to understand the biosynthetic mechanism of these enzymes from their structures. These structural studies have facilitated the elucidation of sequence-function relationship of PKSs and will ultimately contribute to the prediction of product structure. This review will focus on the current knowledge of type I PKS structures and the protein-protein interactions in this system. PMID:23249187

  2. Crystallization and preliminary crystallographic analysis of a novel plant type III polyketide synthase that produces pentaketide chromone

    SciTech Connect

    Morita, Hiroyuki; Kondo, Shin; Abe, Tsuyoshi; Noguchi, Hiroshi; Sugio, Shigetoshi; Abe, Ikuro; Kohno, Toshiyuki

    2006-09-01

    Pentaketide chromone synthase from A. arborescens has been overexpressed in E. coli, purified and crystallized. Diffraction data have been collected to 1.6 Å. Pentaketide chromone synthase (PCS) from Aloe arborescens is a novel plant-specific type III polyketide synthase that catalyzes the formation of 5,7-dihydroxy-2-methylchromone from five molecules of malonyl-CoA. Recombinant PCS expressed in Escherichia coli was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to space group P2{sub 1}, with unit-cell parameters a = 73.2, b = 88.4, c = 70.0 Å, α = γ = 90.0, β = 95.6°. Diffraction data were collected to 1.6 Å resolution using synchrotron radiation at BL24XU of SPring-8.

  3. Glycogen Synthase Kinase 3β Interaction Protein Functions as an A-kinase Anchoring Protein*

    PubMed Central

    Hundsrucker, Christian; Skroblin, Philipp; Christian, Frank; Zenn, Hans-Michael; Popara, Viola; Joshi, Mangesh; Eichhorst, Jenny; Wiesner, Burkhard; Herberg, Friedrich W.; Reif, Bernd; Rosenthal, Walter; Klussmann, Enno

    2010-01-01

    A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3β interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3β (glycogen synthase kinase 3β). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3β by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3β and thereby provides a mechanism for the integration of PKA and GSK3β signaling pathways. PMID:20007971

  4. Mitochondrial protein sorting as a therapeutic target for ATP synthase disorders

    PubMed Central

    Aiyar, Raeka S.; Bohnert, Maria; Duvezin-Caubet, Stéphane; Voisset, Cécile; Gagneur, Julien; Fritsch, Emilie S.; Couplan, Elodie; von der Malsburg, Karina; Funaya, Charlotta; Soubigou, Flavie; Courtin, Florence; Suresh, Sundari; Kucharczyk, Roza; Evrard, Justine; Antony, Claude; St.Onge, Robert P.; Blondel, Marc; di Rago, Jean-Paul; van der Laan, Martin; Steinmetz, Lars M.

    2014-01-01

    Mitochondrial diseases are systemic, prevalent and often fatal; yet treatments remain scarce. Identifying molecular intervention points that can be therapeutically targeted remains a major challenge, which we confronted via a screening assay we developed. Using yeast models of mitochondrial ATP synthase disorders, we screened a drug repurposing library, and applied genomic and biochemical techniques to identify pathways of interest. Here we demonstrate that modulating the sorting of nuclear-encoded proteins into mitochondria, mediated by the TIM23 complex, proves therapeutic in both yeast and patient-derived cells exhibiting ATP synthase deficiency. Targeting TIM23-dependent protein sorting improves an array of phenotypes associated with ATP synthase disorders, including biogenesis and activity of the oxidative phosphorylation machinery. Our study establishes mitochondrial protein sorting as an intervention point for ATP synthase disorders, and because of the central role of this pathway in mitochondrial biogenesis, it holds broad value for the treatment of mitochondrial diseases. PMID:25519239

  5. Mitochondrial protein sorting as a therapeutic target for ATP synthase disorders.

    PubMed

    Aiyar, Raeka S; Bohnert, Maria; Duvezin-Caubet, Stéphane; Voisset, Cécile; Gagneur, Julien; Fritsch, Emilie S; Couplan, Elodie; von der Malsburg, Karina; Funaya, Charlotta; Soubigou, Flavie; Courtin, Florence; Suresh, Sundari; Kucharczyk, Roza; Evrard, Justine; Antony, Claude; St Onge, Robert P; Blondel, Marc; di Rago, Jean-Paul; van der Laan, Martin; Steinmetz, Lars M

    2014-01-01

    Mitochondrial diseases are systemic, prevalent and often fatal; yet treatments remain scarce. Identifying molecular intervention points that can be therapeutically targeted remains a major challenge, which we confronted via a screening assay we developed. Using yeast models of mitochondrial ATP synthase disorders, we screened a drug repurposing library, and applied genomic and biochemical techniques to identify pathways of interest. Here we demonstrate that modulating the sorting of nuclear-encoded proteins into mitochondria, mediated by the TIM23 complex, proves therapeutic in both yeast and patient-derived cells exhibiting ATP synthase deficiency. Targeting TIM23-dependent protein sorting improves an array of phenotypes associated with ATP synthase disorders, including biogenesis and activity of the oxidative phosphorylation machinery. Our study establishes mitochondrial protein sorting as an intervention point for ATP synthase disorders, and because of the central role of this pathway in mitochondrial biogenesis, it holds broad value for the treatment of mitochondrial diseases. PMID:25519239

  6. Biocatalytic role of potato starch synthase III for α-glucan biosynthesis in Synechocystis sp. PCC6803 mutants.

    PubMed

    Yoo, Sang-Ho; Lee, Byung-Hoo; Li, Li; Perris, Shayani D N; Spalding, Martin H; Han, Sang Yun; Jane, Jay-lin

    2015-11-01

    A potato starch synthase III (PSSIII) was expressed in the Synechocystis mutants deficient in either glycogen synthase I (M1) or II (M2) to replenish α-(1,4) linkage synthesizing activity, resulting in new mutants, PM1 and PM2, respectively. These mutants were applied to study the role of exogenous plant starch synthase for starch/glycogen biosynthesis mechanism established in the cyanobacteria. The remaining glycogen synthase genes in PM1 and PM2 were further disrupted to make the mutants PM12 and PM21 which contained PSSIII as the sole glycogen/starch synthase. Among wild type and mutants, there were no significant differences in the amount of α-glucan produced. All the mutants harboring active PSSIII produced α-glucans with relatively much shorter and less longer α-1,4 chains than wild-type glycogen, which was exactly in accordance with the increase in glycogen branching enzyme activity. In fact, α-glucan structure of PM1 was very similar to those of PM12 and PM21, and PM2 had more intermediate chains than M2. This result suggests PSSIII may have distributive elongation property during α-glucan synthesis. In conclusion, the Synechocystis as an expression model system of plant enzymes can be applied to determine the role of starch synthesizing enzymes and their association during α-glucan synthesis. PMID:26358554

  7. Congenital erythropoietic porphyria: identification and expression of 10 mutations in the uroporphyrinogen III synthase gene.

    PubMed Central

    Xu, W; Warner, C A; Desnick, R J

    1995-01-01

    To investigate the molecular basis of the phenotypic heterogeneity in congenital erythropoietic porphyria, the mutations in the uroporphyrinogen III synthase gene from unrelated patients were determined. Six missense (L4F, Y19C, V82F, V99A, A104V, and G225S), a nonsense (Q249X), a frameshift (633insA), and two splicing mutations (IVS2+1 and IVS9 delta A + 4) were identified. When L4F, Y19C, V82F, V99A, A104V, 633insA, G225S, and Q249X were expressed in Escherichia coli, only the V82F, V99A, and A104V alleles expressed residual enzymatic activity. Of note, the V82F mutation, which occurs adjacent to the 5' donor site of intron 4, resulted in approximately 54% aberrantly spliced transcripts with exon 4 deleted. Thus, this novel exonic single-base substitution caused two lesions, a missense mutation and an aberrantly spliced transcript. Of the splicing mutations, the IVS2+1 allele produced a single transcript with exon 2 deleted, whereas the IVS9 delta A+4 allele was alternatively spliced, approximately 26% being normal transcripts and the remainder with exon 9 deleted. The amount of residual activity expressed by each allele provided a basis to correlate genotype with disease severity, thereby permitting genotype/phenotype predictions in this clinically heterogeneous disease. Images PMID:7860775

  8. Composition of clusters and building blocks in amylopectins from maize mutants deficient in starch synthase III.

    PubMed

    Zhu, Fan; Bertoft, Eric; Seetharaman, Koushik

    2013-12-18

    Branches in amylopectin are distributed along the backbone. Units of the branches are building blocks (smaller) and clusters (larger) based on the distance between branches. In this study, composition of clusters and building blocks of amylopectins from dull1 maize mutants deficient in starch synthase III (SSIII) with a common genetic background (W64A) were characterized and compared with the wild type. Clusters were produced from amylopectins by partial hydrolysis using α-amylase of Bacillus amyloliquefaciens and were subsequently treated with phosphorylase a and β-amylase to produce φ,β-limit dextrins. Clusters were further extensively hydrolyzed with the α-amylase to produce building blocks. Structures of clusters and building blocks were analyzed by diverse chromatographic techniques. The results showed that the dull1 mutation resulted in larger clusters with more singly branched building blocks. The average cluster contained ~5.4 blocks in dull1 mutants and ~4.2 blocks in the wild type. The results are compared with previous results from SSIII-deficient amo1 barley and suggest fundamental differences in the cluster structures. PMID:24229421

  9. Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases.

    PubMed

    Klaus, Maja; Ostrowski, Matthew P; Austerjost, Jonas; Robbins, Thomas; Lowry, Brian; Cane, David E; Khosla, Chaitan

    2016-07-29

    The potential for recombining intact polyketide synthase (PKS) modules has been extensively explored. Both enzyme-substrate and protein-protein interactions influence chimeric PKS activity, but their relative contributions are unclear. We now address this issue by studying a library of 11 bimodular and 8 trimodular chimeric PKSs harboring modules from the erythromycin, rifamycin, and rapamycin synthases. Although many chimeras yielded detectable products, nearly all had specific activities below 10% of the reference natural PKSs. Analysis of selected bimodular chimeras, each with the same upstream module, revealed that turnover correlated with the efficiency of intermodular chain translocation. Mutation of the acyl carrier protein (ACP) domain of the upstream module in one chimera at a residue predicted to influence ketosynthase-ACP recognition led to improved turnover. In contrast, replacement of the ketoreductase domain of the upstream module by a paralog that produced the enantiomeric ACP-bound diketide caused no changes in processing rates for each of six heterologous downstream modules compared with those of the native diketide. Taken together, these results demonstrate that protein-protein interactions play a larger role than enzyme-substrate recognition in the evolution or design of catalytically efficient chimeric PKSs. PMID:27246853

  10. Enhanced acetohydroxy acid synthase III activity in an ilvH mutant of Escherichia coli K-12.

    PubMed Central

    Ricca, E; Limauro, D; Lago, C T; de Felice, M

    1988-01-01

    The acetohydroxy acid synthase III isozyme, which catalyzes the first common step in the biosynthesis of isoleucine, leucine, and valine in Escherichia coli K-12, is composed of two subunits, the ilvI and ilvH gene products. A missense mutation in ilvH (ilvH612), which reduced the sensitivity of the enzyme to the end product inhibition by valine, also increased its specific activity and lowered the Km for alpha-acetolactate synthesis. The mutation increased the sensitivity of acetohydroxy acid synthase III to dialysis and heat treatment and reduced the requirement for thiamine pyrophosphate addition to the assay mixture for activity. A strain carrying the ilvH612 mutation grew better than a homologous ilvH+ strain in the presence of leucine. The data indicate that this is a consequence of a more active acetohydroxy acid synthase III isozyme rather than the result of an alteration of the leucine-mediated repression of the ilvIH operon. PMID:3053650

  11. Cloning and Structure-Function Analyses of Quinolone- and Acridone-producing Novel Type III Polyketide Synthases from Citrus microcarpa*

    PubMed Central

    Mori, Takahiro; Shimokawa, Yoshihiko; Matsui, Takashi; Kinjo, Keishi; Kato, Ryohei; Noguchi, Hiroshi; Sugio, Shigetoshi; Morita, Hiroyuki; Abe, Ikuro

    2013-01-01

    Two novel type III polyketide synthases, quinolone synthase (QNS) and acridone synthase (ACS), were cloned from Citrus microcarpa (Rutaceae). The deduced amino acid sequence of C. microcarpa QNS is unique, and it shared only 56–60% identities with C. microcarpa ACS, Medicago sativa chalcone synthase (CHS), and the previously reported Aegle marmelos QNS. In contrast to the quinolone- and acridone-producing A. marmelos QNS, C. microcarpa QNS produces 4-hydroxy-N-methylquinolone as the “single product” by the one-step condensation of N-methylanthraniloyl-CoA and malonyl-CoA. However, C. microcarpa ACS shows broad substrate specificities and produces not only acridone and quinolone but also chalcone, benzophenone, and phloroglucinol from 4-coumaroyl-CoA, benzoyl-CoA, and hexanoyl-CoA, respectively. Furthermore, the x-ray crystal structures of C. microcarpa QNS and ACS, solved at 2.47- and 2.35-Å resolutions, respectively, revealed wide active site entrances in both enzymes. The wide active site entrances thus provide sufficient space to facilitate the binding of the bulky N-methylanthraniloyl-CoA within the catalytic centers. However, the active site cavity volume of C. microcarpa ACS (760 Å3) is almost as large as that of M. sativa CHS (750 Å3), and ACS produces acridone by employing an active site cavity and catalytic machinery similar to those of CHS. In contrast, the cavity of C. microcarpa QNS (290 Å3) is significantly smaller, which makes this enzyme produce the diketide quinolone. These results as well as mutagenesis analyses provided the first structural bases for the anthranilate-derived production of the quinolone and acridone alkaloid by type III polyketide synthases. PMID:23963450

  12. The Saccharomyces cerevisiae FKS1 (ETG1) gene encodes an integral membrane protein which is a subunit of 1,3-beta-D-glucan synthase.

    PubMed Central

    Douglas, C M; Foor, F; Marrinan, J A; Morin, N; Nielsen, J B; Dahl, A M; Mazur, P; Baginsky, W; Li, W; el-Sherbeini, M

    1994-01-01

    In Saccharomyces cerevisiae, mutations in FKS1 confer hypersensitivity to the immunosuppressants FK506 and cyclosporin A, while mutations in ETG1 confer resistance to the cell-wall-active echinocandins (inhibitors of 1,3-beta-D-glucan synthase) and, in some cases, concomitant hypersensitivity to the chitin synthase inhibitor nikkomycin Z. The FKS1 and ETG1 genes were cloned by complementation of these phenotypes and were found to be identical. Disruption of the gene results in (i) a pronounced slow-growth phenotype, (ii) hypersensitivity to FK506 and cyclosporin A, (iii) a slight increase in sensitivity to echinocandin, and (iv) a significant reduction in 1,3-beta-D-glucan synthase activity in vitro. The nucleotide sequence encodes a 215-kDa polypeptide predicted to be an integral membrane protein with 16 transmembrane helices, consistent with previous observations that the etg1-1 mutation results in echinocandin-resistant glucan synthase activity associated with the nonextractable membrane fraction of the enzyme. These results suggest that FKS1 encodes a subunit of 1,3-beta-D-glucan synthase. The residual activity present in the disruption mutant, the nonessential nature of the gene, and results of Southern blot hybridization analysis point to the existence of a glucan synthase isozyme. Images Fig. 1 Fig. 3 Fig. 5 PMID:7528927

  13. Identification of a cellulose synthase-associated protein required for cellulose biosynthesis

    PubMed Central

    Gu, Ying; Kaplinsky, Nick; Bringmann, Martin; Cobb, Alex; Carroll, Andrew; Sampathkumar, Arun; Baskin, Tobias I.; Persson, Staffan; Somerville, Chris R.

    2010-01-01

    Cellulose synthase-interactive protein 1 (CSI1) was identified in a two-hybrid screen for proteins that interact with cellulose synthase (CESA) isoforms involved in primary plant cell wall synthesis. CSI1 encodes a 2,150-amino acid protein that contains 10 predicted Armadillo repeats and a C2 domain. Mutations in CSI1 cause defective cell elongation in hypocotyls and roots and reduce cellulose content. CSI1 is associated with CESA complexes, and csi1 mutants affect the distribution and movement of CESA complexes in the plasma membrane. PMID:20616083

  14. Identification of a type III polyketide synthase involved in the biosynthesis of spirolaxine.

    PubMed

    Sun, Lei; Wang, Siyuan; Zhang, Shuwei; Yu, Dayu; Qin, Yuhui; Huang, Huiyong; Wang, Wei; Zhan, Jixun

    2016-08-01

    Spirolaxine is a natural product isolated from Sporotrichum laxum ATCC 15155, which has shown a variety of biological activities including promising anti-Helicobacter pylori property. To understand how this compound is biosynthesized, the genome of S. laxum was sequenced. Analysis of the genome sequence revealed two putative type III polyketide synthase (PKS) genes in this strain, Sl-pks1 and Sl-pks2, which are located adjacent to each other (~2.0 kb apart) in a tail-to-tail arrangement. Disruption of these two genes revealed that Sl-PKS2 is the dedicated PKS involved in the biosynthesis of spirolaxine. The intron-free Sl-pks2 gene was amplified from the cDNA of S. laxum and ligated into the expression vector pET28a for expression in Escherichia coli BL21-CodonPlus (DE3)-RIL. The major products of Sl-PKS2 in E. coli were characterized as alkylresorcinols that contain a C13-C17 saturated or unsaturated hydrocarbon side chain based on the spectral data. This enzyme was purified and reacted with malonyl-CoA and a series of fatty acyl-SNACs (C6-C10). Corresponding alkylresorcinols were formed from the decarboxylation of the synthesized tetraketide resorcylic acids, together with fatty acyl-primed triketide and tetraketide pyrones as byproducts. This work provides important information about the PKS involved in the biosynthesis of spirolaxine, which will facilitate further understanding and engineering of the biosynthetic pathway of this medicinally important molecule. PMID:27023915

  15. Bacteriocin release protein-mediated secretory expression of recombinant chalcone synthase in Escherichia coli.

    PubMed

    Zakaria, Iffah Izzati; Rahman, Raja Noor Zaliha Raja Abdul; Salleh, Abu Bakar; Basri, Mahiran

    2011-09-01

    Flavonoids are secondary metabolites synthesized by plants shown to exhibit health benefits such as anti-inflammatory, antioxidant, and anti-tumor effects. Thus, due to the importance of this compound, several enzymes involved in the flavonoid pathway have been cloned and characterized in Escherichia coli. However, the formation of inclusion bodies has become a major disadvantage of this approach. As an alternative, chalcone synthase from Physcomitrella patens was secreted into the medium using a bacteriocin release protein expression vector. Secretion of P. patens chalcone synthase into the culture media was achieved by co-expression with a psW1 plasmid encoding bacteriocin release protein in E. coli Tuner (DE3) plysS. The optimized conditions, which include the incubation of cells for 20 h with 40 ng/ml mitomycin C at OD(600) induction time of 0.5 was found to be the best condition for chalcone synthase secretion. PMID:21633820

  16. Novel protein-protein interaction between spermidine synthase and S-adenosylmethionine decarboxylase from Leishmania donovani.

    PubMed

    Mishra, Arjun K; Agnihotri, Pragati; Srivastava, Vijay Kumar; Pratap, J Venkatesh

    2015-01-01

    Polyamine biosynthesis pathway has long been considered an essential drug target for trypanosomatids including Leishmania. S-adenosylmethionine decarboxylase (AdoMetDc) and spermidine synthase (SpdSyn) are enzymes of this pathway that catalyze successive steps, with the product of the former, decarboxylated S-adenosylmethionine (dcSAM), acting as an aminopropyl donor for the latter enzyme. Here we have explored the possibility of and identified the protein-protein interaction between SpdSyn and AdoMetDc. The protein-protein interaction has been identified using GST pull down assay. Isothermal titration calorimetry reveals that the interaction is thermodynamically favorable. Fluorescence spectroscopy studies also confirms the interaction, with SpdSyn exhibiting a change in tertiary structure with increasing concentrations of AdoMetDc. Size exclusion chromatography suggests the presence of the complex as a hetero-oligomer. Taken together, these results suggest that the enzymes indeed form a heteromer. Computational analyses suggest that this complex differs significantly from the corresponding human complex, implying that this complex could be a better therapeutic target than the individual enzymes. PMID:25511700

  17. Study of Class I and Class III Polyhydroxyalkanoate (PHA) Synthases with Substrates Containing a Modified Side Chain.

    PubMed

    Jia, Kaimin; Cao, Ruikai; Hua, Duy H; Li, Ping

    2016-04-11

    Polyhydroxyalkanoates (PHAs) are carbon and energy storage polymers produced by a variety of microbial organisms under nutrient-limited conditions. They have been considered as an environmentally friendly alternative to oil-based plastics due to their renewability, versatility, and biodegradability. PHA synthase (PhaC) plays a central role in PHA biosynthesis, in which its activity and substrate specificity are major factors in determining the productivity and properties of the produced polymers. However, the effects of modifying the substrate side chain are not well understood because of the difficulty to accessing the desired analogues. In this report, a series of 3-(R)-hydroxyacyl coenzyme A (HACoA) analogues were synthesized and tested with class I synthases from Chromobacterium sp. USM2 (PhaCCs and A479S-PhaCCs) and Caulobacter crescentus (PhaCCc) as well as class III synthase from Allochromatium vinosum (PhaECAv). It was found that, while different PHA synthases displayed distinct preference with regard to the length of the alkyl side chains, they could withstand moderate side chain modifications such as terminal unsaturated bonds and the azide group. Specifically, the specific activity of PhaCCs toward propynyl analogue (HHxyCoA) was only 5-fold less than that toward the classical substrate HBCoA. The catalytic efficiency (kcat/Km) of PhaECAv toward azide analogue (HABCoA) was determined to be 2.86 × 10(5) M(-1) s(-1), which was 6.2% of the value of HBCoA (4.62 × 10(6) M(-1) s(-1)) measured in the presence of bovine serum albumin (BSA). These side chain modifications may be employed to introduce new material functions to PHAs as well as to study PHA biogenesis via click-chemistry, in which the latter remains unknown and is important for metabolic engineering to produce PHAs economically. PMID:26974339

  18. Engineered biosynthesis of plant polyketides: chain length control in an octaketide-producing plant type III polyketide synthase.

    PubMed

    Abe, Ikuro; Oguro, Satoshi; Utsumi, Yoriko; Sano, Yukie; Noguchi, Hiroshi

    2005-09-14

    The chalcone synthase (CHS) superfamily of type III polyketide synthases (PKSs) produces a variety of plant secondary metabolites with remarkable structural diversity and biological activities (e.g., chalcones, stilbenes, benzophenones, acrydones, phloroglucinols, resorcinols, pyrones, and chromones). Here we describe an octaketide-producing novel plant-specific type III PKS from aloe (Aloe arborescens) sharing 50-60% amino acid sequence identity with other plant CHS-superfamily enzymes. A recombinant enzyme expressed in Escherichia coli catalyzed seven successive decarboxylative condensations of malonyl-CoA to yield aromatic octaketides SEK4 and SEK4b, the longest polyketides known to be synthesized by the structurally simple type III PKS. Surprisingly, site-directed mutagenesis revealed that a single residue Gly207 (corresponding to the CHS's active site Thr197) determines the polyketide chain length and product specificity. Small-to-large substitutions (G207A, G207T, G207M, G207L, G207F, and G207W) resulted in loss of the octaketide-forming activity and concomitant formation of shorter chain length polyketides (from triketide to heptaketide) including a pentaketide chromone, 2,7-dihydroxy-5-methylchromone, and a hexaketide pyrone, 6-(2,4-dihydroxy-6-methylphenyl)-4-hydroxy-2-pyrone, depending on the size of the side chain. Notably, the functional diversity of the type III PKS was shown to evolve from simple steric modulation of the chemically inert single residue lining the active-site cavity accompanied by conservation of the Cys-His-Asn catalytic triad. This provided novel strategies for the engineered biosynthesis of pharmaceutically important plant polyketides. PMID:16144421

  19. The molecular motor F-ATP synthase is targeted by the tumoricidal protein HAMLET.

    PubMed

    Ho, James; Sielaff, Hendrik; Nadeem, Aftab; Svanborg, Catharina; Grüber, Gerhard

    2015-05-22

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes. PMID:25681694

  20. Promotion of beta-glucan synthase activity in corn microsomal membranes by calcium and protein phosphorylation

    NASA Technical Reports Server (NTRS)

    Paliyath, G.; Poovaiah, B. W.

    1988-01-01

    Regulation of the activity of beta-glucan synthase was studied using microsomal preparations from corn coleoptiles. The specific activity as measured by the incorporation of glucose from uridine diphospho-D-[U-14C]glucose varied between 5 to 15 pmol (mg protein)-1 min-1. Calcium promoted beta-glucan synthase activity and the promotion was observed at free calcium concentrations as low as 1 micromole. Kinetic analysis of substrate-velocity curve showed an apparent Km of 1.92 x 10(-4) M for UDPG. Calcium increased the Vmax from 5.88 x 10(-7) mol liter-1 min-1 in the absence of calcium to 9.52 x 10(-7) mol liter-1 min-1 and 1.66 x 10(-6) mol liter-1 min-1 in the presence of 0.5 mM and 1 mM calcium, respectively. The Km values remained the same under these conditions. Addition of ATP further increased the activity above the calcium-promoted level. Sodium fluoride, a phosphoprotein phosphatase inhibitor, promoted glucan synthase activity indicating that phosphorylation and dephosphorylation are involved in the regulation of the enzyme activity. Increasing the concentration of sodium fluoride from 0.25 mM to 10 mM increased glucan synthase activity five-fold over the + calcium + ATP control. Phosphorylation of membrane proteins also showed a similar increase under these conditions. Calmodulin, in the presence of calcium and ATP stimulated glucan synthase activity substantially, indicating that calmodulin could be involved in the calcium-dependent phosphorylation and promotion of beta-glucan synthase activity. The role of calcium in mediating auxin action is discussed.

  1. Structure of 3-oxoacyl-(acyl-carrier protein) synthase II from Thermus thermophilus HB8

    SciTech Connect

    Bagautdinov, Bagautdin Ukita, Yoko; Miyano, Masashi; Kunishima, Naoki

    2008-05-01

    The crystal structure of 3-oxoacyl-(acyl-carrier protein) synthase II from T. thermophilus HB8 has been determined at 2.0 Å resolution and compared with the structures of β-keto-ACP synthases from other sources. The β-ketoacyl-(acyl carrier protein) synthases (β-keto-ACP synthases; KAS) catalyse the addition of two-carbon units to the growing acyl chain during the elongation phase of fatty-acid synthesis. As key regulators of bacterial fatty-acid synthesis, they are promising targets for the development of new antibacterial agents. The crystal structure of 3-oxoacyl-ACP synthase II from Thermus thermophilus HB8 (TtKAS II) has been solved by molecular replacement and refined at 2.0 Å resolution. The crystal is orthorhombic, space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 72.07, b = 185.57, c = 62.52 Å, and contains one homodimer in the asymmetric unit. The subunits adopt the well known α-β-α-β-α thiolase fold that is common to ACP synthases. The structural and sequence similarities of TtKAS II to KAS I and KAS II enzymes of known structure from other sources support the hypothesis of comparable enzymatic activity. The dimeric state of TtKAS II is important to create each fatty-acid-binding pocket. Closer examination of KAS structures reveals that compared with other KAS structures in the apo form, the active site of TtKAS II is more accessible because of the ‘open’ conformation of the Phe396 side chain.

  2. The Dictyostelium discoideum cellulose synthase: Structure/function analysis and identification of interacting proteins

    SciTech Connect

    Richard L. Blanton

    2004-02-19

    OAK-B135 The major accomplishments of this project were: (1) the initial characterization of dcsA, the gene for the putative catalytic subunit of cellulose synthase in the cellular slime mold Dictyostelium discoideum; (2) the detection of a developmentally regulated event (unidentified, but perhaps a protein modification or association with a protein partner) that is required for cellulose synthase activity (i.e., the dcsA product is necessary, but not sufficient for cellulose synthesis); (3) the continued exploration of the developmental context of cellulose synthesis and DcsA; (4) the isolation of a GFP-DcsA-expressing strain (work in progress); and (5) the identification of Dictyostelium homologues for plant genes whose products play roles in cellulose biosynthesis. Although our progress was slow and many of our results negative, we did develop a number of promising avenues of investigation that can serve as the foundation for future projects.

  3. The Oligomycin-Sensitivity Conferring Protein of Mitochondrial ATP Synthase: Emerging New Roles in Mitochondrial Pathophysiology

    PubMed Central

    Antoniel, Manuela; Giorgio, Valentina; Fogolari, Federico; Glick, Gary D.; Bernardi, Paolo; Lippe, Giovanna

    2014-01-01

    The oligomycin-sensitivity conferring protein (OSCP) of the mitochondrial FOF1 ATP synthase has long been recognized to be essential for the coupling of proton transport to ATP synthesis. Located on top of the catalytic F1 sector, it makes stable contacts with both F1 and the peripheral stalk, ensuring the structural and functional coupling between FO and F1, which is disrupted by the antibiotic, oligomycin. Recent data have established that OSCP is the binding target of cyclophilin (CyP) D, a well-characterized inducer of the mitochondrial permeability transition pore (PTP), whose opening can precipitate cell death. CyPD binding affects ATP synthase activity, and most importantly, it decreases the threshold matrix Ca2+ required for PTP opening, in striking analogy with benzodiazepine 423, an apoptosis-inducing agent that also binds OSCP. These findings are consistent with the demonstration that dimers of ATP synthase generate Ca2+-dependent currents with features indistinguishable from those of the PTP and suggest that ATP synthase is directly involved in PTP formation, although the underlying mechanism remains to be established. In this scenario, OSCP appears to play a fundamental role, sensing the signal(s) that switches the enzyme of life in a channel able to precipitate cell death. PMID:24786291

  4. Structure of 3-oxoacyl-(acyl-carrier protein) synthase II from Thermus thermophilus HB8

    PubMed Central

    Bagautdinov, Bagautdin; Ukita, Yoko; Miyano, Masashi; Kunishima, Naoki

    2008-01-01

    The β-ketoacyl-(acyl carrier protein) synthases (β-keto-ACP synthases; KAS) catalyse the addition of two-carbon units to the growing acyl chain during the elongation phase of fatty-acid synthesis. As key regulators of bacterial fatty-acid synthesis, they are promising targets for the development of new antibacterial agents. The crystal structure of 3-oxoacyl-ACP synthase II from Thermus thermophilus HB8 (TtKAS II) has been solved by molecular replacement and refined at 2.0 Å resolution. The crystal is orthorhombic, space group P21212, with unit-cell parameters a = 72.07, b = 185.57, c = 62.52 Å, and contains one homodimer in the asymmetric unit. The subunits adopt the well known α-β-α-β-α thiolase fold that is common to ACP synthases. The structural and sequence similarities of TtKAS II to KAS I and KAS II enzymes of known structure from other sources support the hypothesis of comparable enzymatic activity. The dimeric state of TtKAS II is important to create each fatty-acid-binding pocket. Closer examination of KAS structures reveals that compared with other KAS structures in the apo form, the active site of TtKAS II is more accessible because of the ‘open’ conformation of the Phe396 side chain. PMID:18453702

  5. Structure of 3-oxoacyl-(acyl-carrier protein) synthase II from Thermus thermophilus HB8.

    PubMed

    Bagautdinov, Bagautdin; Ukita, Yoko; Miyano, Masashi; Kunishima, Naoki

    2008-05-01

    The beta-ketoacyl-(acyl carrier protein) synthases (beta-keto-ACP synthases; KAS) catalyse the addition of two-carbon units to the growing acyl chain during the elongation phase of fatty-acid synthesis. As key regulators of bacterial fatty-acid synthesis, they are promising targets for the development of new antibacterial agents. The crystal structure of 3-oxoacyl-ACP synthase II from Thermus thermophilus HB8 (TtKAS II) has been solved by molecular replacement and refined at 2.0 A resolution. The crystal is orthorhombic, space group P2(1)2(1)2, with unit-cell parameters a = 72.07, b = 185.57, c = 62.52 A, and contains one homodimer in the asymmetric unit. The subunits adopt the well known alpha-beta-alpha-beta-alpha thiolase fold that is common to ACP synthases. The structural and sequence similarities of TtKAS II to KAS I and KAS II enzymes of known structure from other sources support the hypothesis of comparable enzymatic activity. The dimeric state of TtKAS II is important to create each fatty-acid-binding pocket. Closer examination of KAS structures reveals that compared with other KAS structures in the apo form, the active site of TtKAS II is more accessible because of the ;open' conformation of the Phe396 side chain. PMID:18453702

  6. Identification of glycogen synthase as a new substrate for stress-activated protein kinase 2b/p38beta.

    PubMed

    Kuma, Yvonne; Campbell, David G; Cuenda, Ana

    2004-04-01

    The endogenous glycogen synthase in extracts from mouse skeletal muscle, liver and brain bound specifically to SAPK2b (stress-activated protein kinase 2b)/p38b, but not to other members of the group of SAPK/p38 kinases. Glycogen synthase was phosphorylated in vitro more efficiently by SAPK2b/p38b than by SAPK2a/p38a, SAPK3/p38g or SAPK4/p38d. SAPK2b/p38b phosphorylated glycogen synthase in vitro at residues Ser644, Ser652, Thr718 and Ser724, two of which (Ser644 and Ser652) are also phosphorylated by glycogen synthase kinase 3. Thr718 and Ser724 are novel sites not known to be phosphorylated by other protein kinases. Glycogen synthase becomes phosphorylated at Ser644 in response to osmotic shock; this phosphorylation is prevented by pretreatment of the cells with SB 203580, which inhibits SAPK2a/p38a and SAPK2b/p38b activity. In vitro, phosphorylation of glycogen synthase by SAPK2b/p38b alone had no significant effect on its activity, indicating that phosphorylation at residue Ser644 itself is insufficient to decrease glycogen synthase activity. However, after phosphorylation by SAPK2b/p38b, subsequent phosphorylation at Ser640 by glycogen synthase kinase 3 decreased the activity of glycogen synthase. This decrease was not observed when SAPK2b/p38b activity was blocked with SB 203580. These results suggest that SAPK2b/p38b may be a priming kinase that allows glycogen synthase kinase 3 to phosphorylate Ser640 and thereby inhibit glycogen synthase activity. PMID:14680475

  7. Aspects of Subunit Interactions in the Chloroplast ATP Synthase (I. Isolation of a Chloroplast Coupling Factor 1-Subunit III Complex from Spinach Thylakoids).

    PubMed Central

    Wetzel, C. M.; McCarty, R. E.

    1993-01-01

    A chloroplast ATP synthase complex (CF1 [chloroplast-coupling factor 1]-CF0 [membrane-spanning portion of chloroplast ATP synthase]) depleted of all CF0 subunits except subunit III (also known as the proteolipid subunit) was purified to study the interaction between CF1 and subunit III. Subunit III has a putative role in proton translocation across the thylakoid membrane during photophosphorylation; therefore, an accurate model of subunit inter-actions involving subunit III will be valuable for elucidating the mechanism and regulation of energy coupling. Purification of the complex from a crude CF1-CF0 preparation from spinach (Spinacia oleracea) thylakoids was accomplished by detergent treatment during anion-exchange chromatography. Subunit III in the complex was positively identified by amino acid analysis and N-terminal sequencing. The association of subunit III with CF1 was verified by linear sucrose gradient centrifugation, immunoprecipitation, and incorporation of the complex into asolectin liposomes. After incorporation into liposomes, CF1 was removed from the CF1-III complex by ethylenediaminetetracetate treatment. The subunit III-proteoliposomes were competent to rebind purified CF1. These results indicate that subunit III directly interacts with CF1 in spinach thylakoids. PMID:12231815

  8. Heterologous gene expression and functional analysis of a type III polyketide synthase from Aspergillus niger NRRL 328.

    PubMed

    Kirimura, Kohtaro; Watanabe, Shotaro; Kobayashi, Keiichi

    2016-05-13

    Type III polyketide synthases (PKSs) catalyze the formation of pyrone- and resorcinol-types aromatic polyketides. The genomic analysis of the filamentous fungus Aspergillus niger NRRL 328 revealed that this strain has a putative gene (chr_8_2: 2978617-2979847) encoding a type III PKS, although its functions are unknown. In this study, for functional analysis of this putative type III PKS designated as An-CsyA, cloning and heterologous expression of the An-CsyA gene (An-csyA) in Escherichia coli were performed. Recombinant His-tagged An-CsyA was successfully expressed in E. coli BL21 (DE3), purified by Ni(2+)-affinity chromatography, and used for in vitro assay. Tests on the substrate specificity of the His-tagged An-CsyA with myriad acyl-CoAs as starter substrates and malonyl-CoA as extender substrate showed that His-tagged An-CsyA accepted fatty acyl-CoAs (C2-C14) and produced triketide pyrones (C2-C14), tetraketide pyrones (C2-C10), and pentaketide resorcinols (C10-C14). Furthermore, acetoacetyl-CoA, malonyl-CoA, isobutyryl-CoA, and benzoyl-CoA were also accepted as starter substrates, and both of triketide pyrones and tetraketide pyrones were produced. It is noteworthy that the His-tagged An-CsyA produced polyketides from malonyl-CoA as starter and extender substrates and produced tetraketide pyrones from short-chain fatty acyl-CoAs as starter substrates. Therefore, this is the first report showing the functional properties of An-CsyA different from those of other fungal type III PKSs. PMID:27060547

  9. Expression of Ribonucleotide Reductase Subunit-2 and Thymidylate Synthase Correlates with Poor Prognosis in Patients with Resected Stages I–III Non-Small Cell Lung Cancer

    PubMed Central

    Grossi, Francesco; Dal Bello, Maria Giovanna; Salvi, Sandra; Puzone, Roberto; Pfeffer, Ulrich; Fontana, Vincenzo; Alama, Angela; Rijavec, Erika; Barletta, Giulia; Genova, Carlo; Sini, Claudio; Ratto, Giovanni Battista; Taviani, Mario; Truini, Mauro; Merlo, Domenico Franco

    2015-01-01

    Biomarkers can help to identify patients with early-stages or locally advanced non-small cell lung cancer (NSCLC) who have high risk of relapse and poor prognosis. To correlate the expression of seven biomarkers involved in DNA synthesis and repair and in cell division with clinical outcome, we consecutively collected 82 tumour tissues from radically resected NSCLC patients. The following biomarkers were investigated using IHC and qRT-PCR: excision repair cross-complementation group 1 (ERCC1), breast cancer 1 (BRCA1), ribonucleotide reductase subunits M1 and M2 (RRM1 and RRM2), subunit p53R2, thymidylate synthase (TS), and class III beta-tubulin (TUBB3). Gene expression levels were also validated in an available NSCLC microarray dataset. Multivariate analysis identified the protein overexpression of RRM2 and TS as independent prognostic factors of shorter overall survival (OS). Kaplan-Meier analysis showed a trend in shorter OS for patients with RRM2, TS, and ERCC1, BRCA1 overexpressed tumours. For all of the biomarkers except TUBB3, the OS trends relative to the gene expression levels were in agreement with those relative to the protein expression levels. The NSCLC microarray dataset showed RRM2 and TS as biomarkers significantly associated with OS. This study suggests that high expression levels of RRM2 and TS might be negative prognostic factors for resected NSCLC patients. PMID:26663950

  10. Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis

    PubMed Central

    Zhang, Yi; Nikolovski, Nino; Sorieul, Mathias; Vellosillo, Tamara; McFarlane, Heather E.; Dupree, Ray; Kesten, Christopher; Schneider, René; Driemeier, Carlos; Lathe, Rahul; Lampugnani, Edwin; Yu, Xiaolan; Ivakov, Alexander; Doblin, Monika S.; Mortimer, Jenny C.; Brown, Steven P.; Persson, Staffan; Dupree, Paul

    2016-01-01

    As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus. PMID:27277162

  11. Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis.

    PubMed

    Zhang, Yi; Nikolovski, Nino; Sorieul, Mathias; Vellosillo, Tamara; McFarlane, Heather E; Dupree, Ray; Kesten, Christopher; Schneider, René; Driemeier, Carlos; Lathe, Rahul; Lampugnani, Edwin; Yu, Xiaolan; Ivakov, Alexander; Doblin, Monika S; Mortimer, Jenny C; Brown, Steven P; Persson, Staffan; Dupree, Paul

    2016-01-01

    As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus. PMID:27277162

  12. Primary structure of a cerulenin-binding. beta. -ketoacyl-(acyl carrier protein) synthase from barley chloroplasts

    SciTech Connect

    Siggaard-Andersen, M.; Kauppinen, S. ); von Wettstein-Knowles, P. Univ. of Copenhagen )

    1991-05-15

    The radioactively labeled {beta}-ketoacyl thioester synthase inhibitor ({sup 3}H)cerulenin was used to tag three dimeric barley chloroplast proteins ({alpha}{alpha}, {alpha}{beta}, and {beta}{beta}) from the stromal fraction. Oligonucleotides corresponding to amino acid sequences obtained from the purified proteins were used to generate with the polymerase chain reaction a probe for cDNAs encoding the {beta} subunit. cDNA sequencing revealed an open reading frame for 462 residues comprising the mature protein and a 35-amino acid transit peptide. The deduced amino acid sequence of the mature protein is homologous to the {beta}-ketoacyl-(acyl carrier protein) (ACP) synthase I (3-oxoacyl-ACP synthase; acyl-ACP:malonyl-ACP C-acyltransferase (decarboxylating), EC 2.3.1.41) of Escherichia coli. Under analogous experimental conditions ({sup 3}H)cerulenin tagged a single dimeric protein from spinach chloroplasts.

  13. Decarboxylation of malonyl-(acyl carrier protein) by 3-oxoacyl-(acyl carrier protein) synthases in plant fatty acid biosynthesis.

    PubMed Central

    Winter, E; Brummel, M; Schuch, R; Spener, F

    1997-01-01

    In order to identify regulatory steps in fatty acid biosynthesis, the influence of intermediate 3-oxoacyl-(acyl carrier proteins) (3-oxoacyl-ACPs) and end-product acyl-ACPs of the fatty acid synthase reaction on the condensation reaction was investigated in vitro, using total fatty acid synthase preparations and purified 3-oxoacyl-ACP synthases (KASs; EC 2.3.1.41) from Cuphea lanceolata seeds. KAS I and II in the fatty acid synthase preparations were assayed for the elongation of octanoyl- and hexadecanoyl-ACP respectively, and the accumulation of the corresponding condensation product 3-oxoacyl-ACP was studied by modulating the content of the reducing equivalentS NADH and NADPH. Complete omission of reducing equivalents resulted with either KAS in the abnormal synthesis of acetyl-ACP from malonyl-ACP by a decarboxylation reaction. Supplementation with NADPH or NADH, separately or in combination with recombinant 3-oxoacyl-ACP reductase (EC 1.1.1.100), led to a decrease in the amount of acetyl-ACP and a simultaneous increase in elongation products. This demonstrates that the accumulation of 3-oxoacyl-ACP inhibits the condensation reaction on the one hand, and induces the decarboxylation of malonyl-ACP on the other. By carrying out similar experiments with purified enzymes, this decarboxylation was attributed to the action of KAS. Our data point to a regulatory mechanism for the degradation of malonyl-ACP in plants which is activated by the accumulation of the fatty acid synthase intermediate 3-oxoacyl-ACP. PMID:9020860

  14. Two for the Price of One: A Neuroprotective Chaperone Kit within NAD Synthase Protein NMNAT2.

    PubMed

    Lavado-Roldán, Angela; Fernández-Chacón, Rafael

    2016-07-01

    One of the most fascinating properties of the brain is the ability to function smoothly across decades of a lifespan. Neurons are nondividing mature cells specialized in fast electrical and chemical communication at synapses. Often, neurons and synapses operate at high levels of activity through sophisticated arborizations of long axons and dendrites that nevertheless stay healthy throughout years. On the other hand, aging and activity-dependent stress strike onto the protein machineries turning proteins unfolded and prone to form pathological aggregates associated with neurodegeneration. How do neurons protect from those insults and remain healthy for their whole life? Ali and colleagues now present a molecular mechanism by which the enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) acts not only as a NAD synthase involved in axonal maintenance but as a molecular chaperone helping neurons to overcome protein unfolding and protein aggregation. PMID:27454736

  15. Two for the Price of One: A Neuroprotective Chaperone Kit within NAD Synthase Protein NMNAT2

    PubMed Central

    2016-01-01

    One of the most fascinating properties of the brain is the ability to function smoothly across decades of a lifespan. Neurons are nondividing mature cells specialized in fast electrical and chemical communication at synapses. Often, neurons and synapses operate at high levels of activity through sophisticated arborizations of long axons and dendrites that nevertheless stay healthy throughout years. On the other hand, aging and activity-dependent stress strike onto the protein machineries turning proteins unfolded and prone to form pathological aggregates associated with neurodegeneration. How do neurons protect from those insults and remain healthy for their whole life? Ali and colleagues now present a molecular mechanism by which the enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) acts not only as a NAD synthase involved in axonal maintenance but as a molecular chaperone helping neurons to overcome protein unfolding and protein aggregation. PMID:27454736

  16. A protein kinase screen of Neurospora crassa mutant strains reveals that the SNF1 protein kinase promotes glycogen synthase phosphorylation.

    PubMed

    Candido, Thiago De Souza; Gonçalves, Rodrigo Duarte; Felício, Ana Paula; Freitas, Fernanda Zanolli; Cupertino, Fernanda Barbosa; De Carvalho, Ana Carolina Gomes Vieira; Bertolini, Maria Célia

    2014-12-15

    Glycogen functions as a carbohydrate reserve in a variety of organisms and its metabolism is highly regulated. The activities of glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes of the synthesis and degradation processes, respectively, are regulated by allosteric modulation and reversible phosphorylation. To identify the protein kinases affecting glycogen metabolism in Neurospora crassa, we performed a screen of 84 serine/threonine kinase knockout strains. We identified multiple kinases that have already been described as controlling glycogen metabolism in different organisms, such as NcSNF1, NcPHO85, NcGSK3, NcPKA, PSK2 homologue and NcATG1. In addition, many hypothetical kinases have been implicated in the control of glycogen metabolism. Two kinases, NcIME-2 and NcNIMA, already functionally characterized but with no functions related to glycogen metabolism regulation, were also identified. Among the kinases identified, it is important to mention the role of NcSNF1. We showed in the present study that this kinase was implicated in glycogen synthase phosphorylation, as demonstrated by the higher levels of glycogen accumulated during growth, along with a higher glycogen synthase (GSN) ±glucose 6-phosphate activity ratio and a lesser set of phosphorylated GSN isoforms in strain Ncsnf1KO, when compared with the wild-type strain. The results led us to conclude that, in N. crassa, this kinase promotes phosphorylation of glycogen synthase either directly or indirectly, which is the opposite of what is described for Saccharomyces cerevisiae. The kinases also play a role in gene expression regulation, in that gdn, the gene encoding the debranching enzyme, was down-regulated by the proteins identified in the screen. Some kinases affected growth and development, suggesting a connection linking glycogen metabolism with cell growth and development. PMID:25253091

  17. Assembly of the Cysteine Synthase Complex and the Regulatory Role of Protein-Protein Interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Macromolecular assemblies play critical roles in regulating cellular functions. The cysteine synthase complex (CSC), which is formed by association of serine O-acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS), functions as a multienzyme complex that responds to changes in intracellul...

  18. Purification and characterization of the acyl carrier protein of the Streptomyces glaucescens tetracenomycin C polyketide synthase.

    PubMed Central

    Shen, B; Summers, R G; Gramajo, H; Bibb, M J; Hutchinson, C R

    1992-01-01

    The acyl carrier protein (ACP) of the tetracenomycin C polyketide synthase, encoded by the tcmM gene, has been expressed in both Streptomyces glaucescens and Escherichia coli and purified to homogeneity. Expression of the tcmM gene in E. coli results mainly in the TcmM apo-ACP, whereas expression in S. glaucescens yields solely the holo-ACP. The purified holo-TcmM is active in a malonyl coenzyme A:ACP transacylase assay and is labeled by radioactive beta-alanine, confirming that it carries a 4'-phosphopantetheine prosthetic group. Images PMID:1592832

  19. Membrane association of sucrose synthase: changes during the graviresponse and possible control by protein phosphorylation

    NASA Technical Reports Server (NTRS)

    Winter, H.; Huber, J. L.; Huber, S. C.; Davies, E. (Principal Investigator)

    1997-01-01

    Sucrose synthase (SuSy) plays an important role in sucrose degradation and occurs both as a soluble and as a membrane-associated enzyme in higher plants. We show that membrane association can vary in vivo in response to gravistimulation, apparently involving SuSy dephosphorylation, and is a reversible process in vitro. Phosphorylation of SuSy has little effect on its activity but decreases its surface hydrophobicity as reported with the fluorescent probe bis-ANS. We postulate that phosphorylation of SuSy (and perhaps other membrane proteins) is involved in the release of the membrane-bound enzyme in part as a result of decreased surface hydrophobicity.

  20. Recognition of acyl carrier proteins by ketoreductases in assembly line polyketide synthases.

    PubMed

    Ostrowski, Matthew P; Cane, David E; Khosla, Chaitan

    2016-07-01

    Ketoreductases (KRs) are the most widespread tailoring domains found in individual modules of assembly line polyketide synthases (PKSs), and are responsible for controlling the configurations of both the α-methyl and β-hydroxyl stereogenic centers in the growing polyketide chain. Because they recognize substrates that are covalently bound to acyl carrier proteins (ACPs) within the same PKS module, we sought to quantify the extent to which protein-protein recognition contributes to the turnover of these oxidoreductive enzymes using stand-alone domains from the 6-deoxyerythronolide B synthase (DEBS). Reduced 2-methyl-3-hydroxyacyl-ACP substrates derived from two enantiomeric acyl chains and four distinct ACP domains were synthesized and presented to four distinct KR domains. Two KRs, from DEBS modules 2 and 5, displayed little preference for oxidation of substrates tethered to their cognate ACP domains over those attached to the other ACP domains tested. In contrast, the KR from DEBS module 1 showed an ~10-50-fold preference for substrate attached to its native ACP domain, whereas the KR from DEBS module 6 actually displayed an ~10-fold preference for the ACP from DEBS module 5. Our findings suggest that recognition of the ACP by a KR domain is unlikely to affect the rate of native assembly line polyketide biosynthesis. In some cases, however, unfavorable KR-ACP interactions may suppress the rate of substrate processing when KR domains are swapped to construct hybrid PKS modules. PMID:27118242

  1. Novel protein–protein interaction between spermidine synthase and S-adenosylmethionine decarboxylase from Leishmania donovani

    SciTech Connect

    Mishra, Arjun K.; Agnihotri, Pragati; Srivastava, Vijay Kumar; Pratap, J. Venkatesh

    2015-01-09

    Highlights: • L. donovani spermidine synthase and S-adenosylmethionine decarboxylase have been cloned and purified. • S-adenosylmethionine decarboxylase has autocatalytic property. • GST pull down assay shows the two proteins to form a metabolon. • Isothermal titration calorimetry shows that binding was exothermic having K{sub d} value of 0.4 μM. • Interaction confirmed by fluorescence spectroscopy and size exclusion chromatography. - Abstract: Polyamine biosynthesis pathway has long been considered an essential drug target for trypanosomatids including Leishmania. S-adenosylmethionine decarboxylase (AdoMetDc) and spermidine synthase (SpdSyn) are enzymes of this pathway that catalyze successive steps, with the product of the former, decarboxylated S-adenosylmethionine (dcSAM), acting as an aminopropyl donor for the latter enzyme. Here we have explored the possibility of and identified the protein–protein interaction between SpdSyn and AdoMetDc. The protein–protein interaction has been identified using GST pull down assay. Isothermal titration calorimetry reveals that the interaction is thermodynamically favorable. Fluorescence spectroscopy studies also confirms the interaction, with SpdSyn exhibiting a change in tertiary structure with increasing concentrations of AdoMetDc. Size exclusion chromatography suggests the presence of the complex as a hetero-oligomer. Taken together, these results suggest that the enzymes indeed form a heteromer. Computational analyses suggest that this complex differs significantly from the corresponding human complex, implying that this complex could be a better therapeutic target than the individual enzymes.

  2. Characterization and crystal structure of lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase (cDHDPS) protein

    SciTech Connect

    Rice, E.A.; Bannon, G.A.; Glenn, K.C.; Jeong, S.S.; Sturman, E.J.; Rydel, T.J.

    2008-11-21

    The lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase enzyme (cDHDPS) was recently successfully introduced into maize plants to enhance the level of lysine in the grain. To better understand lysine insensitivity of the cDHDPS, we expressed, purified, kinetically characterized the protein, and solved its X-ray crystal structure. The cDHDPS enzyme has a fold and overall structure that is highly similar to other DHDPS proteins. A noteworthy feature of the active site is the evidence that the catalytic lysine residue forms a Schiff base adduct with pyruvate. Analyses of the cDHDPS structure in the vicinity of the putative binding site for S-lysine revealed that the allosteric binding site in the Escherichia coli DHDPS protein does not exist in cDHDPS due to three non-conservative amino acids substitutions, and this is likely why cDHDPS is not feedback inhibited by lysine.

  3. Evolution of acyl-ACP-thioesterases and β-ketoacyl-ACP-synthases revealed by protein-protein interactions

    PubMed Central

    Beld, Joris; Blatti, Jillian L.; Behnke, Craig; Mendez, Michael; Burkart, Michael D.

    2014-01-01

    The fatty acid synthase (FAS) is a conserved primary metabolic enzyme complex capable of tolerating cross-species engineering of domains for the development of modified and overproduced fatty acids. In eukaryotes, acyl-acyl carrier protein thioesterases (TEs) off-load mature cargo from the acyl carrier protein (ACP), and plants have developed TEs for short/medium-chain fatty acids. We showed that engineering plant TEs into the green microalga Chlamydomonas reinhardtii does not result in the predicted shift in fatty acid profile. Since fatty acid biosynthesis relies on substrate recognition and protein-protein interactions between the ACP and its partner enzymes, we hypothesized that plant TEs and algal ACP do not functionally interact. Phylogenetic analysis revealed major evolutionary differences between FAS enzymes, including TEs and ketoacyl synthases (KSs), in which the former is present only in some species, whereas the latter is present in all, and has a common ancestor. In line with these results, TEs appeared to be selective towards their ACP partners whereas KSs showed promiscuous behavior across bacterial, plant and algal species. Based on phylogenetic analyses, in silico docking, in vitro mechanistic crosslinking and in vivo algal engineering, we propose that phylogeny can predict effective interactions between ACPs and partner enzymes. PMID:25110394

  4. Colonic Fatty Acid Synthase is Down-regulated in Sprague-Dawley Rats Fed Soy Protein Isolate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fatty Acid Synthase (FAS), a key enzyme in the fatty acid biosynthetic pathway, is over-expressed in multiple cancers. The aim of this study was to evaluate the effects of dietary proteins [soy protein isolate (SPI) and casein (CAS), latter is the control] on the expression of FAS in the colonic muc...

  5. Dietary Soy Protein Inhibits DNA Damage and Cell Survival of Colon Epithelial Cells through Attenuated Expression of Fatty Acid Synthase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dietary intake of soy protein decreases tumor incidence in rat models of chemically induced colon cancer. We hypothesized that decreased expression of Fatty Acid Synthase (FASN) underlies, in part, the tumor preventive effects of soy protein, since FASN over-expression characterizes early tumorigene...

  6. Synthesis of Unnatural 2-Substituted Quinolones and 1,3-Diketones by a Member of Type III Polyketide Synthases from Huperzia serrata.

    PubMed

    Wang, Juan; Wang, Xiao-Hui; Liu, Xiao; Li, Jun; Shi, Xiao-Ping; Song, Yue-Lin; Zeng, Ke-Wu; Zhang, Le; Tu, Peng-Fei; Shi, She-Po

    2016-08-01

    A curcuminoids, benzalacetone-, and quinolone-producing type III polyketide synthase (HsPKS3) from Huperzia serrata uniquely catalyzes the formation of unnatural 2-substituted quinolones and 1,3-diketones via head-to-head condensation of two completely different substrates. The broad range of substrate tolerance of HsPKS3 facilitates accessing structurally diverse 2-substituted quinolones and 1,3-diketones. PMID:27399835

  7. TORC2-dependent protein kinase Ypk1 phosphorylates ceramide synthase to stimulate synthesis of complex sphingolipids.

    PubMed

    Muir, Alexander; Ramachandran, Subramaniam; Roelants, Françoise M; Timmons, Garrett; Thorner, Jeremy

    2014-01-01

    Plasma membrane lipid composition must be maintained during growth and under environmental insult. In yeast, signaling mediated by TOR Complex 2 (TORC2)-dependent protein kinase Ypk1 controls lipid abundance and distribution in response to membrane stress. Ypk1, among other actions, alleviates negative regulation of L-serine:palmitoyl-CoA acyltransferase, upregulating production of long-chain base precursors to sphingolipids. To explore other roles for TORC2-Ypk1 signaling in membrane homeostasis, we devised a three-tiered genome-wide screen to identify additional Ypk1 substrates, which pinpointed both catalytic subunits of the ceramide synthase complex. Ypk1-dependent phosphorylation of both proteins increased upon either sphingolipid depletion or heat shock and was important for cell survival. Sphingolipidomics, other biochemical measurements and genetic analysis demonstrated that these modifications of ceramide synthase increased its specific activity and stimulated channeling of long-chain base precursors into sphingolipid end-products. Control at this branch point also prevents accumulation of intermediates that could compromise cell growth by stimulating autophagy. PMID:25279700

  8. Erythroid 5-aminolevulinate synthase mediates the upregulation of membrane band 3 protein expression by iron.

    PubMed

    Huang, Qianchuan; Li, Jinying; Feng, Weihua; Xu, Yanqun; Huang, Zhenxia; Lv, Shuqing; Zhou, Hong; Gao, Lei

    2010-03-01

    Iron deficiency leads to abnormal expression and function of band 3 protein in erythrocytes, but the underlying mechanisms remain elusive. The mRNA of erythroid-specific 5-aminolevulinate synthase (eALAS) contains an iron response element and the eALAS protein is an important mediator of iron utilization by erythrocytes. In this study, we investigated the effect of short hairpin RNA (shRNA) mediated silencing of eALAS on the expression of band 3 protein induced by iron. By real-time RT-PCR and Western blot we showed that at mRNA and protein level iron-induced expression of band 3 protein was lower in eALAS-shRNA transfected K562 cells than in control cells. Of note, the lowest expression was detected in K562 cells cultured in iron deficiency condition (p < 0.01). Thus either iron deficiency or depletion of eALAS could suppress the expression of erythroid band 3 protein. These results demonstrated for the first time that iron and the iron-regulatory system regulate the expression of the erythrocyte membrane proteins. PMID:20087844

  9. A Remote Mutation Affects the Hydride Transfer by Disrupting Concerted Protein Motions in Thymidylate Synthase

    PubMed Central

    Wang, Zhen; Abeysinghe, Thelma; Finer-Moore, Janet S.; Stroud, Robert M.; Kohen, Amnon

    2012-01-01

    The role of protein flexibility in enzyme-catalyzed activation of chemical bonds is an evolving perspective in enzymology. Here we examine the role of protein motions in the hydride transfer reaction catalyzed by thymidylate synthase (TSase). Being remote from the chemical reaction site, the Y209W mutation of E. coli TSase significantly reduces the protein activity, despite the remarkable similarity between the crystal structures of the wild type and mutant enzymes with ligands representing their Michaelis complexes. The most conspicuous difference between those two crystal structures is in the anisotropic B-factors, which indicates disruption of the correlated atomic vibrations of protein residues in the mutant. This dynamically altered mutant allows a variety of small thiols to compete for the reaction intermediate that precedes the hydride transfer, indicating disruption of motions that preorganize the protein environment for this chemical step. Although the mutation causes higher enthalpy of activation of the hydride transfer, it only shows a small effect on the temperature-dependence of the intrinsic KIE, suggesting marginal changes in the geometry and dynamics of the H-donor and acceptor at the tunneling ready state. These observations suggest that that the mutation disrupts the concerted motions that bring the H-donor and acceptor together during the pre- and re-organization of the protein environment. The integrated structural and kinetic data allow us to probe the impact of protein motions on different timescales on the hydride transfer reaction within a complex enzymatic mechanism. PMID:23034004

  10. Protein phosphorylation as a mechanism for regulation of spinach leaf sucrose-phosphate synthase activity

    SciTech Connect

    Huber, J.L.A.; Huber, S.C. )

    1989-04-01

    Protein phosphorylation has been identified as a mechanism for the light-dark regulation of spinach sucrose-phosphate synthase (SPS) activity, previously shown to involve some type of covalent modification of the enzyme. The 120 kD subunit of SPS in extracts of light-treated leaves was labeled with {sup 32}P in the presence of ({gamma}-{sup 32}P) ATP. In this in vitro system, {sup 32}P incorporation into light-activated SPS was dependent upon ATP and magnesium concentrations as well as time, and was closely paralleled by inactivation of the enzyme. The soluble protein kinase involved in the interconversion of SPS between activated and deactivated forms may be specific for SPS as it co-purifies with SPS during partial purification of the enzyme. The kinase appears not to be calcium activated and no evidence has been obtained for metabolite control of SPS phosphorylation/inactivation.

  11. AMP-Activated Protein Kinase and Glycogen Synthase Kinase 3β Modulate the Severity of Sepsis-Induced Lung Injury

    PubMed Central

    Liu, Zhongyu; Bone, Nathaniel; Jiang, Shaoning; Park, Dae Won; Tadie, Jean-Marc; Deshane, Jessy; Rodriguez, Cilina Ann; Pittet, Jean-Francois; Abraham, Edward; Zmijewski, Jaroslaw W

    2015-01-01

    Alterations in metabolic and bioenergetic homeostasis contribute to sepsis-mediated organ injury. However, how AMP-activated protein kinase (AMPK), a major sensor and regulator of energy expenditure and production, affects development of organ injury and loss of innate capacity during polymicrobial sepsis remains unclear. In the present experiments, we found that cross-talk between the AMPK and GSK3β signaling pathways controls chemotaxis and the ability of neutrophils and macrophages to kill bacteria ex vivo. In mice with polymicrobial abdominal sepsis or more severe sepsis induced by the combination of hemorrhage and intraabdominal infection, administration of the AMPK activator metformin or the GSK3β inhibitor SB216763 reduced the severity of acute lung injury (ALI). Improved survival in metformin-treated septic mice was correlated with preservation of mitochondrial complex V (ATP synthase) function and increased amounts of ETC complex III and IV. Although immunosuppression is a consequence of sepsis, metformin effectively increased innate immune capacity to eradicate P. aeruginosa in the lungs of septic mice. We also found that AMPK activation diminished accumulation of the immunosuppressive transcriptional factor HIF-1α as well as the development of endotoxin tolerance in LPS-treated macrophages. Furthermore, AMPK-dependent preservation of mitochondrial membrane potential also prevented LPS-mediated dysfunction of neutrophil chemotaxis. These results indicate that AMPK activation reduces the severity of polymicrobial sepsis-induced lung injury and prevents the development of sepsis-associated immunosuppression. PMID:26650187

  12. Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases

    NASA Technical Reports Server (NTRS)

    Boo, Yong Chool; Jo, Hanjoong

    2003-01-01

    Vascular endothelial cells are directly and continuously exposed to fluid shear stress generated by blood flow. Shear stress regulates endothelial structure and function by controlling expression of mechanosensitive genes and production of vasoactive factors such as nitric oxide (NO). Though it is well known that shear stress stimulates NO production from endothelial nitric oxide synthase (eNOS), the underlying molecular mechanisms remain unclear and controversial. Shear-induced production of NO involves Ca2+/calmodulin-independent mechanisms, including phosphorylation of eNOS at several sites and its interaction with other proteins, including caveolin and heat shock protein-90. There have been conflicting results as to which protein kinases-protein kinase A, protein kinase B (Akt), other Ser/Thr protein kinases, or tyrosine kinases-are responsible for shear-dependent eNOS regulation. The functional significance of each phosphorylation site is still unclear. We have attempted to summarize the current status of understanding in shear-dependent eNOS regulation.

  13. Identification of protein-protein interactions of isoflavonoid biosynthetic enzymes with 2-hydroxyisoflavanone synthase in soybean (Glycine max (L.) Merr.).

    PubMed

    Waki, Toshiyuki; Yoo, DongChan; Fujino, Naoto; Mameda, Ryo; Denessiouk, Konstantin; Yamashita, Satoshi; Motohashi, Reiko; Akashi, Tomoyoshi; Aoki, Toshio; Ayabe, Shin-ichi; Takahashi, Seiji; Nakayama, Toru

    2016-01-15

    Metabolic enzymes, including those involved in flavonoid biosynthesis, are proposed to form weakly bound, ordered protein complexes, called "metabolons". Some hypothetical models of flavonoid biosynthetic metabolons have been proposed, in which metabolic enzymes are believed to anchor to the cytoplasmic surface of the endoplasmic reticulum (ER) via ER-bound cytochrome P450 isozymes (P450s). However, no convincing evidence for the interaction of flavonoid biosynthetic enzymes with P450s has been reported previously. Here, we analyzed binary protein-protein interactions of 2-hydroxyisoflavanone synthase 1 (GmIFS1), a P450 (CYP93C), with cytoplasmic enzymes involved in isoflavone biosynthesis in soybean. We identified binary interactions between GmIFS1 and chalcone synthase 1 (GmCHS1) and between GmIFS1 and chalcone isomerases (GmCHIs) by using a split-ubiquitin membrane yeast two-hybrid system. These binary interactions were confirmed in planta by means of bimolecular fluorescence complementation (BiFC) using tobacco leaf cells. In these BiFC analyses, fluorescence signals that arose from the interaction of these cytoplasmic enzymes with GmIFS1 generated sharp, network-like intracellular patterns, which was very similar to the ER-localized fluorescence patterns of GmIFS1 labeled with a fluorescent protein. These observations provide strong evidence that, in planta, interaction of GmCHS1 and GmCHIs with GmIFS1 takes place on ER on which GmIFS1 is located, and also provide important clues to understand how enzymes and proteins form metabolons to establish efficient metabolic flux of (iso)flavonoid biosynthesis. PMID:26694697

  14. Citrate synthase proteins in extremophilic organisms: Studies within a structure-based model

    SciTech Connect

    Różycki, Bartosz Cieplak, Marek

    2014-12-21

    We study four citrate synthase homodimeric proteins within a structure-based coarse-grained model. Two of these proteins come from thermophilic bacteria, one from a cryophilic bacterium and one from a mesophilic organism; three are in the closed and two in the open conformations. Even though the proteins belong to the same fold, the model distinguishes the properties of these proteins in a way which is consistent with experiments. For instance, the thermophilic proteins are more stable thermodynamically than their mesophilic and cryophilic homologues, which we observe both in the magnitude of thermal fluctuations near the native state and in the kinetics of thermal unfolding. The level of stability correlates with the average coordination number for amino acid contacts and with the degree of structural compactness. The pattern of positional fluctuations along the sequence in the closed conformation is different than in the open conformation, including within the active site. The modes of correlated and anticorrelated movements of pairs of amino acids forming the active site are very different in the open and closed conformations. Taken together, our results show that the precise location of amino acid contacts in the native structure appears to be a critical element in explaining the similarities and differences in the thermodynamic properties, local flexibility, and collective motions of the different forms of the enzyme.

  15. Citrate synthase proteins in extremophilic organisms: Studies within a structure-based model

    NASA Astrophysics Data System (ADS)

    RóŻycki, Bartosz; Cieplak, Marek

    2014-12-01

    We study four citrate synthase homodimeric proteins within a structure-based coarse-grained model. Two of these proteins come from thermophilic bacteria, one from a cryophilic bacterium and one from a mesophilic organism; three are in the closed and two in the open conformations. Even though the proteins belong to the same fold, the model distinguishes the properties of these proteins in a way which is consistent with experiments. For instance, the thermophilic proteins are more stable thermodynamically than their mesophilic and cryophilic homologues, which we observe both in the magnitude of thermal fluctuations near the native state and in the kinetics of thermal unfolding. The level of stability correlates with the average coordination number for amino acid contacts and with the degree of structural compactness. The pattern of positional fluctuations along the sequence in the closed conformation is different than in the open conformation, including within the active site. The modes of correlated and anticorrelated movements of pairs of amino acids forming the active site are very different in the open and closed conformations. Taken together, our results show that the precise location of amino acid contacts in the native structure appears to be a critical element in explaining the similarities and differences in the thermodynamic properties, local flexibility, and collective motions of the different forms of the enzyme.

  16. Citrate synthase proteins in extremophilic organisms: studies within a structure-based model.

    PubMed

    Różycki, Bartosz; Cieplak, Marek

    2014-12-21

    We study four citrate synthase homodimeric proteins within a structure-based coarse-grained model. Two of these proteins come from thermophilic bacteria, one from a cryophilic bacterium and one from a mesophilic organism; three are in the closed and two in the open conformations. Even though the proteins belong to the same fold, the model distinguishes the properties of these proteins in a way which is consistent with experiments. For instance, the thermophilic proteins are more stable thermodynamically than their mesophilic and cryophilic homologues, which we observe both in the magnitude of thermal fluctuations near the native state and in the kinetics of thermal unfolding. The level of stability correlates with the average coordination number for amino acid contacts and with the degree of structural compactness. The pattern of positional fluctuations along the sequence in the closed conformation is different than in the open conformation, including within the active site. The modes of correlated and anticorrelated movements of pairs of amino acids forming the active site are very different in the open and closed conformations. Taken together, our results show that the precise location of amino acid contacts in the native structure appears to be a critical element in explaining the similarities and differences in the thermodynamic properties, local flexibility, and collective motions of the different forms of the enzyme. PMID:25527961

  17. Borna disease virus P protein inhibits nitric oxide synthase gene expression in astrocytes

    SciTech Connect

    Peng Guiqing; Zhang Fengmin; Zhang Qi; Wu Kailang; Zhu Fan; Wu Jianguo

    2007-09-30

    Borna disease virus (BDV) is one of the potential infectious agents involved in the development of central nervous system (CNS) diseases. Neurons and astrocytes are the main targets of BDV infection, but little is known about the roles of BDV infection in the biological effects of astrocytes. Here we reported that BDV inhibits the activation of inducible nitric oxide synthase (iNOS) in murine astrocytes induced by bacterial LPS and PMA. To determine which protein of BDV is responsible for the regulation of iNOS expression, we co-transfected murine astrocytes with reporter plasmid iNOS-luciferase and plasmid expressing individual BDV proteins. Results from analyses of reporter activities revealed that only the phosphoprotein (P) of BDV had an inhibitory effect on the activation of iNOS. In addition, P protein inhibits nitric oxide production through regulating iNOS expression. We also reported that the nuclear factor kappa B (NF-{kappa}B) binding element, AP-1 recognition site, and interferon-stimulated response element (ISRE) on the iNOS promoter were involved in the repression of iNOS gene expression regulated by the P protein. Functional analysis indicated that sequences from amino acids 134 to 174 of the P protein are necessary for the regulation of iNOS. These data suggested that BDV may suppress signal transduction pathways, which resulted in the inhibition of iNOS activation in astrocytes.

  18. Architectures of Whole-Module and Bimodular Proteins from the 6-Deoxyerythronolide B Synthase

    PubMed Central

    Edwards, Andrea L.; Matsui, Tsutomu; Weiss, Thomas M.; Khosla, Chaitan

    2014-01-01

    The 6-deoxyerythronolide B synthase (DEBS) is a prototypical assembly line polyketide synthase (PKS) produced by the actinomycete Saccharopolyspora erythraea that synthesizes the macrocyclic core of the antibiotic erythromycin, 6-deoxyerythronolide B (6-dEB). The megasynthase is a 2 MDa trimeric complex comprised of three unique homodimers assembled from the gene products DEBS1, DEBS2, and DEBS3, which are housed within the erythromycin biosynthetic gene cluster. Each homodimer contains two clusters of catalytically independent enzymatic domains, each referred to as a module, which catalyzes one round of polyketide chain extension and modification. Modules are named sequentially to indicate the order in which they are utilized during synthesis of 6-dEB. We report small angle X-ray scattering (SAXS) analyses of a whole module and bimodule from DEBS as well as a set of domains for which high-resolution structures are available. In all cases, the solution state was probed under previously established conditions that ensure each protein is catalytically active. SAXS data are consistent with atomic-resolution structures of DEBS fragments. Therefore, we used the available high-resolution structures of DEBS domains to model the architectures of the larger protein assemblies using rigid body refinement. Our data supports a model in which, the third module of DEBS forms a disc-shaped structure capable of caging the acyl carrier protein domain proximal to each active site. The molecular envelope of DEBS3 is a thin, elongated ellipsoid, and the results of rigid body modeling suggest that modules 5 and 6 stack colinearly along the 2-fold axis of symmetry. PMID:24704088

  19. Automating gene library synthesis by structure-based combinatorial protein engineering: examples from plant sesquiterpene synthases.

    PubMed

    Dokarry, Melissa; Laurendon, Caroline; O'Maille, Paul E

    2012-01-01

    Structure-based combinatorial protein engineering (SCOPE) is a homology-independent recombination method to create multiple crossover gene libraries by assembling defined combinations of structural elements ranging from single mutations to domains of protein structure. SCOPE was originally inspired by DNA shuffling, which mimics recombination during meiosis, where mutations from parental genes are "shuffled" to create novel combinations in the resulting progeny. DNA shuffling utilizes sequence identity between parental genes to mediate template-switching events (the annealing and extension of one parental gene fragment on another) in PCR reassembly reactions to generate crossovers and hence recombination between parental genes. In light of the conservation of protein structure and degeneracy of sequence, SCOPE was developed to enable the "shuffling" of distantly related genes with no requirement for sequence identity. The central principle involves the use of oligonucleotides to encode for crossover regions to choreograph template-switching events during PCR assembly of gene fragments to create chimeric genes. This approach was initially developed to create libraries of hybrid DNA polymerases from distantly related parents, and later developed to create a combinatorial mutant library of sesquiterpene synthases to explore the catalytic landscapes underlying the functional divergence of related enzymes. This chapter presents a simplified protocol of SCOPE that can be integrated with different mutagenesis techniques and is suitable for automation by liquid-handling robots. Two examples are presented to illustrate the application of SCOPE to create gene libraries using plant sesquiterpene synthases as the model system. In the first example, we outline how to create an active-site library as a series of complex mixtures of diverse mutants. In the second example, we outline how to create a focused library as an array of individual clones to distil minimal combinations of

  20. Methyl-accepting chemotaxis protein III and transducer gene trg.

    PubMed Central

    Hazelbauer, G L; Engström, P; Harayama, S

    1981-01-01

    A comparison of the two-dimensional gel patterns of methyl-3H- and 35S-labeled membrane proteins from trg+ and trg null mutant strains of Escherichia coli indicated that the product of trg is probably methyl-accepting chemotaxis protein III. Like the other known methyl-accepting chemotaxis proteins, the trg product is a membrane protein that migrates as more than one species in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, implying that it too is multiple methylated. It appears likely that all chemoreceptors are linked to the tumble regulator through a single class of membrane protein transducers which are methyl-accepting proteins. Three transducers are coded for by genes tsr, tar, and, probably, trg. Another methyl-accepting protein, which is not related to any of these genes, was observed. Images PMID:7007323

  1. Isolation and characterization of the orotidine 5'-monophosphate decarboxylase domain of the multifunctional protein uridine 5'-monophosphate synthase.

    PubMed

    Floyd, E E; Jones, M E

    1985-08-01

    The multifunctional protein uridine 5'-monophosphate (UMP) synthase catalyzes the final two reactions of the de novo biosynthesis of UMP in mammalian cells by the sequential action of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate (OMP) decarboxylase (EC 4.1.1.23). This protein is composed of one or two identical subunits; the monomer weighs of 51,500 daltons. UMP synthase from mouse Ehrlich ascites cells can exist as three distinct species as determined by sucrose density gradient centrifugation: a 3.6 S monomer, a 5.1 S dimer, and a 5.6 S conformationally altered dimer. Limited digestion of each of these three species with trypsin produced a 28,500-dalton peptide that was relatively resistant to further proteolysis. The peptide appears to be one of the two enzyme domains of UMP synthase for it retained only OMP decarboxylase activity. Similar results were obtained when UMP synthase was digested with elastase. OMP decarboxylase activity was less stable for the domain than for UMP synthase; the domain can rapidly lose activity upon storage or upon dilution. The size of the mammalian OMP decarboxylase domain is similar to that of yeast OMP decarboxylase. If the polypeptides which are cleaved from UMP synthase by trypsin are derived exclusively from either the amino or the carboxyl end of UMP synthase, then the size of a fragment possessing the orotate phosphoribosyltransferase domain could be as large as 23,000 daltons which is similar in size to the orotate phosphoribosyltransferase of yeast and of Escherichia coli. PMID:3839509

  2. Circadian Regulation of Sucrose Phosphate Synthase Activity in Tomato by Protein Phosphatase Activity.

    PubMed Central

    Jones, T. L.; Ort, D. R.

    1997-01-01

    Sucrose phosphate synthase (SPS), a key enzyme in sucrose biosynthesis, is regulated by protein phosphorylation and shows a circadian pattern of activity in tomato. SPS is most active in its dephosphorylated state, which normally coincides with daytime. Applying okadaic acid, a potent protein phosphatase inhibitor, prevents SPS activation. More interesting is that a brief treatment with cycloheximide, a cytoplasmic translation inhibitor, also prevents the light activation of SPS without any effect on the amount of SPS protein. Cordycepin, an inhibitor of transcript synthesis and processing, has the same effect. Both of these inhibitors also prevent the activation phase of the circadian rhythm in SPS activity. Conversely, cycloheximide and cordycepin do not prevent the decline in circadian SPS activity that normally occurs at night. These observations indicate that SPS phosphatase activity but not SPS kinase activity is controlled, directly or indirectly, at the level of gene expression. Taken together, these data imply that there is a circadian rhythm controlling the transcription of a protein phosphatase that subsequently dictates the circadian rhythm in SPS activity via effects on this enzyme's phosphorylation state. PMID:12223667

  3. Mycobacterium tuberculosis acyl carrier protein synthase adopts two different pH-dependent structural conformations

    SciTech Connect

    Gokulan, Kuppan; Aggarwal, Anup; Shipman, Lance; Besra, Gurdyal S.; Sacchettini, James C.

    2011-07-01

    Bacterial acyl carrier protein synthase plays an essential role in the synthesis of fatty acids, nonribosomal peptides and polyketides. In Mycobacterium tuberculosis, AcpS or group I phosphopentatheine transferase exhibits two different structural conformations depending upon the pH. The crystal structures of acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) and Corynebacterium ammoniagenes determined at pH 5.3 and pH 6.5, respectively, are reported. Comparison of the Mtb apo-AcpS structure with the recently reported structure of the Mtb AcpS–ADP complex revealed that AcpS adopts two different conformations: the orthorhombic and trigonal space-group structures show structural differences in the α2 helix and in the conformation of the α3–α4 connecting loop, which is in a closed conformation. The apo-AcpS structure shows electron density for the entire model and was obtained at lower pH values (4.4–6.0). In contrast, at a higher pH value (6.5) AcpS undergoes significant conformational changes, resulting in disordered regions that show no electron density in the AcpS model. The solved structures also reveal that C. ammoniagenes AcpS undergoes structural rearrangement in two regions, similar to the recently reported Mtb AcpS–ADP complex structure. In vitro reconstitution experiments show that AcpS has a higher post-translational modification activity between pH 4.4 and 6.0 than at pH values above 6.5, where the activity drops owing to the change in conformation. The results show that apo-AcpS and AcpS–ADP adopt different conformations depending upon the pH conditions of the crystallization solution.

  4. Arabidopsis OR proteins are the major post-transcriptional regulators of phytoene synthase in mediating carotenoid biosynthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Carotenoids are indispensable natural pigments to plants and humans. Phytoene synthase (PSY), the rate-limiting enzyme in carotenoid biosynthetic pathway, and ORANGE (OR), a regulator of chromoplast differentiation and enhancer of carotenoid biosynthesis, represent two key proteins that control caro...

  5. Thymidylate synthase protein and p53 mRNA form an in vivo ribonucleoprotein complex.

    PubMed

    Chu, E; Copur, S M; Ju, J; Chen, T M; Khleif, S; Voeller, D M; Mizunuma, N; Patel, M; Maley, G F; Maley, F; Allegra, C J

    1999-02-01

    A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS-overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS-overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression. PMID:9891091

  6. Thymidylate Synthase Protein and p53 mRNA Form an In Vivo Ribonucleoprotein Complex

    PubMed Central

    Chu, Edward; Copur, Sitki M.; Ju, Jingfang; Chen, Tian-men; Khleif, Samir; Voeller, Donna M.; Mizunuma, Nobuyuki; Patel, Mahendra; Maley, Gladys F.; Maley, Frank; Allegra, Carmen J.

    1999-01-01

    A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS-overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS-overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression. PMID:9891091

  7. Fusion protein of retinol-binding protein and albumin domain III reduces liver fibrosis

    PubMed Central

    Lee, Hongsik; Jeong, Hyeyeun; Park, Sangeun; Yoo, Wonbaek; Choi, Soyoung; Choi, Kyungmin; Lee, Min-Goo; Lee, Mihwa; Cha, DaeRyong; Kim, Young-Sik; Han, Jeeyoung; Kim, Wonkon; Park, Sun-Hwa; Oh, Junseo

    2015-01-01

    Activated hepatic stellate cells (HSCs) play a key role in liver fibrosis, and inactivating HSCs has been considered a promising therapeutic approach. We previously showed that albumin and its derivative designed for stellate cell-targeting, retinol-binding protein–albumin domain III fusion protein (referred to as R-III), inactivate cultured HSCs. Here, we investigated the mechanism of action of albumin/R-III in HSCs and examined the anti-fibrotic potential of R-III in vivo. R-III treatment and albumin expression downregulated retinoic acid (RA) signaling which was involved in HSC activation. RA receptor agonist and retinaldehyde dehydrogenase overexpression abolished the anti-fibrotic effect of R-III and albumin, respectively. R-III uptake into cultured HSCs was significantly decreased by siRNA-STRA6, and injected R-III was localized predominantly in HSCs in liver. Importantly, R-III administration reduced CCl4- and bile duct ligation-induced liver fibrosis. R-III also exhibited a preventive effect against CCl4-inducd liver fibrosis. These findings suggest that the anti-fibrotic effect of albumin/R-III is, at least in part, mediated by downregulation of RA signaling and that R-III is a good candidate as a novel anti-fibrotic drug. PMID:25864124

  8. Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria

    SciTech Connect

    Halavaty, Andrei S.; Kim, Youngchang; Minasov, George; Shuvalova, Ludmilla; Dubrovska, Ievgeniia; Winsor, James; Zhou, Min; Onopriyenko, Olena; Skarina, Tatiana; Papazisi, Leka; Kwon, Keehwan; Peterson, Scott N.; Joachimiak, Andrzej; Savchenko, Alexei; Anderson, Wayne F.

    2012-10-01

    The structural characterization of acyl-carrier-protein synthase (AcpS) from three different pathogenic microorganisms is reported. One interesting finding of the present work is a crystal artifact related to the activity of the enzyme, which fortuitously represents an opportunity for a strategy to design a potential inhibitor of a pathogenic AcpS. Some bacterial type II fatty-acid synthesis (FAS II) enzymes have been shown to be important candidates for drug discovery. The scientific and medical quest for new FAS II protein targets continues to stimulate research in this field. One of the possible additional candidates is the acyl-carrier-protein synthase (AcpS) enzyme. Its holo form post-translationally modifies the apo form of an acyl carrier protein (ACP), which assures the constant delivery of thioester intermediates to the discrete enzymes of FAS II. At the Center for Structural Genomics of Infectious Diseases (CSGID), AcpSs from Staphylococcus aureus (AcpS{sub SA}), Vibrio cholerae (AcpS{sub VC}) and Bacillus anthracis (AcpS{sub BA}) have been structurally characterized in their apo, holo and product-bound forms, respectively. The structure of AcpS{sub BA} is emphasized because of the two 3′, 5′-adenosine diphosphate (3′, 5′-ADP) product molecules that are found in each of the three coenzyme A (CoA) binding sites of the trimeric protein. One 3′, 5′-ADP is bound as the 3′, 5′-ADP part of CoA in the known structures of the CoA–AcpS and 3′, 5′-ADP–AcpS binary complexes. The position of the second 3′, 5′-ADP has never been described before. It is in close proximity to the first 3′, 5′-ADP and the ACP-binding site. The coordination of two ADPs in AcpS{sub BA} may possibly be exploited for the design of AcpS inhibitors that can block binding of both CoA and ACP.

  9. Effects of starch synthase IIa gene dosage on grain, protein and starch in endosperm of wheat.

    PubMed

    Konik-Rose, Christine; Thistleton, Jenny; Chanvrier, Helene; Tan, Ihwa; Halley, Peter; Gidley, Michael; Kosar-Hashemi, Behjat; Wang, Hong; Larroque, Oscar; Ikea, Joseph; McMaugh, Steve; Regina, Ahmed; Rahman, Sadequr; Morell, Matthew; Li, Zhongyi

    2007-11-01

    Starch synthases (SS) are responsible for elongating the alpha-1,4 glucan chains of starch. A doubled haploid population was generated by crossing a line of wheat, which lacks functional ssIIa genes on each genome (abd), and an Australian wheat cultivar, Sunco, with wild type ssIIa alleles on each genome (ABD). Evidence has been presented previously indicating that the SGP-1 (starch granule protein-1) proteins present in the starch granule in wheat are products of the ssIIa genes. Analysis of 100 progeny lines demonstrated co-segregation of the ssIIa alleles from the three genomes with the SGP-1 proteins, providing further evidence that the SGP-1 proteins are the products of the ssIIa genes. From the progeny lines, 40 doubled haploid lines representing the eight possible genotypes for SSIIa (ABD, aBD, AbD, ABd, abD, aBd, Abd, abd) were characterized for their grain weight, protein content, total starch content and starch properties. For some properties (chain length distribution, pasting properties, swelling power, and gelatinization properties), a progressive change was observed across the four classes of genotypes (wild type, single nulls, double nulls and triple nulls). However, for other grain properties (seed weight and protein content) and starch properties (total starch content, granule morphology and crystallinity, granule size distribution, amylose content, amylose-lipid dissociation properties), a statistically significant change only occurred for the triple nulls, indicating that all three genes had to be missing or inactive for a change to occur. These results illustrate the importance of SSIIa in controlling grain and starch properties and the importance of amylopectin fine structure in controlling starch granule properties in wheat. PMID:17721773

  10. KOJAK encodes a cellulose synthase-like protein required for root hair cell morphogenesis in Arabidopsis

    PubMed Central

    Favery, Bruno; Ryan, Eoin; Foreman, Julia; Linstead, Paul; Boudonck, Kurt; Steer, Martin; Shaw, Peter; Dolan, Liam

    2001-01-01

    The cell wall is an important determinant of plant cell form. Here we define a class of Arabidopsis root hair mutants with defective cell walls. Plants homozygous for kojak (kjk) mutations initiate root hairs that rupture at their tip soon after initiation. The KJK gene was isolated by positional cloning, and its identity was confirmed by the molecular complementation of the Kjk− phenotype and the sequence of three kjk mutant alleles. KOJAK encodes a cellulose synthase-like protein, AtCSLD3. KOJAK/AtCSLD3 is the first member of this subfamily of proteins to be shown to have a function in cell growth. Subcellular localization of the KOJAK/AtCSLD3 protein using a GFP fusion shows that KOJAK/AtCSLD3 is located on the endoplasmic reticulum, indicating that KOJAK/AtCSLD3 is required for the synthesis of a noncellulosic wall polysaccharide. Consistent with the cell specific defect in the roots of kjk mutants, KOJAK/AtCSDL3 is preferentially expressed in hair cells of the epidermis. The Kjk− phenotype and the pattern of KOJAK/AtCSLD3 expression suggest that this gene acts early in the process of root hair outgrowth. These results suggest that KOJAK/AtCSLD3 is involved in the biosynthesis of β-glucan-containing polysaccharides that are required during root hair elongation. PMID:11156607

  11. Dietary whey protein hydrolysates increase skeletal muscle glycogen levels via activation of glycogen synthase in mice.

    PubMed

    Kanda, Atsushi; Morifuji, Masashi; Fukasawa, Tomoyuki; Koga, Jinichiro; Kanegae, Minoru; Kawanaka, Kentaro; Higuchi, Mitsuru

    2012-11-14

    Previously, we have shown that consuming carbohydrate plus whey protein hydrolysates (WPHs) replenished muscle glycogen after exercise more effectively than consuming intact whey protein or branched-chain amino acids (BCAAs). The mechanism leading to superior glycogen replenishment after consuming WPH is unclear. In this 5 week intervention, ddY mice were fed experimental diets containing WPH, a mixture of whey amino acids (WAAs), or casein (control). After the intervention, gastrocnemius muscle glycogen levels were significantly higher in the WPH group (4.35 mg/g) than in the WAA (3.15 mg/g) or control (2.51 mg/g) groups. In addition, total glycogen synthase (GS) protein levels were significantly higher in the WPH group (153%) than in the WAA (89.2%) or control groups, and phosphorylated GS levels were significantly decreased in the WPH group (51.4%). These results indicate that dietary WPH may increase the muscle glycogen content through increased GS activity. PMID:23113736

  12. NOSTRIN: A protein modulating nitric oxide release and subcellular distribution of endothelial nitric oxide synthase

    PubMed Central

    Zimmermann, Kirstin; Opitz, Nils; Dedio, Jürgen; Renné, Christoph; Müller-Esterl, Werner; Oess, Stefanie

    2002-01-01

    Activity and localization of endothelial nitric oxide synthase (eNOS) is regulated in a remarkably complex fashion, yet the complex molecular machinery mastering stimulus-induced eNOS translocation and trafficking is poorly understood. In a search by the yeast two-hybrid system using the eNOS oxygenase domain as bait, we have identified a previously uncharacterized eNOS-interacting protein, dubbed NOSTRIN (for eNOS traffic inducer). NOSTRIN contains a single polypeptide chain of 506-aa residues of 58 kDa with an N-terminal cdc15 domain and a C-terminal SH3 domain. NOSTRIN mRNA is abundant in highly vascularized tissues such as placenta, kidney, lung, and heart, and NOSTRIN protein is expressed in vascular endothelial cells. Coimmunoprecipitation experiments demonstrated the eNOS–NOSTRIN interaction in vitro and in vivo, and NOSTRIN's SH3 domain was essential and sufficient for eNOS binding. NOSTRIN colocalized extensively with eNOS at the plasma membrane of confluent human umbilical venous endothelial cells and in punctate cytosolic structures of CHO-eNOS cells. NOSTRIN overexpression induced a profound redistribution of eNOS from the plasma membrane to vesicle-like structures matching the NOSTRIN pattern and at the same time led to a significant inhibition of NO release. We conclude that NOSTRIN contributes to the intricate protein network controlling activity, trafficking, and targeting of eNOS. PMID:12446846

  13. Type III polyketide synthase beta-ketoacyl-ACP starter unit and ethylmalonyl-CoA extender unit selectivity discovered by Streptomyces coelicolor genome mining.

    PubMed

    Song, Lijiang; Barona-Gomez, Francisco; Corre, Christophe; Xiang, Longkuan; Udwary, Daniel W; Austin, Michael B; Noel, Joseph P; Moore, Bradley S; Challis, Gregory L

    2006-11-22

    Polyketide synthases (PKSs) are involved in the biosynthesis of many important natural products. In bacteria, type III PKSs typically catalyze iterative decarboxylation and condensation reactions of malonyl-CoA building blocks in the biosynthesis of polyhydroxyaromatic products. Here it is shown that Gcs, a type III PKS encoded by the sco7221 ORF of the bacterium Streptomyces coelicolor, is required for biosynthesis of the germicidin family of 3,6-dialkyl-4-hydroxypyran-2-one natural products. Evidence consistent with Gcs-catalyzed elongation of specific beta-ketoacyl-ACP products of the fatty acid synthase FabH with ethyl- or methylmalonyl-CoA in the biosynthesis of germicidins is presented. Selectivity for beta-ketoacyl-ACP starter units and ethylmalonyl-CoA as an extender unit is unprecedented for type III PKSs, suggesting these enzymes may be capable of utilizing a far wider range of starter and extender units for natural product assembly than believed until now. PMID:17105255

  14. Slow onset inhibition of bacterial beta-ketoacyl-acyl carrier protein synthases by thiolactomycin.

    PubMed

    Machutta, Carl A; Bommineni, Gopal R; Luckner, Sylvia R; Kapilashrami, Kanishk; Ruzsicska, Bela; Simmerling, Carlos; Kisker, Caroline; Tonge, Peter J

    2010-02-26

    Thiolactomycin (TLM), a natural product thiolactone antibiotic produced by species of Nocardia and Streptomyces, is an inhibitor of the beta-ketoacyl-acyl carrier protein synthase (KAS) enzymes in the bacterial fatty acid synthase pathway. Using enzyme kinetics and direct binding studies, TLM has been shown to bind preferentially to the acyl-enzyme intermediates of the KASI and KASII enzymes from Mycobacterium tuberculosis and Escherichia coli. These studies, which utilized acyl-enzyme mimics in which the active site cysteine was replaced by a glutamine, also revealed that TLM is a slow onset inhibitor of the KASI enzymes KasA and ecFabB but not of the KASII enzymes KasB and ecFabF. The differential affinity of TLM for the acyl-KAS enzymes is proposed to result from structural change involving the movement of helices alpha5 and alpha6 that prepare the enzyme to bind malonyl-AcpM or TLM and that is initiated by formation of hydrogen bonds between the acyl-enzyme thioester and the oxyanion hole. The finding that TLM is a slow onset inhibitor of ecFabB supports the proposal that the long residence time of TLM on the ecFabB homologues in Serratia marcescens and Klebsiella pneumonia is an important factor for the in vivo antibacterial activity of TLM against these two organisms despite the fact that the in vitro MIC values are only 100-200 microg/ml. The mechanistic data on the interaction of TLM with KasA will provide an important foundation for the rational development of high affinity KasA inhibitors based on the thiolactone skeleton. PMID:20018879

  15. Thioaptamers Targeting Dengue Virus Type-2 Envelope Protein Domain III

    PubMed Central

    Gandham, Sai Hari A.; Volk, David E.; Rao, Lokesh G. L.; Neerathilingam, Muniasamy; Gorenstein, David G.

    2014-01-01

    Thioaptamers targeting the dengue-2 virus (DENV-2) envelope protein domain III (EDIII) were developed. EDIII, which contains epitopes for binding neutralizing antibodies, is the putative host-receptor binding domain and is thus an attractive target for development of vaccines, anti-viral therapeutic and diagnostic agents. Thioaptamer DENTA-1 bound to DENV-2 EDIII adjacent to a known neutralizing antibody binding site with a dissociation constant of 154 nM. PMID:25261724

  16. Cruentaren A Binds F1F0 ATP Synthase To Modulate the Hsp90 Protein Folding Machinery

    PubMed Central

    2015-01-01

    The molecular chaperone Hsp90 requires the assistance of immunophilins, co-chaperones, and partner proteins for the conformational maturation of client proteins. Hsp90 inhibition represents a promising anticancer strategy due to the dependence of numerous oncogenic signaling pathways upon Hsp90 function. Historically, small molecules have been designed to inhibit ATPase activity at the Hsp90 N-terminus; however, these molecules also induce the pro-survival heat shock response (HSR). Therefore, inhibitors that exhibit alternative mechanisms of action that do not elicit the HSR are actively sought. Small molecules that disrupt Hsp90-co-chaperone interactions can destabilize the Hsp90 complex without induction of the HSR, which leads to inhibition of cell proliferation. In this article, selective inhibition of F1F0 ATP synthase by cruentaren A was shown to disrupt the Hsp90-F1F0 ATP synthase interaction and result in client protein degradation without induction of the HSR. PMID:24450340

  17. Seasonal, tissue-specific regulation of Akt/protein kinase B and glycogen synthase in hibernators.

    PubMed

    Hoehn, Kyle L; Hudachek, Susan F; Summers, Scott A; Florant, Gregory L

    2004-03-01

    Yellow-bellied marmots (Marmota flaviventris) exhibit a circannual cycle of hyperphagia and nutrient storage in the summer followed by hibernation in the winter. This annual cycle of body mass gain and loss is primarily due to large-scale accumulation of lipid in the summer, which is then mobilized and oxidized for energy during winter. The rapid and predictable change in body mass makes these animals ideal for studies investigating the molecular basis for body weight regulation. In the study described herein, we monitored seasonal changes in the protein levels and activity of a central regulator of anabolic metabolism, the serine-threonine kinase Akt-protein kinase B (Akt/PKB), during the months accompanying maximal weight gain and entry into hibernation (June-November). Interestingly, under fasting conditions, Akt/PKB demonstrated a tissue-specific seasonal activation. Specifically, although Akt/PKB levels did not change, the activity of Akt/PKB (isoforms 1/alpha and 2/beta) in white adipose tissue (WAT) increased significantly in July. Moreover, glycogen synthase, which lies downstream of Akt/PKB on a linear pathway linking the enzyme to the stimulation of glycogen synthesis, demonstrated a similar pattern of seasonal activation. By contrast, Akt/PKB activity in skeletal muscle peaked much later (i.e., September). These data suggest the existence of a novel, tissue-specific mechanism regulating Akt/PKB activation during periods of marked anabolism. PMID:14656767

  18. The Arabidopsis Protein CGLD11 Is Required for Chloroplast ATP Synthase Accumulation.

    PubMed

    Grahl, Sabine; Reiter, Bennet; Gügel, Irene Luise; Vamvaka, Evgenia; Gandini, Chiara; Jahns, Peter; Soll, Jürgen; Leister, Dario; Rühle, Thilo

    2016-06-01

    ATP synthases in chloroplasts (cpATPase) and mitochondria (mtATPase) are responsible for ATP production during photosynthesis and oxidative phosphorylation, respectively. Both enzymes consist of two multisubunit complexes, the membrane-bound coupling factor O and the soluble coupling factor 1. During cpATPase biosynthesis, several accessory factors facilitate subunit production and orchestrate complex assembly. Here, we describe a new auxiliary protein in Arabidopsis thaliana, which is required for cpATPase accumulation. AtCGLD11 (CONSERVED IN THE GREEN LINEAGE AND DIATOMS 11) is a protein without any known functional domain and shows dual localization to chloroplasts and mitochondria. Loss of AtCGLD11 function results in reduced levels of cpATPase and impaired photosynthetic performance with lower rates of ATP synthesis. In yeast two-hybrid experiments, AtCGLD11 interacts with the β subunits of the cpATPase and mtATPase. Our results suggest that AtCGLD11 functions in F1 assembly during cpATPase biogenesis, while its role in mtATPase biosynthesis may not, or not yet, be essential. PMID:26979383

  19. Assembly of the cysteine synthase complex and the regulatory role of protein-protein interactions.

    PubMed

    Kumaran, Sangaralingam; Yi, Hankuil; Krishnan, Hari B; Jez, Joseph M

    2009-04-10

    Macromolecular assemblies play critical roles in regulating cellular functions. The cysteine synthase complex (CSC), which is formed by association of serine O-acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS), acts as a sensor and modulator of thiol metabolism by responding to changes in nutrient conditions. Here we examine the oligomerization and energetics of formation of the soybean CSC. Biophysical examination of the CSC by size exclusion chromatography and sedimentation ultracentrifugation indicates that this assembly (complex M(r) approximately 330,000) consists of a single SAT trimer (trimer M(r) approximately 110,000) and three OASS dimers (dimer M(r) approximately 70,000). Analysis of the SAT-OASS interaction by isothermal titration calorimetry reveals negative cooperativity with three distinct binding events during CSC formation with K(d) values of 0.3, 7.5, and 78 nm. The three binding events are also observed using surface plasmon resonance with comparable affinities. The stability of the CSC derives from rapid association and extremely slow dissociation of OASS with SAT and requires the C terminus of SAT for the interaction. Steady-state kinetic analysis shows that CSC formation enhances SAT activity and releases SAT from substrate inhibition and feedback inhibition by cysteine, the final product of the biosynthesis pathway. Cysteine inhibits SAT and the CSC with K(i) values of 2 and 70 microm, respectively. These results suggest a new model for the architecture of this regulatory complex and additional control mechanisms for biochemically controlling plant cysteine biosynthesis. Based on previous work and our results, we suggest that OASS acts as an enzyme chaperone of SAT in the CSC. PMID:19213732

  20. Fatty Acid Biosynthesis in Pseudomonas aeruginosa Is Initiated by the FabY Class of β-Ketoacyl Acyl Carrier Protein Synthases

    PubMed Central

    Yuan, Yanqiu; Sachdeva, Meena; Leeds, Jennifer A.

    2012-01-01

    The prototypical type II fatty acid synthesis (FAS) pathway in bacteria utilizes two distinct classes of β-ketoacyl synthase (KAS) domains to assemble long-chain fatty acids, the KASIII domain for initiation and the KASI/II domain for elongation. The central role of FAS in bacterial viability and virulence has stimulated significant effort toward developing KAS inhibitors, particularly against the KASIII domain of the β-acetoacetyl-acyl carrier protein (ACP) synthase FabH. Herein, we show that the opportunistic pathogen Pseudomonas aeruginosa does not utilize a FabH ortholog but rather a new class of divergent KAS I/II enzymes to initiate the FAS pathway. When a P. aeruginosa cosmid library was used to rescue growth in a fabH downregulated strain of Escherichia coli, a single unannotated open reading frame, PA5174, complemented fabH depletion. While deletion of all four KASIII domain-encoding genes in the same P. aeruginosa strain resulted in a wild-type growth phenotype, deletion of PA5174 alone specifically attenuated growth due to a defect in de novo FAS. Siderophore secretion and quorum-sensing signaling, particularly in the rhl and Pseudomonas quinolone signal (PQS) systems, was significantly muted in the absence of PA5174. The defect could be repaired by intergeneric complementation with E. coli fabH. Characterization of recombinant PA5174 confirmed a preference for short-chain acyl coenzyme A (acyl-CoA) substrates, supporting the identification of PA5174 as the predominant enzyme catalyzing the condensation of acetyl coenzyme A with malonyl-ACP in P. aeruginosa. The identification of the functional role for PA5174 in FAS defines the new FabY class of β-ketoacyl synthase KASI/II domain condensation enzymes. PMID:22753059

  1. Induction of nitric oxide synthase by protein synthesis inhibition in aortic smooth muscle cells

    PubMed Central

    Marczin, Nándor; Go, Carolyn Y; Papapetropoulos, Andreas; Catravas, John D

    1998-01-01

    The role of de novo protein synthesis in inducible NO synthase (iNOS) activation was investigated in vitro by evaluating the effects of protein synthesis inhibitors cycloheximide (CH) and anisomycin (ANI) on iNOS activity, protein and mRNA levels in rat aortic smooth muscle cells (RASMC).As determined by cyclic GMP accumulation, substrate (L-arginine)- and inhibitor (NG-monomethyl-L-arginine, NMMA)-sensitive iNOS activity was significantly elevated in CH- or ANI-treated RASMC after 24 h.Lipopolysaccharide (LPS) produced a time-dependent increase in cyclic GMP levels with maximal stimulation at 6 h and a decline to near baseline at 24 h. CH attenuated LPS-induced cyclic GMP accumulation at 3 and 6 h. However, cyclic GMP levels were superinduced at later times by CH. The concentration-dependence of cyclic GMP stimulation by cycloheximide was biphasic both in the absence and presence of LPS, with maximal stimulation at 10 μM and inhibition at higher concentrations.Increased iNOS activity by CH was associated with elevated levels of immunoreactive iNOS protein as judged by Western blotting in LPS- and CH-treated cells.CH-induced iNOS activity and superinduction of iNOS by CH in cells treated with LPS were both significantly inhibited by actinomycin D, a transcription inhibitor.RT-PCR revealed elevated iNOS mRNA levels after 12 h of exposure to CH. The combination of LPS and CH caused a significant increase in iNOS gene expression relative to LPS- or CH stimulation alone.These results show that partial protein synthesis inhibition by CH alone upregulates iNOS mRNA and superinduces iNOS mRNA in cytokine-treated RASMC, which is translated to the functional enzyme generating biologically active NO. Thus iNOS activation in these cells not only requires new protein synthesis but it also appears to be negatively regulated by newly synthesized proteins. PMID:9535031

  2. Protein-protein interactions within the Fatty Acid Synthase-II system of Mycobacterium tuberculosis are essential for mycobacterial viability.

    PubMed

    Veyron-Churlet, Romain; Guerrini, Olivier; Mourey, Lionel; Daffé, Mamadou; Zerbib, Didier

    2004-12-01

    Despite the existence of efficient chemotherapy, tuberculosis remains a leading cause of mortality worldwide. New drugs are urgently needed to reduce the potential impact of the emergence of multidrug-resistant strains of the causative agent Mycobacterium tuberculosis (Mtb). The front-line antibiotic isoniazid (INH), and several other drugs, target the biosynthesis of mycolic acids and especially the Fatty Acid Synthase-II (FAS-II) elongation system. This biosynthetic pathway is essential and specific for mycobacteria and still represents a valuable system for the search of new anti-tuberculous agents. Several data, in the literature, suggest the existence of protein-protein interactions within the FAS-II system. These interactions themselves might serve as targets for a new generation of drugs directed against Mtb. By using an extensive in vivo yeast two-hybrid approach and in vitro co-immunoprecipitation, we have demonstrated the existence of both homotypic and heterotypic interactions between the known components of FAS-II. The condensing enzymes KasA, KasB and mtFabH interact with each other and with the reductases MabA and InhA. Furthermore, we have designed and constructed point mutations of the FAS-II reductase MabA, able to disrupt its homotypic interactions and perturb the interaction pattern of this protein within FAS-II. Finally, we showed by a transdominant genetic approach that these mutants are dominant negative in both non-pathogenic and pathogenic mycobacteria. These data allowed us to draw a dynamic model of the organization of FAS-II. They also represent an important step towards the design of a new generation of anti-tuberculous agents, as being inhibitors of essential protein-protein interactions. PMID:15554959

  3. Occurrence, structure, and evolution of nitric oxide synthase-like proteins in the plant kingdom.

    PubMed

    Jeandroz, Sylvain; Wipf, Daniel; Stuehr, Dennis J; Lamattina, Lorenzo; Melkonian, Michael; Tian, Zhijian; Zhu, Ying; Carpenter, Eric J; Wong, Gane Ka-Shu; Wendehenne, David

    2016-01-01

    Nitric oxide (NO) signaling regulates various physiological processes in both animals and plants. In animals, NO synthesis is mainly catalyzed by NO synthase (NOS) enzymes. Although NOS-like activities that are sensitive to mammalian NOS inhibitors have been detected in plant extracts, few bona fide plant NOS enzymes have been identified. We searched the data set produced by the 1000 Plants (1KP) international consortium for the presence of transcripts encoding NOS-like proteins in over 1000 species of land plants and algae. We also searched for genes encoding NOS-like enzymes in 24 publicly available algal genomes. We identified no typical NOS sequences in 1087 sequenced transcriptomes of land plants. In contrast, we identified NOS-like sequences in 15 of the 265 algal species analyzed. Even if the presence of NOS enzymes assembled from multipolypeptides in plants cannot be conclusively discarded, the emerging data suggest that, instead of generating NO with evolutionarily conserved NOS enzymes, land plants have evolved finely regulated nitrate assimilation and reduction processes to synthesize NO through a mechanism different than that in animals. PMID:26933064

  4. Plastic adaptation toward mutations in proteins: structural comparison of thymidylate synthases.

    PubMed

    Perry, K M; Fauman, E B; Finer-Moore, J S; Montfort, W R; Maley, G F; Maley, F; Stroud, R M

    1990-01-01

    The structure of thymidylate synthase (TS) from Escherichia coli was solved from cubic crystals with a = 133 A grown under reducing conditions at pH 7.0, and refined to R = 22% at 2.1 A resolution. The structure is compared with that from Lactobacillus casei solved to R = 21% at 2.3 A resolution. The structures are compared using a difference distance matrix, which identifies a common core of residues that retains the same relationship to one another in both species. After subtraction of the effects of a 50 amino acid insert present in Lactobacillus casei, differences in position of atoms correlate with temperature factors and with distance from the nearest substituted residue. The dependence of structural difference on thermal factor is parameterized and reflects both errors in coordinates that correlate with thermal factor, and the increased width of the energy well in which atoms of high thermal factor lie. The dependence of structural difference on distance from the nearest substitution also depends on thermal factors and shows an exponential dependence with half maximal effect at 3.0 A from the substitution. This represents the plastic accommodation of the protein which is parameterized in terms of thermal B factor and distance from a mutational change. PMID:2128651

  5. Sphingomyelin synthase-related protein SMSr controls ceramide homeostasis in the ER.

    PubMed

    Vacaru, Ana M; Tafesse, Fikadu G; Ternes, Philipp; Kondylis, Vangelis; Hermansson, Martin; Brouwers, Jos F H M; Somerharju, Pentti; Rabouille, Catherine; Holthuis, Joost C M

    2009-06-15

    Ceramides are central intermediates of sphingolipid metabolism with critical functions in cell organization and survival. They are synthesized on the cytosolic surface of the endoplasmic reticulum (ER) and transported by ceramide transfer protein to the Golgi for conversion to sphingomyelin (SM) by SM synthase SMS1. In this study, we report the identification of an SMS1-related (SMSr) enzyme, which catalyses the synthesis of the SM analogue ceramide phosphoethanolamine (CPE) in the ER lumen. Strikingly, SMSr produces only trace amounts of CPE, i.e., 300-fold less than SMS1-derived SM. Nevertheless, blocking its catalytic activity causes a substantial rise in ER ceramide levels and a structural collapse of the early secretory pathway. We find that the latter phenotype is not caused by depletion of CPE but rather a consequence of ceramide accumulation in the ER. Our results establish SMSr as a key regulator of ceramide homeostasis that seems to operate as a sensor rather than a converter of ceramides in the ER. PMID:19506037

  6. Role of long-range protein dynamics in different thymidylate synthase catalyzed reactions.

    PubMed

    Abeysinghe, Thelma; Kohen, Amnon

    2015-01-01

    Recent studies of Escherichia coli thymidylate synthase (ecTSase) showed that a highly conserved residue, Y209, that is located 8 Å away from the reaction site, plays a key role in the protein's dynamics. Those crystallographic studies indicated that Y209W mutant is a structurally identical but dynamically altered relative to the wild type (WT) enzyme, and that its turnover catalytic rate governed by a slow hydride-transfer has been affected. The most challenging test of an examination of a fast chemical conversion that precedes the rate-limiting step has been achieved here. The physical nature of both fast and slow C-H bond activations have been compared between the WT and mutant by means of observed and intrinsic kinetic isotope effects (KIEs) and their temperature dependence. The findings indicate that the proton abstraction step has not been altered as much as the hydride transfer step. Additionally, the comparison indicated that other kinetic steps in the TSase catalyzed reaction were substantially affected, including the order of the substrate binding. Enigmatically, although Y209 is H-bonded to 3'-OH of 2'-deoxyuridine-5'-mono-phosphate (dUMP), its altered dynamics is more pronounced on the binding of the remote cofactor, (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate), revealing the importance of long-range dynamics of the enzymatic complex and its catalytic function. PMID:25837629

  7. A cellulose synthase-like protein is required for osmotic stress tolerance in Arabidopsis

    PubMed Central

    Zhu, Jianhua; Lee, Byeong-Ha; Dellinger, Mike; Cui, Xinping; Zhang, Changqing; Wu, Shang; Nothnagel, Eugene A.; Zhu, Jian-Kang

    2011-01-01

    SUMMARY Osmotic stress imposed by soil salinity and drought stress significantly affects plant growth and development, but osmotic stress sensing and tolerance mechanisms are not well understood. Forward genetic screens using a root-bending assay have previously identified salt overly sensitive (sos) mutants of Arabidopsis that fall into five loci, SOS1 to SOS5. These loci are required for the regulation of ion homeostasis or cell expansion under salt stress, but do not play a major role in plant tolerance to the osmotic stress component of soil salinity or drought. Here we report an additional sos mutant, sos6-1, which defines a locus essential for osmotic stress tolerance. sos6-1 plants are hypersensitive to salt stress and osmotic stress imposed by mannitol or polyethylene glycol in culture media or by water deficit in the soil. SOS6 encodes a cellulose synthase-like protein, AtCSLD5. Only modest differences in cell wall chemical composition could be detected, but we found that sos6-1 mutant plants accumulate high levels of reactive oxygen species (ROS) under osmotic stress and are hypersensitive to the oxidative stress reagent methyl viologen. The results suggest that SOS6/AtCSLD5 is not required for normal plant growth and development but has a critical role in osmotic stress tolerance and this function likely involves its regulation of ROS under stress. PMID:20409003

  8. Structure/Function Analysis of a Type III Polyketide Synthase in the Brown Alga Ectocarpus siliculosus Reveals a Biochemical Pathway in Phlorotannin Monomer Biosynthesis[W

    PubMed Central

    Meslet-Cladière, Laurence; Delage, Ludovic; Leroux, Cédric J.-J.; Goulitquer, Sophie; Leblanc, Catherine; Creis, Emeline; Gall, Erwan Ar; Stiger-Pouvreau, Valérie; Czjzek, Mirjam; Potin, Philippe

    2013-01-01

    Brown algal phlorotannins are structural analogs of condensed tannins in terrestrial plants and, like plant phenols, they have numerous biological functions. Despite their importance in brown algae, phlorotannin biosynthetic pathways have been poorly characterized at the molecular level. We found that a predicted type III polyketide synthase in the genome of the brown alga Ectocarpus siliculosus, PKS1, catalyzes a major step in the biosynthetic pathway of phlorotannins (i.e., the synthesis of phloroglucinol monomers from malonyl-CoA). The crystal structure of PKS1 at 2.85-Å resolution provided a good quality electron density map showing a modified Cys residue, likely connected to a long chain acyl group. An additional pocket not found in other known type III PKSs contains a reaction product that might correspond to a phloroglucinol precursor. In vivo, we also found a positive correlation between the phloroglucinol content and the PKS III gene expression level in cells of a strain of Ectocarpus adapted to freshwater during its reacclimation to seawater. The evolution of the type III PKS gene family in Stramenopiles suggests a lateral gene transfer event from an actinobacterium. PMID:23983220

  9. Protein kinase Cδ regulates endothelial nitric oxide synthase expression via Akt activation and nitric oxide generation

    PubMed Central

    Sud, Neetu; Wedgwood, Stephen; Black, Stephen M.

    2008-01-01

    In this study, we explore the roles of the delta isoform of PKC (PKCδ) in the regulation of endothelial nitric oxide synthase (eNOS) activity in pulmonary arterial endothelial cells isolated from fetal lambs (FPAECs). Pharmacological inhibition of PKCδ with either rottlerin or with the peptide, δV1-1, acutely attenuated NO production, and this was associated with a decrease in phosphorylation of eNOS at Ser1177 (S1177). The chronic effects of PKCδ inhibition using either rottlerin or the overexpression of a dominant negative PKCδ mutant included the downregulation of eNOS gene expression that was manifested by a decrease in both eNOS promoter activity and protein expression after 24 h of treatment. We also found that PKCδ inhibition blunted Akt activation as observed by a reduction in phosphorylated Akt at position Ser473. Thus, we conclude that PKCδ is actively involved in the activation of Akt. To determine the effect of Akt on eNOS signaling, we overexpressed a dominant negative mutant of Akt and determined its effect of NO generation, eNOS expression, and phosphorylation of eNOS at S1177. Our results demonstrated that Akt inhibition was associated with decreased NO production that correlated with reduced phosphorylation of eNOS at S1177, and decreased eNOS promoter activity. We next evaluated the effect of endogenously produced NO on eNOS expression by incubating FPAECs with the eNOS inhibitor 2-ethyl-2-thiopseudourea (ETU). ETU significantly inhibited NO production, eNOS promoter activity, and eNOS protein levels. Together, our data indicate involvement of PKCδ-mediated Akt activation and NO generation in maintaining eNOS expression. PMID:18192589

  10. Sticky swinging arm dynamics: studies of an acyl carrier protein domain from the mycolactone polyketide synthase

    PubMed Central

    Vance, Steven; Tkachenko, Olga; Thomas, Ben; Bassuni, Mona; Hong, Hui; Nietlispach, Daniel; Broadhurst, William

    2016-01-01

    Type I modular polyketide synthases (PKSs) produce polyketide natural products by passing a growing acyl substrate chain between a series of enzyme domains housed within a gigantic multifunctional polypeptide assembly. Throughout each round of chain extension and modification reactions, the substrate stays covalently linked to an acyl carrier protein (ACP) domain. In the present study we report on the solution structure and dynamics of an ACP domain excised from MLSA2, module 9 of the PKS system that constructs the macrolactone ring of the toxin mycolactone, cause of the tropical disease Buruli ulcer. After modification of apo ACP with 4′-phosphopantetheine (Ppant) to create the holo form, 15N nuclear spin relaxation and paramagnetic relaxation enhancement (PRE) experiments suggest that the prosthetic group swings freely. The minimal chemical shift perturbations displayed by Ppant-attached C3 and C4 acyl chains imply that these substrate-mimics remain exposed to solvent at the end of a flexible Ppant arm. By contrast, hexanoyl and octanoyl chains yield much larger chemical shift perturbations, indicating that they interact with the surface of the domain. The solution structure of octanoyl-ACP shows the Ppant arm bending to allow the acyl chain to nestle into a nonpolar pocket, whereas the prosthetic group itself remains largely solvent exposed. Although the highly reduced octanoyl group is not a natural substrate for the ACP from MLSA2, similar presentation modes would permit partner enzyme domains to recognize an acyl group while it is bound to the surface of its carrier protein, allowing simultaneous interactions with both the substrate and the ACP. PMID:26920023

  11. Phosphorylation of sites 3 and 2 in rabbit skeletal muscle glycogen synthase by a multifunctional protein kinase (ATP-citrate lyase kinase)

    SciTech Connect

    Sheorain, V.S.; Ramakrishna, S.; Benjamin, W.B.; Soderling, T.R.

    1985-10-05

    A multifunctional protein kinase, purified from rat liver as ATP-citrate lyase kinase, has been identified as a glycogen synthase kinase. This kinase catalyzed incorporation of up to 1.5 mol of and)2numberSPO4/mol of synthase subunit associated with a decrease in the glycogen synthase activity ratio from 0.85 to a value of 0.15. Approximately 65-70% of the TUPO4 was incorporated into site 3 and 30-35% into site 2 as determined by reverse phase high performance liquid chromatography. This multifunctional kinase was distinguished from glycogen synthase kinase-3 on the basis of nucleotide and protein substrate specificities. Since the phosphate contents in glycogen synthase of sites 3 and 2 are altered in diabetes and by insulin administration, the possible involvement of the multifunctional kinase was explored. Glycogen synthase purified from diabetic rabbits was phosphorylated in vitro by this multifunctional kinase at only 10% of the rate compared to synthase purified from control rabbits. Treatment of the diabetics with insulin restored the synthase to a form that was readily phosphorylated in vitro.

  12. Biochemical analysis of the substrate specificity of the beta-ketoacyl-acyl carrier protein synthase domain of module 2 of the erythromycin polyketide synthase.

    PubMed

    Wu, Jiaquan; Kinoshita, Kenji; Khosla, Chaitan; Cane, David E

    2004-12-28

    The beta-ketoacyl-acyl carrier protein synthase (KS) domain of the modular 6-deoxyerythronolide B synthase (DEBS) catalyzes the fundamental chain building reaction of polyketide biosynthesis. The KS-catalyzed reaction involves two discrete steps consisting of formation of an acyl-enzyme intermediate generated from the incoming acylthioester substrate and an active site cysteine residue, and the conversion of this intermediate to the beta-ketoacyl-acyl carrier protein product by a decarboxylative condensation with a paired methylmalonyl-SACP. We have determined the rate constants for the individual biochemical steps by a combination of protein acylation and transthioesterification experiments. The first-order rate constant (k(2)) for formation of the acyl-enzyme intermediate from [1-(14)C]-(2S,3R)-2-methyl-3-hydroxypentanoyl-SNAC (2) and recombinant DEBS module 2 is 5.8 +/- 2.6 min(-)(1), with a dissociation constant (K(S)) of 3.5 +/- 2.8 mM. The acyl-enzyme adduct was formed at a near-stoichiometric ratio of approximately 0.8:1. Transthioesterification between unlabeled diketide-SNAC 2 and N-[1-(14)C-acetyl]cysteamine gave a k(exch) of 0.15 +/- 0.06 min(-)(1), with a K(m) for HSNAC of 5.7 +/- 4.9 mM and a K(m) for 2 of 5.3 +/- 0.9 mM. Under the conditions that were used, k(exch) was equal to k(-)(2), the first-order rate constant for reversal of the acyl-enzyme-forming reaction. Since the rate of the decarboxylative condensation is much greater that the rate of reversion to the starting material (k(3) > k(-)(2)), formation of the acyl-enzyme adduct is effectively irreversible, thereby establishing that the observed value of the specificity constant (k(cat)/K(m)) is solely a reflection of the intrinsic substrate specificity of the KS-catalyzed acyl-enzyme-forming reaction. These findings were also extended to a panel of diketide- and triketide-SNAC analogues, revealing that some substrate analogues that are not converted to product by DEBS module 2 form dead

  13. Retinol Binding Protein-Albumin Domain III Fusion Protein Deactivates Hepatic Stellate Cells

    PubMed Central

    Park, Sangeun; Choi, Soyoung; Lee, Min-Goo; Lim, Chaeseung; Oh, Junseo

    2012-01-01

    Liver fibrosis is characterized by accumulation of extracellular matrix, and activated hepatic stellate cells (HSCs) are the primary source of the fibrotic neomatrix and considered as therapeutic target cells. We previously showed that albumin in pancreatic stellate cells (PSCs), the key cell type for pancreatic fibrogenesis, is directly involved in the formation of vitamin A-containing lipid droplets, inhibiting PSC activation. In this study, we evaluated the anti-fibrotic activity of both albumin and retinol binding protein-albumin domain III fusion protein (R-III), designed for stellate cell-targeted delivery of albumin III, in rat primary HSCs and investigated the underlying mechanism. Forced expression of albumin or R-III in HSCs after passage 2 (activated HSCs) induced lipid droplet formation and deactivated HSCs, whereas point mutations in high-affinity fatty acid binding sites of albumin domain III abolished their activities. Exogenous R-III, but not albumin, was successfully internalized into and deactivated HSC-P2. When HSCs at day 3 after plating (pre-activated HSCs) were cultured in the presence of purified R-III, spontaneous activation of HSCs was inhibited even after passage 2, suggestive of a potential for preventive effect. Furthermore, treatment of HSCs-P2 with R-III led to a significant reduction in both cytoplasmic levels of all-trans retinoic acid and the subsequent retinoic acid signaling. Therefore, our data suggest that albumin deactivates HSCs with reduced retinoic acid levels and that R-III may have therapeutic and preventive potentials on liver fibrosis. PMID:23161170

  14. Inhibition of Type III Interferon Activity by Orthopoxvirus Immunomodulatory Proteins

    PubMed Central

    2010-01-01

    The type III interferon (IFN) family elicits an antiviral response that is nearly identical to that evoked by IFN-α/β. However, these cytokines (known as IFN-λ1, 2, and 3) signal through a distinct receptor, and thus may be resistant to the evasion strategies used by some viruses to avoid the IFN-α/β response. Orthopoxviruses are highly resistant to IFN-α/β because they encode well-characterized immunomodulatory proteins that inhibit IFN activity. These include a secreted receptor (B18R) that neutralizes IFN-α/β, and a cytoplasmic protein (E3L) that blocks IFN-α/β effector functions in infected cells. We therefore determined the ability of these immunomodulators to abrogate the IFN-λ–induced antiviral response. We found that (i) vaccinia virus (VACV) replication is resistant to IFN-λ antiviral activity; (ii) neither VACV B18R nor the variola virus homolog B20R neutralizes IFN-λ; (iii) VACV E3L inhibits the IFN-λ–mediated antiviral response through a PKR-dependent pathway; (iv) VACV infection inhibits IFN-λR–mediated signal transduction and gene expression. These results demonstrate differential sensitivity of IFN-λ to multiple distinct evasion mechanisms employed by a single virus. PMID:20038204

  15. Brassica juncea HMG-CoA synthase: localization of mRNA and protein.

    PubMed

    Nagegowda, Dinesh A; Ramalingam, Sathishkumar; Hemmerlin, Andréa; Bach, Thomas J; Chye, Mee-Len

    2005-08-01

    3-Hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA) synthase (HMGS; EC 2.3.3.10) synthesizes HMG-CoA, a substrate for mevalonate biosynthesis in the isoprenoid pathway. It catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA (AcAc-CoA) to yield S-HMG-CoA and HS-CoA. In Brassica juncea (Indian mustard), HMGS is encoded by four isogenes (BjHMGS1-BjHMGS4). We have already enzymatically characterized recombinant BjHMGS1 expressed in Escherichia coli, and have identified its residues that are significant in catalysis. To further study HMGS mRNA expression that is developmentally regulated in flowers and seedlings, we have examined its mRNA distribution by in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR). We observed predominant localization of HMGS mRNA in the stigmas and ovules of flower buds and in the piths of seedling hypocotyls. RT-PCR analysis revealed that BjHMGS1 and BjHMGS2 but not BjHMGS3 and BjHMGS4were expressed in floral buds. To investigate the subcellular localization of BjHMGS1, we fused BjHMGS1 translationally in-frame either to the N- or C-terminus of green fluorescent protein (GFP). BjHMGS1-GFP and GFP-BjHMGS1 fusions were used in particle gun bombardment of onion epidermal cells and tobacco BY-2 cells. The GFP-BjHMGS1 construct was also used in agroinfiltration of tobacco leaves. Both GFP-fusion proteins were observed transiently expressed in the cytosol on confocal microscopy of onion epidermal cells, tobacco BY-2 cells, and agroinfiltrated tobacco leaves. Further, subcellular fractionation of total proteins from transgenic plants expressing GFP-BjHMGS1 derived from Agrobacterium-mediated transformation confirmed that BjHMGS1 is a cytosolic enzyme. We suggest that the presence of BjHMGS isoforms is likely related to the specialization of each in different cellular and metabolic processes rather than to a different intracellular compartmentation of the enzyme. PMID:15770484

  16. Cloning, expression, and characterization of recombinant nitric oxide synthase-like protein from Bacillus anthracis

    SciTech Connect

    Midha, Shuchi; Mishra, Rajeev; Aziz, M.A.; Sharma, Meenakshi; Mishra, Ashish; Khandelwal, Puneet; Bhatnagar, Rakesh . E-mail: rakbhat01@yahoo.com

    2005-10-14

    Nitric oxide synthase (NOS) is amongst a family of evolutionarily conserved enzymes, involved in a multi-turnover process that results in NO as a product. The significant role of NO in various pathological and physiological processes has created an interest in this enzyme from several perspectives. This study describes for the first time, cloning and expression of a NOS-like protein, baNOS, from Bacillus anthracis, a pathogenic bacterium responsible for causing anthrax. baNOS was expressed in Escherichia coli as a soluble and catalytically active enzyme. Homology models generated for baNOS indicated that the key structural features that are involved in the substrate and active site interaction have been highly conserved. Further, the behavior of baNOS in terms of heme-substrate interactions and heme-transitions was studied in detail. The optical perturbation spectra of the heme domain demonstrated that the ligands perturb the heme site in a ligand specific manner. baNOS forms a five-coordinate, high-spin complex with L-arginine analogs and a six-coordinate low-spin complex with inhibitor imidazole. Studies indicated that the binding of L-arginine, N {sup {omega}}-hydroxy-L-arginine, and imidazole produces various spectroscopic species that closely correspond to the equivalent complexes of mammalian NOS. The values of spectral binding constants further corroborated these results. The overall conservation of the key structural features and the correlation of heme-substrate interactions in baNOS and mammalian NOS, thus, point towards an interesting phenomenon of convergent evolution. Importantly, the NO generated by NOS of mammalian macrophages plays a potent role in antimicrobicidal activity. Because of the existence of high structural and behavioral similarity between mammalian NOS and baNOS, we propose that NO produced by B. anthracis may also have a pivotal pathophysiological role in anthrax infection. Therefore, this first report of characterization of a NOS

  17. Mycobacterium tuberculosis acyl carrier protein synthase adopts two different pH-dependent structural conformations

    SciTech Connect

    Gokulan, Kuppan; Aggarwal, Anup; Shipman, Lance; Besra, Gurdyal S.; Sacchettini, James C.

    2011-09-20

    The crystal structures of acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) and Corynebacterium ammoniagenes determined at pH 5.3 and pH 6.5, respectively, are reported. Comparison of the Mtb apo-AcpS structure with the recently reported structure of the Mtb AcpS-ADP complex revealed that AcpS adopts two different conformations: the orthorhombic and trigonal space-group structures show structural differences in the {alpha}2 helix and in the conformation of the {alpha}3-{alpha}4 connecting loop, which is in a closed conformation. The apo-AcpS structure shows electron density for the entire model and was obtained at lower pH values (4.4-6.0). In contrast, at a higher pH value (6.5) AcpS undergoes significant conformational changes, resulting in disordered regions that show no electron density in the AcpS model. The solved structures also reveal that C. ammoniagenes AcpS undergoes structural rearrangement in two regions, similar to the recently reported Mtb AcpS-ADP complex structure. In vitro reconstitution experiments show that AcpS has a higher post-translational modification activity between pH 4.4 and 6.0 than at pH values above 6.5, where the activity drops owing to the change in conformation. The results show that apo-AcpS and AcpS-ADP adopt different conformations depending upon the pH conditions of the crystallization solution.

  18. The first example of a centro-symmetrical bis(imido)-bridged dinuclear cobalt(III) complex: synthesis via oxidative dehydrogenation and phenoxazinone synthase activity.

    PubMed

    Panja, Anangamohan; Guionneau, Philippe

    2013-04-14

    A bis(imido)-bridged dinuclear cobalt(III) complex, [Co2(amp)2(μ-imp)2Cl2]Cl2·2H2O () [amp = 2-aminomethylpyridine; imp = 2-iminomethylpyridine anion], was synthesized by the reaction of cobalt(II) chloride with 2-aminomethylpyridine in the presence of alkaline hydrogen peroxide at room temperature. X-ray crystallography reveals that both the metal centres in the molecule are related to each other through an inversion centre, and the geometry of each of the Co(III) ions is a distorted octahedral structure having a CoN5Cl coordination environment. The most important feature of the structure is the modification of half of the coordinated amines by the oxidative dehydrogenation process which involves double bridging in the complex cation. To the best of our knowledge, this is the first example of a bis(imido)-bridged dinuclear cobalt(III) complex derived from metal-assisted oxidative dehydrogenation of the coordinated primary amine ligand. Complex was found to be an excellent functional model for the phenoxazinone synthase, catalyzing the oxidative coupling of 2-aminophenol to the corresponding 2-aminophenoxazinone chromophore in dioxygen saturated methanol. The detailed kinetic investigations reveal that the phenoxazinone chromophore is produced via a potential complex-substrate intermediate. PMID:23396321

  19. Cobalamin in inflammation III — glutathionylcobalamin and methylcobalamin/adenosylcobalamin coenzymes: the sword in the stone? How cobalamin may directly regulate the nitric oxide synthases

    PubMed Central

    Wheatley, Carmen

    2007-01-01

    Several mysteries surround the structure and function of the nitric oxide synthases (NOS). The NOS oxygenase domain structure is unusually open with a large area of solvent that could accommodate an unidentified ligand. The exact mechanism of the two-step five-electron monoxygenation of arginine to NG-hydroxy-L-arginine, thence to citrulline and nitric oxide (NO), is not clear, particularly as arginine/NG-hydroxy-L-arginine is bound at a great distance to the supposed catalytic heme Fe [III], as the anti-stereoisomer. The Return of the Scarlet Pimpernel Paper proposed that cobalamin is a primary indirect regulator of the NOS. An additional direct regulatory effect of the ‘base-off’ dimethylbenzimidazole of glutathionylcobalamin (GSCbl), which may act as a sixth ligand to the heme iron, promote Co-oriented, BH4/BH3 radical catalysed oxidation of L-arginine to NO, and possibly regulate the rate of inducible NOS/NO production by the NOS dimers, is further advanced. The absence of homology between the NOS and methionine synthase/methylmalonyl CoA mutase may enable GSCbl to regulate both sets of enzymes simultaneously by completely separate mechanisms. Thus, cobalamin may exert central control over both pro-and anti-inflammatory systems. PMID:18923642

  20. Cobalamin in inflammation III - glutathionylcobalamin and methylcobalamin/adenosylcobalamin coenzymes: the sword in the stone? How cobalamin may directly regulate the nitric oxide synthases.

    PubMed

    Wheatley, Carmen

    2007-09-01

    Several mysteries surround the structure and function of the nitric oxide synthases (NOS). The NOS oxygenase domain structure is unusually open with a large area of solvent that could accommodate an unidentified ligand. The exact mechanism of the two-step five-electron monoxygenation of arginine to N(G)-hydroxy-L-arginine, thence to citrulline and nitric oxide (NO), is not clear, particularly as arginine/N(G)-hydroxy-L-arginine is bound at a great distance to the supposed catalytic heme Fe [III], as the anti-stereoisomer. The Return of the Scarlet Pimpernel Paper proposed that cobalamin is a primary indirect regulator of the NOS. An additional direct regulatory effect of the 'base-off' dimethylbenzimidazole of glutathionylcobalamin (GSCbl), which may act as a sixth ligand to the heme iron, promote Co-oriented, BH(4)/BH(3) radical catalysed oxidation of L-arginine to NO, and possibly regulate the rate of inducible NOS/NO production by the NOS dimers, is further advanced. The absence of homology between the NOS and methionine synthase/methylmalonyl CoA mutase may enable GSCbl to regulate both sets of enzymes simultaneously by completely separate mechanisms. Thus, cobalamin may exert central control over both pro-and anti-inflammatory systems. PMID:18923642

  1. Identification of proteins associated with RNA polymerase III using a modified tandem chromatin affinity purification.

    PubMed

    Nguyen, Ngoc-Thuy-Trinh; Saguez, Cyril; Conesa, Christine; Lefebvre, Olivier; Acker, Joël

    2015-02-01

    To identify the proteins associated with the RNA polymerase III (Pol III) machinery in exponentially growing yeast cells, we developed our own tandem chromatin affinity purification procedure (TChAP) after in vivo cross-link, allowing a reproducible and good recovery of the protein bait and its associated partners. In contrast to TFIIIA that could only be purified as a free protein, this protocol allows us to capture free Pol III together with Pol III bound on its target genes. Transcription factors, elongation factors, RNA-associated proteins and proteins involved in Pol III biogenesis were identified by mass spectrometry. Interestingly, the presence of all the TFIIIB subunits found associated with Pol III together with the absence of TFIIIC and chromatin factors including histones suggest that DNA-bound Pol III purified using TChAP is mainly engaged in transcription reinitiation. PMID:25086199

  2. Protein export through the bacterial flagellar type III export pathway.

    PubMed

    Minamino, Tohru

    2014-08-01

    For construction of the bacterial flagellum, which is responsible for bacterial motility, the flagellar type III export apparatus utilizes both ATP and proton motive force across the cytoplasmic membrane and exports flagellar proteins from the cytoplasm to the distal end of the nascent structure. The export apparatus consists of a membrane-embedded export gate made of FlhA, FlhB, FliO, FliP, FliQ, and FliR and a water-soluble ATPase ring complex consisting of FliH, FliI, and FliJ. FlgN, FliS, and FliT act as substrate-specific chaperones that do not only protect their cognate substrates from degradation and aggregation in the cytoplasm but also efficiently transfer the substrates to the export apparatus. The ATPase ring complex facilitates the initial entry of the substrates into the narrow pore of the export gate. The export gate by itself is a proton-protein antiporter that uses the two components of proton motive force, the electric potential difference and the proton concentration difference, for different steps of the export process. A specific interaction of FlhA with FliJ located in the center of the ATPase ring complex allows the export gate to efficiently use proton motive force to drive protein export. The ATPase ring complex couples ATP binding and hydrolysis to its assembly-disassembly cycle for rapid and efficient protein export cycle. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey. PMID:24064315

  3. CESA TRAFFICKING INHIBITOR inhibits cellulose deposition and interferes with the trafficking of cellulose synthase complexes and their associated proteins KORRIGAN1 and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1.

    PubMed

    Worden, Natasha; Wilkop, Thomas E; Esteve, Victor Esteva; Jeannotte, Richard; Lathe, Rahul; Vernhettes, Samantha; Weimer, Bart; Hicks, Glenn; Alonso, Jose; Labavitch, John; Persson, Staffan; Ehrhardt, David; Drakakaki, Georgia

    2015-02-01

    Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls. PMID:25535279

  4. CESA TRAFFICKING INHIBITOR Inhibits Cellulose Deposition and Interferes with the Trafficking of Cellulose Synthase Complexes and Their Associated Proteins KORRIGAN1 and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN11[OPEN

    PubMed Central

    Wilkop, Thomas E.; Esteve, Victor Esteva; Jeannotte, Richard; Lathe, Rahul; Vernhettes, Samantha; Weimer, Bart; Hicks, Glenn; Alonso, Jose; Labavitch, John; Persson, Staffan; Ehrhardt, David; Drakakaki, Georgia

    2015-01-01

    Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls. PMID:25535279

  5. PROTEIN III, A NEURON-SPECIFIC PHOSPHOPROTEIN: VARIANT FORMS FOUND IN HUMAN BRAIN

    EPA Science Inventory

    Recent work in the laboratory has shown the presence of many neuron-specific phosphoproteins in the mammalian nervous system. Two of these proteins, Protein III and Synapsin I, are specifically associated with synaptic vesicles in neurons throughout the brain. Protein III consist...

  6. ATP synthase.

    PubMed

    Junge, Wolfgang; Nelson, Nathan

    2015-01-01

    Oxygenic photosynthesis is the principal converter of sunlight into chemical energy. Cyanobacteria and plants provide aerobic life with oxygen, food, fuel, fibers, and platform chemicals. Four multisubunit membrane proteins are involved: photosystem I (PSI), photosystem II (PSII), cytochrome b6f (cyt b6f), and ATP synthase (FOF1). ATP synthase is likewise a key enzyme of cell respiration. Over three billion years, the basic machinery of oxygenic photosynthesis and respiration has been perfected to minimize wasteful reactions. The proton-driven ATP synthase is embedded in a proton tight-coupling membrane. It is composed of two rotary motors/generators, FO and F1, which do not slip against each other. The proton-driven FO and the ATP-synthesizing F1 are coupled via elastic torque transmission. Elastic transmission decouples the two motors in kinetic detail but keeps them perfectly coupled in thermodynamic equilibrium and (time-averaged) under steady turnover. Elastic transmission enables operation with different gear ratios in different organisms. PMID:25839341

  7. Starch synthase 4 is located in the thylakoid membrane and interacts with plastoglobule-associated proteins in Arabidopsis.

    PubMed

    Gámez-Arjona, Francisco M; Raynaud, Sandy; Ragel, Paula; Mérida, Angel

    2014-10-01

    Starch synthesis requires the formation of a primer that can be subsequently elongated and branched. How this primer is produced, however, remains unknown. The control of the number of starch granules produced per chloroplast is also a matter of debate. We previously showed starch synthase 4 (SS4) to be involved in both processes, although the mechanisms involved are yet to be fully characterised. The present work shows that SS4 displays a specific localization different from other starch synthases. Thus, this protein is located in specific areas of the thylakoid membrane and interacts with the proteins fibrillin 1a (FBN1a) and 1b (FBN1b), which are mainly located in plastoglobules. SS4 would seem to be associated with plastoglobules attached to the thylakoids (or to that portion of the thylakoids where plastoglobules have originated), forming a complex that includes the FBN1s and other as-yet unidentified proteins. The present results also indicate that the localization pattern of SS4, and its interactions with the FBN1 proteins, are mediated through its N-terminal region, which contains two long coiled-coil motifs. The localization of SS4 in specific areas of the thylakoid membrane suggests that starch granules are originated at specific regions of the chloroplast. PMID:25088399

  8. Substrate Ambiguity of 3-Deoxy-d-manno-Octulosonate 8-Phosphate Synthase from Neisseria gonorrhoeae in the Context of Its Membership in a Protein Family Containing a Subset of 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthases†

    PubMed Central

    Subramaniam, Prem S.; Xie, Gang; Xia, Tianhui; Jensen, Roy A.

    1998-01-01

    3-Deoxy-d-manno-octulosonate 8-phosphate (KDOP) synthase and 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase catalyze similar phosphoenolpyruvate-utilizing reactions. The genome of Neisseria gonorrhoeae contains one gene encoding KDOP synthase and one gene encoding DAHP synthase. Of the two nonhomologous DAHP synthase families known, the N. gonorrhoeae protein belongs to the family I assemblage. KDOP synthase exhibited an ability to replace arabinose-5-P with either erythrose-4-P or ribose-5-P as alternative substrates. The results of periodate oxidation studies suggested that the product formed by KDOP synthase with erythrose-4-P as the substrate was 3-deoxy-d-ribo-heptulosonate 7-P, an isomer of DAHP. As expected, this product was not utilized as a substrate by dehydroquinate synthase. The significance of the ability of KDOP synthase to substitute erythrose-4-P for arabinose-5-P is (i) recognition of the possibility that the KDOP synthase might otherwise be mistaken for a species of DAHP synthase and (ii) the possibility that the broad-specificity type of KDOP synthase might be a relatively vulnerable target for antimicrobial agents which mimic the normal substrates. An analysis of sequences in the database indicates that the family I group of DAHP synthase has a previously unrecognized membership which includes the KDOP synthases. The KDOP synthases fall into a subfamily grouping which includes a small group of DAHP synthases. Thus, family I DAHP synthases separate into two subfamilies, one of which includes the KDOP synthases. The two subfamilies appear to have diverged prior to the acquisition of allosteric-control mechanisms for DAHP synthases. These allosteric control specificities are highly diverse and correlate with the presence of N-terminal extensions which lack homology with one another. PMID:9422601

  9. Structure of Yellow Fever Virus Envelope Protein Domain III

    PubMed Central

    Volk, David E.; May, Fiona J.; Gandham, Sai H. A.; Anderson, Anjenique; Von Lindern, Jana J.; Beasley, David W. C.; Barrett, Alan D. T.; Gorenstein, David G.

    2009-01-01

    The structure of recombinant domain III of the envelope protein (rED3) of yellow fever virus (YFV), containing the major neutralization site, was determined using NMR spectroscopy. The amino acid sequence and structure of the YFV-rED3 shows differences from ED3s of other mosquito-borne flaviviruses; in particular, the partially surface-exposed BC loop where methionine-304 and valine-324 were identified as being critical for the structure of the loop. Variations in the structure and surface chemistry of ED3 between flaviviruses affect neutralization sites and may affect host cell receptor interactions and play a role in the observed variations in viral pathogenesis and tissue tropism. PMID:19818466

  10. A Chitin Synthase and Its Regulator Protein Are Critical for Chitosan Production and Growth of the Fungal Pathogen Cryptococcus neoformans†

    PubMed Central

    Banks, Isaac R.; Specht, Charles A.; Donlin, Maureen J.; Gerik, Kimberly J.; Levitz, Stuart M.; Lodge, Jennifer K.

    2005-01-01

    Chitin is an essential component of the cell wall of many fungi. Chitin also can be enzymatically deacetylated to chitosan, a more flexible and soluble polymer. Cryptococcus neoformans is a fungal pathogen that causes cryptococcal meningoencephalitis, particularly in immunocompromised patients. In this work, we show that both chitin and chitosan are present in the cell wall of vegetatively growing C. neoformans yeast cells and that the levels of both rise dramatically as cells grow to higher density in liquid culture. C. neoformans has eight putative chitin synthases, and strains with any one chitin synthase deleted are viable at 30°C. In addition, C. neoformans genes encode three putative regulator proteins, which are homologs of Saccharomyces cerevisiae Skt5p. None of these three is essential for viability. However, one of the chitin synthases (Chs3) and one of the regulators (Csr2) are important for growth. Cells with deletions in either CHS3 or CSR2 have several shared phenotypes, including sensitivity to growth at 37°C. The similarity of their phenotypes also suggests that Csr2 specifically regulates chitin synthesis by Chs3. Lastly, both chs3Δ and the csr2Δ mutants are defective in chitosan production, predicting that Chs3-Csr2 complex with chitin deacetylases for conversion of chitin to chitosan. These data suggest that chitin synthesis could be an excellent antifungal target. PMID:16278457

  11. Protein extraction by Winsor-III microemulsion systems.

    PubMed

    Gomez del Rio, Javier A; Hayes, Douglas G

    2011-07-01

    Proteins (bovine serum albumin (BSA), α-chymotrypsin, cytochrome c, and lysozyme) were extracted from 0.5 to 2.0 g L(-1) aqueous solution by adding an equal volume of isooctane solution that contained a surfactant mixture (Aerosol-OT, or AOT, and a 1,3-dioxolane (or cyclic ketal) alkyl ethoxylate, CK-2,13-E5.6), producing a three-phase (Winsor-III) microemulsion with a middle, bicontinuous microemulsion, phase highly concentrated in protein (5-13 g L(-1)) and small in volume (12-20% of entire volume). Greater than 90% forward extraction was achieved within a few minutes. Robust W-III microemulsion systems were formulated at 40°C, or at 25°C by including a surfactant with shorter ethoxylate length, CK-2,13-E3 , or 1.5% NaCl (aq). Successful forward extraction correlated with high partitioning of AOT in the middle phase (>95%). The driving force for forward extraction was mainly electrostatic attractions imposed by the anionic surfactant AOT, with the exception of BSA at high ionic strength, which interacted via hydrophobic interactions. Through use of aqueous stripping solutions of high ionic strength (5.0 wt %) and/or pH 12.0 (to negate the electrostatic attractive driving force), cytochrome c and α-chymotrypsin were back extracted from the middle phase at >75% by mass, with the specific activity of recovered α-chymotrypsin being >90% of its original value. PMID:21695808

  12. Phosphorylation of inhibitor-2 and activation of MgATP-dependent protein phosphatase by rat skeletal muscle glycogen synthase kinase

    SciTech Connect

    Hegazy, M.G.; Reimann, E.M.; Thysseril, T.J.; Schlender, K.K.

    1986-05-01

    Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca/sup 2 +/ or cAMP. This kinase has an apparent Mr of 62,000 and uses ATP but not GTP as a phosphoryl donor. GSK-M phosphorylated glycogen synthase at sites 2 and 3. It phosphorylated ATP-citrate lyase and activated MgATP-dependent phosphatase in the presence of ATP but not GTP. As expected, the kinase also phosphorylated phosphatase inhibitor 2 (I-2). Phosphatase incorporation reached approximately 0.3 mol/mol of I-2. Phosphopeptide maps were obtained by digesting /sup 32/P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated /sup 32/P-labeled peaks were obtained when I-2 was phosphorylated with either GSK-M or glycogen synthase kinase 3 (GSK-3) and these peptides were different from those obtained when I-2 was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase (CSU) or casein kinase II (CK-II). When I-2 was phosphorylated with GSK-M or GSK-3 and cleaved by CNBr, a single radioactive peak was obtained. Phosphoamino acid analysis showed that I-2 was phosphorylated by GSK-M or GSK-3 predominately in Thr whereas CSU and CK-II phosphorylated I-2 exclusively in Ser. These results indicate that GSK-M is similar to GSK-3 and to ATP-citrate lyase kinase. However, it appears to differ in Mr from ATP-citrate lyase kinase and it differs from GSK-3 in that it phosphorylates glycogen synthase at site 2 and it does not use GTP as a phosphoryl donor.

  13. Acyl-carrier protein - Phosphopantetheinyltransferase partnerships in fungal fatty acid synthases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The synthesis of fatty acids is an essential primary metabolic process for energy storage and cellular structural integrity. Assembly of saturated fatty acids is achieved by fatty acid synthases (FASs) that combine acetyl- and malonyl-CoAs by repetitive decarboxylative Claisen condensations with su...

  14. BIOGENESIS FACTOR REQUIRED FOR ATP SYNTHASE 3 Facilitates Assembly of the Chloroplast ATP Synthase Complex.

    PubMed

    Zhang, Lin; Duan, Zhikun; Zhang, Jiao; Peng, Lianwei

    2016-06-01

    Thylakoid membrane-localized chloroplast ATP synthases use the proton motive force generated by photosynthetic electron transport to produce ATP from ADP. Although it is well known that the chloroplast ATP synthase is composed of more than 20 proteins with α3β3γ1ε1δ1I1II1III14IV1 stoichiometry, its biogenesis process is currently unclear. To unravel the molecular mechanisms underlying the biogenesis of chloroplast ATP synthase, we performed extensive screening for isolating ATP synthase mutants in Arabidopsis (Arabidopsis thaliana). In the recently identified bfa3 (biogenesis factors required for ATP synthase 3) mutant, the levels of chloroplast ATP synthase subunits were reduced to approximately 25% of wild-type levels. In vivo labeling analysis showed that assembly of the CF1 component of chloroplast ATP synthase was less efficient in bfa3 than in the wild type, indicating that BFA3 is required for CF1 assembly. BFA3 encodes a chloroplast stromal protein that is conserved in higher plants, green algae, and a few species of other eukaryotic algae, and specifically interacts with the CF1β subunit. The BFA3 binding site was mapped to a region in the catalytic site of CF1β. Several residues highly conserved in eukaryotic CF1β are crucial for the BFA3-CF1β interaction, suggesting a coevolutionary relationship between BFA3 and CF1β. BFA3 appears to function as a molecular chaperone that transiently associates with unassembled CF1β at its catalytic site and facilitates subsequent association with CF1α during assembly of the CF1 subcomplex of chloroplast ATP synthase. PMID:27208269

  15. Crystal Structure of the Human Fatty Acid Synthase Enoyl-Acyl Carrier Protein-Reductase Domain Complexed with Triclosan Reveals Allosteric Protein-Protein Interface Inhibition*

    PubMed Central

    Sippel, Katherine H.; Vyas, Nand K.; Zhang, Wei; Sankaran, Banumathi; Quiocho, Florante A.

    2014-01-01

    Human fatty acid synthase (FAS) is a large, multidomain protein that synthesizes long chain fatty acids. Because these fatty acids are primarily provided by diet, FAS is normally expressed at low levels; however, it is highly up-regulated in many cancers. Human enoyl-acyl carrier protein-reductase (hER) is one of the FAS catalytic domains, and its inhibition by drugs like triclosan (TCL) can increase cytotoxicity and decrease drug resistance in cancer cells. We have determined the structure of hER in the presence and absence of TCL. TCL was not bound in the active site, as predicted, but rather at the protein-protein interface (PPI). TCL binding induces a dimer orientation change that causes downstream structural rearrangement in critical active site residues. Kinetics studies indicate that TCL is capable of inhibiting the isolated hER domain with an IC50 of ∼55 μm. Given the hER-TCL structure and the inhibition observed in the hER domain, it seems likely that TCL is observed in the physiologically relevant binding site and that it acts as an allosteric PPI inhibitor. TCL may be a viable scaffold for the development of anti-cancer PPI FAS inhibitors. PMID:25301948

  16. Structural basis for the formation of acylalkylpyrones from two β-ketoacyl units by the fungal type III polyketide synthase CsyB.

    PubMed

    Mori, Takahiro; Yang, Dengfeng; Matsui, Takashi; Hashimoto, Makoto; Morita, Hiroyuki; Fujii, Isao; Abe, Ikuro

    2015-02-20

    The acylalkylpyrone synthase CsyB from Aspergillus oryzae catalyzes the one-pot formation of the 3-acyl-4-hydroxy-6-alkyl-α-pyrone scaffold from acetoacetyl-CoA, fatty acyl-CoA, and malonyl-CoA. This is the first type III polyketide synthase that performs not only the polyketide chain elongation but also the condensation of two β-ketoacyl units. The crystal structures of wild-type CsyB and its I375F and I375W mutants were solved at 1.7-, 2.3-, and 2.0-Å resolutions, respectively. The crystal structures revealed a unique active site architecture featuring a hitherto unidentified novel pocket for accommodation of the acetoacetyl-CoA starter in addition to the conventional elongation/cyclization pocket with the Cys-His-Asn catalytic triad and the long hydrophobic tunnel for binding the fatty acyl chain. The structures also indicated the presence of a putative nucleophilic water molecule activated by the hydrogen bond networks with His-377 and Cys-155 at the active site center. Furthermore, an in vitro enzyme reaction confirmed that the (18)O atom of the H2(18)O molecule is enzymatically incorporated into the final product. These observations suggested that the enzyme reaction is initiated by the loading of acetoacetyl-CoA onto Cys-155, and subsequent thioester bond cleavage by the nucleophilic water generates the β-keto acid intermediate, which is placed within the novel pocket. The second β-ketoacyl unit is then produced by polyketide chain elongation of fatty acyl-CoA with one molecule of malonyl-CoA, and the condensation with the β-keto acid generates the final products. Indeed, steric modulation of the novel pocket by the structure-based I375F and I375W mutations resulted in altered specificities for the chain lengths of the substrates. PMID:25564614

  17. Cloning and functional analysis of the promoter of a maize starch synthase III gene (ZmDULL1).

    PubMed

    Wu, J D; Jiang, C P; Zhu, H S; Jiang, H Y; Cheng, B J; Zhu, S W

    2015-01-01

    The ZmDULL1 gene encodes a starch synthase and is a determinant of the structure of endosperm starch in maize (Zea mays L.). However, little is known regarding the regulatory mechanism of the ZmDULL1 gene. In this study, we isolated and characterized the ZmDULL1 promoter (PDULL1), which is the 5' flanking region of ZmDULL1 in maize. Sequence analysis showed that several cis-acting elements important for endosperm expression (GCN4_motif and AACA-element) were located within the promoter. A series of PDULL1 deletion derivatives, PDULL1-1-PDULL1-4, from the translation start code (-1676, -1216, -740, and -343) were fused to the β-glucuronidase (GUS) reporter gene. Each deletion construct was transformed into rice using the Agrobacterium-mediated method, and then GUS activity was measured in transgenic plants. The results showed that PDULL1 was an endosperm-specific promoter. Further analysis showed that the promoter sequence (-343 to -1 base pairs) was sufficient for mediating GUS gene expression in endosperm. These results indicate that the region from -343 to -1 base pairs of PDULL1 is valuable for transgenic rice breeding and genetic engineering studies. PMID:26125743

  18. Arabidopsis MYC2 Interacts with DELLA Proteins in Regulating Sesquiterpene Synthase Gene Expression[W][OA

    PubMed Central

    Hong, Gao-Jie; Xue, Xue-Yi; Mao, Ying-Bo; Wang, Ling-Jian; Chen, Xiao-Ya

    2012-01-01

    Arabidopsis thaliana flowers emit volatile terpenes, which may function in plant–insect interactions. Here, we report that Arabidopsis MYC2, a basic helix-loop-helix transcription factor, directly binds to promoters of the sesquiterpene synthase genes TPS21 and TPS11 and activates their expression. Expression of TPS21 and TPS11 can be induced by the phytohormones gibberellin (GA) and jasmonate (JA), and both inductions require MYC2. The induction of TPS21 and TPS11 results in increased emission of sesquiterpene, especially (E)-β-caryophyllene. DELLAs, the GA signaling repressors, negatively affect sesquiterpene biosynthesis, as the sesquiterpene synthase genes were repressed in plants overaccumulating REPRESSOR OF GA1-3 (RGA), one of the Arabidopsis DELLAs, and upregulated in a penta DELLA-deficient mutant. Yeast two-hybrid and coimmunoprecipitation assays demonstrated that DELLAs, represented by RGA, directly interact with MYC2. In yeast cells, the N terminus of MYC2 was responsible for binding to RGA. MYC2 has been proposed as a major mediator of JA signaling and crosstalk with abscisic acid, ethylene, and light signaling pathways. Our results demonstrate that MYC2 is also connected to GA signaling in regulating a subset of genes. In Arabidopsis inflorescences, it integrates both GA and JA signals into transcriptional regulation of sesquiterpene synthase genes and promotes sesquiterpene production. PMID:22669881

  19. Homology analyses of the protein sequences of fatty acid synthases from chicken liver, rat mammary gland, and yeast

    SciTech Connect

    Chang, Soo-Ik ); Hammes, G.G. )

    1989-11-01

    Homology analyses of the protein sequences of chicken liver and rat mammary gland fatty acid synthases were carried out. The amino acid sequences of the chicken and rat enzymes are 67% identical. If conservative substitutions are allowed, 78% of the amino acids are matched. A region of low homologies exists between the functional domains, in particular around amino acid residues 1059-1264 of the chicken enzyme. Homologies between the active sites of chicken and rat and of chicken and yeast enzymes have been analyzed by an alignment method. A high degree of homology exists between the active sites of the chicken and rat enzymes. However, the chicken and yeast enzymes show a lower degree of homology. The DADPH-binding dinucleotide folds of the {beta}-ketoacyl reductase and the enoyl reductase sites were identified by comparison with a known consensus sequence for the DADP- and FAD-binding dinucleotide folds. The active sites of all of the enzymes are primarily in hydrophobic regions of the protein. This study suggests that the genes for the functional domains of fatty acid synthase were originally separated, and these genes were connected to each other by using different connecting nucleotide sequences in different species. An alternative explanation for the differences in rat and chicken is a common ancestry and mutations in the joining regions during evolution.

  20. ROLE OF TYPE III PROTEIN SECRETION SYSTEM IN SINORHIZOBIUM FREDII USDA257 AND SOYBEAN INTERACTIONS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant and animal pathogenic bacteria have evolved a specialized protein secretion system called type III to directly inject proteins into their host cells. The Type III secretion system (TTSS) plays an important role in plant-microbe interactions since mutation in TTSS causes a loss of bacterial pa...

  1. Stilbene Synthase and Chalcone Synthase 1

    PubMed Central

    Rolfs, Claus-Henning; Kindl, Helmut

    1984-01-01

    Cultured cells of Picea excelsa capable of forming stilbenes and flavanoids have been established. Unlike needles of intact plants containing piceatannol (3,3′,4′,5-tetrahydroxystilbene) and stilbene glycosides the cultured cells converted phenylalanine and p-coumaric acid primarily into resveratrol monomethyl ether (3,4′-dihydroxy-5-methoxystilbene) and naringenin. Partially purified enzyme preparations were assayed for chalcone synthase as well as for stilbene synthase activity converting malonyl-CoA plus p-coumaroyl-CoA into 3,4′,5-trihydroxystilbene (resveratrol). Although stilbene synthase and chalcone synthase use the same substrates and exhibit similar molecular properties, i.e. molecular weight and subunit molecular weight, they are two different proteins. This difference was demonstrated by gel electrophoresis and by means of monospecific antibodies. PMID:16663649

  2. Site-specific regulatory interaction between spinach leaf sucrose-phosphate synthase and 14-3-3 proteins

    NASA Technical Reports Server (NTRS)

    Toroser, D.; Athwal, G. S.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    We report an Mg2+-dependent interaction between spinach leaf sucrose-phosphate synthase (SPS) and endogenous 14-3-3 proteins, as evidenced by co-elution during gel filtration and co-immunoprecipitation. The content of 14-3-3s associated with an SPS immunoprecipitate was inversely related to activity, and was specifically reduced when tissue was pretreated with 5-aminoimidazole-4-carboxamide riboside, suggesting metabolite control in vivo. A synthetic phosphopeptide based on Ser-229 was shown by surface plasmon resonance to bind a recombinant plant 14-3-3, and addition of the phosphorylated SPS-229 peptide was found to stimulate the SPS activity of an SPS:14-3-3 complex. Taken together, the results suggest a regulatory interaction of 14-3-3 proteins with Ser-229 of SPS.

  3. Polypeptide composition of bacterial cyclic diguanylic acid-dependent cellulose synthase and the occurrence of immunologically crossreacting proteins in higher plants

    SciTech Connect

    Mayer, R.; Ross, P.; Weinhouse, H.; Amikam, D.; Volman, G.; Ohana, P.; Benziman, M. ); Calhoon, R.D.; Wong, Hing C.; Emerick, A.W. )

    1991-06-15

    To comprehend the catalytic and regulatory mechanism of the cyclic diguanylic acid (c-di-GMP)-dependent cellulose synthase of Acetobacter xylinum and its relatedness to similar enzymes in other organisms, the structure of this enzyme was analyzed at the polypeptide level. The enzyme, purified 350-fold by enzyme-product entrapment, contains three major peptides (90, 67, and 54 kDa), which, based on direct photoaffinity and immunochemical labeling and amino acid sequence analysis, are constituents of the native cellulose synthase. Labeling of purified synthase with either ({sup 32}P)c-di-GMP or ({alpha}-{sup 32}P)UDP-glucose indicates that activator- and substrate-specific binding sites are most closely associated with the 67- and 54-kDa peptides, respectively, whereas marginal photolabeling is detected in the 90-k-Da peptide. However, antibodies raised against a protein derived from the cellulose synthase structural gene (bcsB) specifically label all three peptides. The authors suggest that the structurally related 67- and 54-kDa peptides are fragments proteolytically derived from the 90-kDa peptide encoded by bcsB. The anti-cellulose synthase antibodies crossreact with a similar set of peptides derived from other cellulose-producing microorganisms and plants such as Agrobacterium tumefaciens, Rhizobium leguminosarum, mung bean, peas, barley, and cotton. The occurrence of such cellulose synthase-like structures in plant species suggests that a common enzymatic mechanism for cellulose biogenesis is employed throughout nature.

  4. From Amino Acid to Glucosinolate Biosynthesis: Protein Sequence Changes in the Evolution of Methylthioalkylmalate Synthase in Arabidopsis[W][OA

    PubMed Central

    de Kraker, Jan-Willem; Gershenzon, Jonathan

    2011-01-01

    Methylthioalkylmalate synthase (MAM) catalyzes the committed step in the side chain elongation of Met, yielding important precursors for glucosinolate biosynthesis in Arabidopsis thaliana and other Brassicaceae species. MAM is believed to have evolved from isopropylmalate synthase (IPMS), an enzyme involved in Leu biosynthesis, based on phylogenetic analyses and an overlap of catalytic abilities. Here, we investigated the changes in protein structure that have occurred during the recruitment of IPMS from amino acid to glucosinolate metabolism. The major sequence difference between IPMS and MAM is the absence of 120 amino acids at the C-terminal end of MAM that constitute a regulatory domain for Leu-mediated feedback inhibition. Truncation of this domain in Arabidopsis IPMS2 results in loss of Leu feedback inhibition and quaternary structure, two features common to MAM enzymes, plus an 8.4-fold increase in the kcat/Km for a MAM substrate. Additional exchange of two amino acids in the active site resulted in a MAM-like enzyme that had little residual IPMS activity. Hence, combination of the loss of the regulatory domain and a few additional amino acid exchanges can explain the evolution of MAM from IPMS during its recruitment from primary to secondary metabolism. PMID:21205930

  5. A novel lumazine synthase molecule from Brucella significantly promotes the immune-stimulation effects of antigenic protein.

    PubMed

    Du, Z Q; Wang, J Y

    2015-01-01

    Brucella, an intracellular parasite that infects some livestock and humans, can damage or destroy the reproductive system of livestock. The syndrome is referred to as brucellosis and often occurs in pastoral areas; it is contagious from livestock to humans. In this study, the intact Brucella suis outer membrane protein 31 (omp31) gene was cloned, recombinantly expressed, and examined as a subunit vaccine candidate. The intact Brucella lumazine synthase (bls) gene was cloned and recombinantly expressed to study polymerization function in vitro. Non-reducing gel electrophoresis showed that rBs-BLS existed in different forms in vitro, including as a dimer and a pentamer. An enzyme-linked immunosorbent assay result showed that rOmp31 protein could induce production of an antibody in rabbits. However, the rOmp31-BLS fusion protein could elicit a much higher antibody titer in rabbits; this construct involved fusion of the Omp31 molecule with the BLS molecule. Our results indicate that Omp31 is involved in immune stimulation, while BLS has a polymerizing function based on rOmp31-BLS fusion protein immunogenicity. These data suggest that Omp31 is an ideal subunit vaccine candidate and that the BLS molecule is a favorable transport vector for antigenic proteins. PMID:26535621

  6. Structural and kinetic analysis of the unnatural fusion protein 4-coumaroyl-CoA ligase::stilbene synthase

    SciTech Connect

    Wang, Yechun; Yi, Hankuil; Wang, Melissa; Yu, Oliver; Jez, Joseph M.

    2012-10-24

    To increase the biochemical efficiency of biosynthetic systems, metabolic engineers have explored different approaches for organizing enzymes, including the generation of unnatural fusion proteins. Previous work aimed at improving the biosynthesis of resveratrol, a stilbene associated a range of health-promoting activities, in yeast used an unnatural engineered fusion protein of Arabidopsis thaliana (thale cress) 4-coumaroyl-CoA ligase (At4CL1) and Vitis vinifera (grape) stilbene synthase (VvSTS) to increase resveratrol levels 15-fold relative to yeast expressing the individual enzymes. Here we present the crystallographic and biochemical analysis of the 4CL::STS fusion protein. Determination of the X-ray crystal structure of 4CL::STS provides the first molecular view of an artificial didomain adenylation/ketosynthase fusion protein. Comparison of the steady-state kinetic properties of At4CL1, VvSTS, and 4CL::STS demonstrates that the fusion protein improves catalytic efficiency of either reaction less than 3-fold. Structural and kinetic analysis suggests that colocalization of the two enzyme active sites within 70 {angstrom} of each other provides the basis for enhanced in vivo synthesis of resveratrol.

  7. Proteomic profiling of cellulase-aid-extracted membrane proteins for functional identification of cellulose synthase complexes and their potential associated- components in cotton fibers

    PubMed Central

    Li, Ao; Wang, Ruyi; Li, Xianliang; Liu, Mingyong; Fan, Jian; Guo, Kai; Luo, Bing; Chen, Tingting; Feng, Shengqiu; Wang, Yanting; Wang, Bingrui; Peng, Liangcai; Xia, Tao

    2016-01-01

    Cotton fibers are an excellent model for understanding of cellulose biosynthesis in higher plants. In this study, we determined a high cellulose biosynthesis activity in vitro by optimizing biochemical reaction conditions in cotton fibers. By adding a commercial cellulase enzyme into fibers extraction process, we extracted markedly higher levels of GhCESA1 and GhCESA8 proteins and observed an increase in β-1,4-glucan and β-1,3-glucan products in vitro. LC-MS/MS analysis of anti-GhCESA8-immunoprecipitated proteins showed that 19 proteins could be found in three independent experiments including four CESAs (GhCESA1,2,7,8), five well-known non-CESA proteins, one callose synthase (CALS) and nine novel proteins. Notably, upon the cellulase treatment, four CESAs, one CALS and four novel proteins were measured at relatively higher levels by calculating total peptide counts and distinct peptide numbers, indicating that the cellulase-aid-extracted proteins most likely contribute to the increase in β-glucan products in vitro. These results suggest that the cellulase treatment may aid to release active cellulose synthases complexes from growing glucan chains and make them more amenable to extraction. To our knowledge, it is the first time report about the functional identification of the potential proteins that were associated with plant cellulose and callose synthases complexes by using the cellulase-aided protein extraction. PMID:27192945

  8. Proteomic profiling of cellulase-aid-extracted membrane proteins for functional identification of cellulose synthase complexes and their potential associated- components in cotton fibers.

    PubMed

    Li, Ao; Wang, Ruyi; Li, Xianliang; Liu, Mingyong; Fan, Jian; Guo, Kai; Luo, Bing; Chen, Tingting; Feng, Shengqiu; Wang, Yanting; Wang, Bingrui; Peng, Liangcai; Xia, Tao

    2016-01-01

    Cotton fibers are an excellent model for understanding of cellulose biosynthesis in higher plants. In this study, we determined a high cellulose biosynthesis activity in vitro by optimizing biochemical reaction conditions in cotton fibers. By adding a commercial cellulase enzyme into fibers extraction process, we extracted markedly higher levels of GhCESA1 and GhCESA8 proteins and observed an increase in β-1,4-glucan and β-1,3-glucan products in vitro. LC-MS/MS analysis of anti-GhCESA8-immunoprecipitated proteins showed that 19 proteins could be found in three independent experiments including four CESAs (GhCESA1,2,7,8), five well-known non-CESA proteins, one callose synthase (CALS) and nine novel proteins. Notably, upon the cellulase treatment, four CESAs, one CALS and four novel proteins were measured at relatively higher levels by calculating total peptide counts and distinct peptide numbers, indicating that the cellulase-aid-extracted proteins most likely contribute to the increase in β-glucan products in vitro. These results suggest that the cellulase treatment may aid to release active cellulose synthases complexes from growing glucan chains and make them more amenable to extraction. To our knowledge, it is the first time report about the functional identification of the potential proteins that were associated with plant cellulose and callose synthases complexes by using the cellulase-aided protein extraction. PMID:27192945

  9. Type III Protein Secretion Systems in Bacterial Pathogens of Animals and Plants

    PubMed Central

    Hueck, Christoph J.

    1998-01-01

    Various gram-negative animal and plant pathogens use a novel, sec-independent protein secretion system as a basic virulence mechanism. It is becoming increasingly clear that these so-called type III secretion systems inject (translocate) proteins into the cytosol of eukaryotic cells, where the translocated proteins facilitate bacterial pathogenesis by specifically interfering with host cell signal transduction and other cellular processes. Accordingly, some type III secretion systems are activated by bacterial contact with host cell surfaces. Individual type III secretion systems direct the secretion and translocation of a variety of unrelated proteins, which account for species-specific pathogenesis phenotypes. In contrast to the secreted virulence factors, most of the 15 to 20 membrane-associated proteins which constitute the type III secretion apparatus are conserved among different pathogens. Most of the inner membrane components of the type III secretion apparatus show additional homologies to flagellar biosynthetic proteins, while a conserved outer membrane factor is similar to secretins from type II and other secretion pathways. Structurally conserved chaperones which specifically bind to individual secreted proteins play an important role in type III protein secretion, apparently by preventing premature interactions of the secreted factors with other proteins. The genes encoding type III secretion systems are clustered, and various pieces of evidence suggest that these systems have been acquired by horizontal genetic transfer during evolution. Expression of type III secretion systems is coordinately regulated in response to host environmental stimuli by networks of transcription factors. This review comprises a comparison of the structure, function, regulation, and impact on host cells of the type III secretion systems in the animal pathogens Yersinia spp., Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhimurium, enteropathogenic Escherichia coli

  10. Norcoclaurine Synthase Is a Member of the Pathogenesis-Related 10/Bet v1 Protein Family[W

    PubMed Central

    Lee, Eun-Jeong; Facchini, Peter

    2010-01-01

    Norcoclaurine synthase (NCS) catalyzes the first committed step in the biosynthesis of benzylisoquinoline alkaloids (BIAs). NCS from Thalictrum flavum (Tf NCS), Papaver somniferum (Ps NCS1 and Ps NCS2), and Coptis japonica (Cj PR10A) share substantial identity with pathogen-related 10 (PR10) and Bet v1 proteins, whose functions are not well understood. A distinct enzyme (Cj NCS1) with similarity to 2-oxoglutarate–dependent dioxygenases was suggested as the bona fide NCS in C. japonica. Here, we validate the exclusive role of PR10/Bet v1-type NCS enzymes in BIA metabolism. Immunolocalization of Ps NCS2 revealed its cell type–specific occurrence in phloem sieve elements, which contain all other known BIA biosynthetic enzymes. In opium poppy, NCS transcripts and proteins were abundant in root and stem, but at low levels in leaf and carpel. Silencing of NCS in opium poppy profoundly reduced alkaloid levels compared with controls. Immunoprecipitation of NCS from total protein extracts of T. flavum cells resulted in a nearly complete attenuation of NCS activity. A Ps NCS2–green fluorescent protein fusion introduced by microprojectile bombardment into opium poppy cells initially localized to the endoplasmic reticulum but subsequently sorted to the vacuole. In our hands, Cj NCS1 did not catalyze the formation of (S)-norcoclaurine from dopamine and 4-hydroxyphenylacetaldehyde. PMID:21037103

  11. Coordination modes of tyrosinate-ligated heme enzymes: magnetic circular dichroism studies of Plexaura homomalla allene oxide synthase, Mycobacterium avium ssp. paratuberculosis protein-2744c, and bovine liver catalase in their ferric and ferrous states

    PubMed Central

    Bandara, D. M. Indika; Sono, Masanori; Bruce, Grant S.; Brash, Alan R.; Dawson, John H.

    2012-01-01

    Bovine liver catalase (BLC), catalase-related allene oxide synthase (cAOS) from Plexaura homomalla, and a recently isolated protein from the cattle pathogen Mycobacterium avium ssp. paratuberculosis (MAP-2744c (MAP)) are all tyrosinate-ligated heme enzymes whose crystal structures have been reported. cAOS and MAP have low (< 20%) sequence similarity to, and significantly different catalytic functions from, BLC. cAOS transforms 8R-hydroperoxy-eicosatetraenoic acid to an allene epoxide, whereas the MAP protein is an organic peroxide-dependent peroxidase. To shed light on the functional differences among these three proteins, we have investigated the heme iron coordination properties of these tyrosinate-ligated heme proteins in their ferric and ferrous states using magnetic circular dichroism and UV-visible absorption spectroscopy. The MAP protein shows remarkable spectral similarities to cAOS and BLC in its native Fe(III) state, but clear differences from ferric His93Tyr Mb, which may be attributed to the presence of an Arg+-Nω-H … O-Tyr (proximal heme axial ligand) hydrogen bond in the first three heme proteins. Furthermore, the spectra of Fe(III)-CN−, Fe(III)-NO, Fe(II)-NO (except for five-coordinate MAP), Fe(II)-CO, and Fe(II)-O2 states of cAOS and MAP, but not H93Y Mb, are also similar to the corresponding six-coordinate complexes of BLC, suggesting that a tyrosinate (Tyr-O−) is the heme axial ligand trans to the bound ligands in these complexes. The Arg+-Nω-H to −O-Tyr hydrogen bond would be expected to modulate the donor properties of the proximal tyrosinate oxyanion and, combined with the subtle differences in the catalytic site structures, affect the activities of cAOS, MAP and BLC. PMID:22104301

  12. Human brain prostaglandin D synthase has been evolutionarily differentiated from lipophilic-ligand carrier proteins.

    PubMed Central

    Nagata, A; Suzuki, Y; Igarashi, M; Eguchi, N; Toh, H; Urade, Y; Hayaishi, O

    1991-01-01

    cDNAs for glutathione-independent prostaglandin D synthase were isolated from cDNA libraries of human brain. The longest cDNA insert was 837 base pairs long and contained a coding region of 570 base pairs corresponding to 190 amino acid residues with a calculated Mr of 21,016. Between two cDNA inserts isolated from the two different libraries, nucleotide substitutions were observed at 16 positions, including conservative amino acid substitutions at 2 positions and nonconservative substitutions at 5 positions, indicating genetic heterogeneity of this enzyme in humans. The computer-assisted homology search revealed that the enzyme is a member of the lipocalin superfamily, comprising secretory hydrophobic molecule transporters, showing the greatest homology (28.8-29.4% identity; 51.3-53.1% similarity) to alpha 1-microglobulin among the members of this superfamily. In a phylogenetic tree of the superfamily, this enzyme, alpha 1-microglobulin, and the gamma chain of the complement component C8 form a cluster separate from the other 14 members. The two distinctive characteristics of glutathione-independent prostaglandin D synthase, as compared to the other members of this superfamily, are its enzymatic properties and its association with membranes that were probably acquired after evolutionary divergence of the two lipocalins. Based on the observed sequence homology, the tertiary structure of the enzyme was deduced to consist of an eight-stranded anti-parallel beta-barrel forming a hydrophobic pocket. Furthermore, the Cys-65 residue in the pocket, which is conserved only in the human and rat enzymes but not in other lipocalins, was considered to be a putative active site of the enzyme. Images PMID:1902577

  13. A novel principle for conferring selectivity to poly(A)-binding proteins: interdependence of two ATP synthase beta-subunit mRNA-binding proteins.

    PubMed

    Andersson, U; Antonicka, H; Houstek, J; Cannon, B

    2000-02-15

    Based on electrophoretic mobility-shift assays and UV cross-linking experiments, we present evidence in the present work for the existence of two mammalian cytosolic proteins that selectively interact with the 3'-untranslated region of the mRNA coding for the catalytic beta-subunit of mitochondrial ATP synthase (beta-mtATPase). One of the proteins, beta-mtATPase mRNA-binding protein (BARB)1, is a novel poly(A)-binding protein that specifically binds the poly(A) tail of the beta-mtATPase transcript. BARB1 achieves this mRNA selectivity through its interaction with a second protein, BARB2, that binds the beta-mtATPase mRNA through a 22-bp element with a uridylate core, located 75 bp upstream of the poly(A) tail. Conversely, in the absence of BARB1, BARB2 is still able to bind the beta-mtATPase mRNA, but does so with lower affinity. Thus the interaction between BARB1 and BARB2 and beta-mtATPase mRNA involves the formation of a complex between the two BARB proteins. We conclude that BARB1 and BARB2 selectively bind the 3'-untranslated region of beta-mtATPase mRNA in a novel and interdependent manner. The complex between these two proteins may be involved in post-transcriptional regulation of gene expression. PMID:10657236

  14. Interaction of yeast repressor-activator protein Ume6p with glycogen synthase kinase 3 homolog Rim11p.

    PubMed Central

    Malathi, K; Xiao, Y; Mitchell, A P

    1997-01-01

    Meiosis and expression of early meiotic genes in the budding yeast Saccharomyces cerevisiae depend upon Rim11p, Ume6p, and Ime1p. Rim11p (also called Mds1p and ScGSK3) is a protein kinase related to glycogen synthase kinase 3 (GSK3); Ume6p is an architectural transcription factor; and Imelp is a Ume6p-binding protein that provides a transcriptional activation domain. Rim11p is required for Ime1p-Ume6p interaction, and prior studies have shown that Rim11p binds to and phosphorylates Ime1p. We show here that Rim11p binds to and phosphorylates Ume6p, as well. Amino acid substitutions in Ume6p that alter a consensus GSK3 site reduce or abolish Rim11p-Ume6p interaction and Rim11p-dependent phosphorylation, and they cause defects in interaction between Ume6p and Ime1p and in meiotic gene expression. Therefore, interaction between Rim11p and Ume6p, resulting in phosphorylation of Ume6p, is required for Ime1p-Ume6p complex formation. Rim11p, like metazoan GSK3beta, phosphorylates both interacting subunits of a target protein complex. PMID:9372955

  15. Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

    SciTech Connect

    Zhu, Wenzhen; Yang, Bingwu; Fu, Huiling; Ma, Long; Liu, Tingting; Chai, Rongfei; Zheng, Zhaodi; Zhang, Qunye; Li, Guorong

    2015-03-13

    As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-induced inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation.

  16. Starch phosphorylation in potato tubers is influenced by allelic variation in the genes encoding glucan water dikinase, starch branching enzymes I and II, and starch synthase III

    PubMed Central

    Carpenter, Margaret A.; Joyce, Nigel I.; Genet, Russell A.; Cooper, Rebecca D.; Murray, Sarah R.; Noble, Alasdair D.; Butler, Ruth C.; Timmerman-Vaughan, Gail M.

    2015-01-01

    Starch phosphorylation is an important aspect of plant metabolism due to its role in starch degradation. Moreover, the degree of phosphorylation of starch determines its physicochemical properties and is therefore relevant for industrial uses of starch. Currently, starch is chemically phosphorylated to increase viscosity and paste stability. Potato cultivars with elevated starch phosphorylation would make this process unnecessary, thereby bestowing economic and environmental benefits. Starch phosphorylation is a complex trait which has been previously shown by antisense gene repression to be influenced by a number of genes including those involved in starch synthesis and degradation. We have used an association mapping approach to discover genetic markers associated with the degree of starch phosphorylation. A diverse collection of 193 potato lines was grown in replicated field trials, and the levels of starch phosphorylation at the C6 and C3 positions of the glucosyl residues were determined by mass spectrometry of hydrolyzed starch from tubers. In addition, the potato lines were genotyped by amplicon sequencing and microsatellite analysis, focusing on candidate genes known to be involved in starch synthesis. As potato is an autotetraploid, genotyping included determination of allele dosage. Significant associations (p < 0.001) were found with SNPs in the glucan water dikinase (GWD), starch branching enzyme I (SBEI) and the starch synthase III (SSIII) genes, and with a SSR allele in the SBEII gene. SNPs in the GWD gene were associated with C6 phosphorylation, whereas polymorphisms in the SBEI and SBEII genes were associated with both C6 and C3 phosphorylation and the SNP in the SSIII gene was associated with C3 phosphorylation. These allelic variants have potential as genetic markers for starch phosphorylation in potato. PMID:25806042

  17. X-Ray Crystal Structure of Mycobacterium Tuberculosis β-Ketoacyl Acyl Carrier Protein Synthase II (mtKasB)

    PubMed Central

    Sridharan, Sudharsan; Wang, Lei; Brown, Alistair K.; Dover, Lynn G.; Kremer, Laurent; Besra, Gurdyal S.; Sacchettini, James C.

    2007-01-01

    Summary Mycolic acids are long chain α-alkyl branched, β-hydroxy fatty acids that represent a characteristic component of the Mycobacterium tuberculosis cell wall. Through their covalent attachment to peptidoglycan via an arabinogalactan polysaccharide, they provide the basis for an essential outer envelope membrane. Mycobacteria possess two fatty acid synthases (FAS); FAS-I carries out de novo synthesis of fatty acids while FAS-II is considered to elongate medium chain length fatty acyl primers to provide long chain (C56) precursors of mycolic acids. Here we report the crystal structure of Mycobacterium tuberculosis β-ketoacyl acyl carrier protein synthase (ACP) II mtKasB, a mycobacterial elongation condensing enzyme involved in FAS-II. This enzyme, along with the M. tuberculosis β-ketoacyl ACP synthase I mtKasA, catalyzes the Claisen-type condensation reaction responsible for fatty acyl elongation in FAS-II and are potential targets for development of novel anti-tubercular drugs. The crystal structure refined to 2.4 Å resolution revealed that, like other KAS-II enzymes, mtKasB adopts a thiolase fold but contains unique structural features in the capping region that may be crucial to its preference for longer fatty acyl chains than its counterparts from other bacteria. Modeling of mtKasA using the mtKasB structure as a template predicts the overall structures to be almost identical, but a larger entrance to the active site tunnel is envisaged that might contribute to the greater sensitivity of mtKasA to the inhibitor thiolactomycin (TLM). Modeling of TLM binding in mtKasB shows that the drug fits the active site poorly and results of enzyme inhibition assays using TLM analogues are wholly consistent with our structural observations. Consequently, the structure described here further highlights the potential of TLM as an anti-tubercular lead compound and will aid further exploration of the TLM scaffold towards the design of novel compounds which inhibit

  18. Coordination modes of tyrosinate-ligated catalase-type heme enzymes: magnetic circular dichroism studies of Plexaura homomalla allene oxide synthase, Mycobacterium avium ssp. paratuberculosis protein-2744c, and bovine liver catalase in their ferric and ferrous states.

    PubMed

    Bandara, D M Indika; Sono, Masanori; Bruce, Grant S; Brash, Alan R; Dawson, John H

    2011-12-01

    Bovine liver catalase (BLC), catalase-related allene oxide synthase (cAOS) from Plexaura homomalla, and a recently isolated protein from the cattle pathogen Mycobacterium avium ssp. paratuberculosis (MAP-2744c (MAP)) are all tyrosinate-ligated heme enzymes whose crystal structures have been reported. cAOS and MAP have low (<20%) sequence similarity to, and significantly different catalytic functions from, BLC. cAOS transforms 8R-hydroperoxy-eicosatetraenoic acid to an allene epoxide, whereas the MAP protein is a putative organic peroxide-dependent peroxidase. To elucidate factors influencing the functions of these and related heme proteins, we have investigated the heme iron coordination properties of these tyrosinate-ligated heme enzymes in their ferric and ferrous states using magnetic circular dichroism and UV-visible absorption spectroscopy. The MAP protein shows remarkable spectral similarities to cAOS and BLC in its native Fe(III) state, but clear differences from ferric proximal heme ligand His93Tyr Mb (myoglobin) mutant, which may be attributed to the presence of an Arg(+)-N(ω)-H···¯O-Tyr (proximal heme axial ligand) hydrogen bond in the first three heme proteins. Furthermore, the spectra of Fe(III)-CN¯, Fe(III)-NO, Fe(II)-NO (except for five-coordinate MAP), Fe(II)-CO, and Fe(II)-O(2) states of cAOS and MAP, but not H93Y Mb, are also similar to the corresponding six-coordinate complexes of BLC, suggesting that a tyrosinate (Tyr-O¯) is the heme axial ligand trans to the bound ligands in these complexes. The Arg(+)-N(ω)-H to ¯O-Tyr hydrogen bond would be expected to modulate the donor properties of the proximal tyrosinate oxyanion and, combined with the subtle differences in the catalytic site structures, affect the activities of cAOS, MAP and BLC. PMID:22104301

  19. Identification of a starter unit acyl-carrier protein transacylase domain in an iterative type I polyketide synthase

    PubMed Central

    Crawford, Jason M.; Dancy, Blair C. R.; Hill, Eric A.; Udwary, Daniel W.; Townsend, Craig A.

    2006-01-01

    Polyketides are a class of natural products that exhibit a wide range of functional and structural diversity. They include antibiotics, immunosuppressants, antifungals, antihypercholesterolemics, and cytotoxins. Polyketide synthases (PKSs) use chemistry similar to fatty acid synthases (FASs), although building block variation and differing extents of reduction of the growing polyketide chain underlie their biosynthetic versatility. In contrast to the well studied sequential modular type I PKSs, less is known about how the iterative type I PKSs carry out and control chain initiation, elongation, folding, and cyclization during polyketide processing. Domain structure analysis of a group of related fungal, nonreducing PKSs has revealed well defined N-terminal domains longer than commonly seen for FASs and modular PKSs. Predicted structure of this domain disclosed a region similar to malonyl-CoA:acyl-carrier protein (ACP) transacylases (MATs). MATs play a key role transferring precursor CoA thioesters from solution onto FASs and PKSs for chain elongation. On the basis of site-directed mutagenesis, radiolabeling, and kinetics experiments carried out with individual domains of the norsolorinic acid PKS, we propose that the N-terminal domain is a starter unit:ACP transacylase (SAT domain) that selects a C6 fatty acid from a dedicated yeast-like FAS and transfers this unit onto the PKS ACP, leading to the production of the aflatoxin precursor, norsolorinic acid. These findings could indicate a much broader role for SAT domains in starter unit selection among nonreducing iterative, fungal PKSs, and they provide a biochemical rationale for the classical acetyl “starter unit effect.” PMID:17071746

  20. Role of Long-Range Protein Dynamics in Different Thymidylate Synthase Catalyzed Reactions

    PubMed Central

    Abeysinghe, Thelma; Kohen, Amnon

    2015-01-01

    Recent studies of Escherichia coli thymidylate synthase (ecTSase) showed that a highly conserved residue, Y209, that is located 8 Å away from the reaction site, plays a key role in the protein’s dynamics. Those crystallographic studies indicated that Y209W mutant is a structurally identical but dynamically altered relative to the wild type (WT) enzyme, and that its turnover catalytic rate governed by a slow hydride-transfer has been affected. The most challenging test of an examination of a fast chemical conversion that precedes the rate-limiting step has been achieved here. The physical nature of both fast and slow C-H bond activations have been compared between the WT and mutant by means of observed and intrinsic kinetic isotope effects (KIEs) and their temperature dependence. The findings indicate that the proton abstraction step has not been altered as much as the hydride transfer step. Additionally, the comparison indicated that other kinetic steps in the TSase catalyzed reaction were substantially affected, including the order of the substrate binding. Enigmatically, although Y209 is H-bonded to 3'-OH of 2'-deoxyuridine-5'-mono­phosphate (dUMP), its altered dynamics is more pronounced on the binding of the remote cofactor, (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate), revealing the importance of long-range dynamics of the enzymatic complex and its catalytic function. PMID:25837629

  1. Cocaine regulates protein kinase B and glycogen synthase kinase-3 activity in selective regions of rat brain

    PubMed Central

    SA, Perrine; JS, Miller; EM, Unterwald

    2008-01-01

    Protein kinase B (Akt) signaling regulates dopamine-mediated locomotor behaviors. Here the ability of cocaine to regulate Akt and glycogen synthase kinase-3 (GSK3) was studied. Rats were injected with cocaine or saline in a binge-pattern, which consisted of 3 daily injections of 15 mg/kg cocaine or 1 ml/kg saline spaced one hour apart for 1, 3 or 14 days. Amygdala, nucleus accumbens, caudate putamen and hippocampus tissues were dissected 30 minutes following the last injection and analyzed for phosphorylated and total Akt and GSK3(α & β) protein levels using Western blot analysis. Phosphorylation of Akt on the threonine-308 residue was significantly reduced in the nucleus accumbens and increased in the amygdala after 1 day of cocaine treatment; however, these effects were not accompanied by a significant decrease in GSK3 phosphorylation. Phosphorylation of Akt and GSK3 were significantly reduced after 14 days of cocaine administration, an effect that was only observed in the amygdala. Cocaine did not alter Akt or GSK3 phosphorylation in the caudate putamen or hippocampus. The findings in nucleus accumbens may reflect dopaminergic motor-stimulant activity caused by acute cocaine, whereas the effects in amygdala may be associated with changes in emotional state that occur after acute and chronic cocaine exposure. PMID:18717814

  2. High hydrostatic pressure induces synthesis of heat-shock proteins and trehalose-6-phosphate synthase in Anastrepha ludens larvae.

    PubMed

    Vargas-Ortiz, Manuel A; Quintana-Castro, Rodolfo; Oliart-Ros, Rosa M; De la Cruz-Medina, Javier; Ramírez de León, José A; Garcia, Hugo S

    2013-04-01

    The Mexican fruit fly (Anastrepha ludens) is responsible for losses of up to 25% of crops such as mango and citrus fruits in Central America and México. The larval life cycle of A. ludens comprises three stages with a duration ranging from 3 to 8 days. Because of the damage caused by A. ludens, several methods of control have been studied and implemented. High hydrostatic pressures (HHP) are currently applied to foods and it is now proposed to be employed to inactivate eggs and larvae of A. ludens. Originally HHP was designed to inactivate microorganisms, since it exerts marked effects on cell morphology, and can affect enzymatic reactions and genetic mechanisms of microbial cells, with no major changes altering the sensory or nutritional quality of the foodstuff. In this study, A. ludens in two larval stages (5- and 8-day-old) were subjected to HHP treatments. The biochemical response of the larvae of A. ludens was dependent on their stage of development. The third larval stage (L3) developed a better protection mechanism based on the synthesis of stress proteins or heat-shock proteins (HSPs) and the enzyme trehalose-6-phosphate synthase, which are linked and possibly act together to achieve greater survivability to stress caused by hydrostatic pressure. PMID:23361865

  3. A Mitochondrial ATP synthase Subunit Interacts with TOR Signaling to Modulate Protein Homeostasis and Lifespan in Drosophila

    PubMed Central

    Sun, Xiaoping; Wheeler, Charles T.; Yolitz, Jason; Laslo, Mara; Alberico, Thomas; Sun, Yaning; Song, Qisheng; Zou, Sige

    2014-01-01

    SUMMARY Diet composition is a critical determinant of lifespan and nutrient imbalance is detrimental health. However, how nutrients interact with genetic factors to modulate lifespan remains elusive. We investigated how diet composition influences mitochondrial ATP synthase subunit d (ATPsyn-d) in modulating lifespan in Drosophila. ATPsyn-d knockdown extended lifespan in females fed low carbohydrate-to-protein (C:P) diets, but not the high C:P ratio diet. This extension was associated with increased resistance to oxidative stress, transcriptional changes in metabolism, proteostasis and immune genes, reduced protein damage and aggregation, and reduced phosphorylation of S6K and ERK in TOR and MAPK signaling, respectively. ATPsyn-d knockdown did not extend lifespan in females with reduced TOR signaling induced genetically by Tsc2 overexpression or pharmacologically by rapamycin. Our data reveal a link among diet, mitochondria, MAPK and TOR signaling in aging and stresses the importance of considering genetic background and diet composition in implementing interventions for promoting healthy aging. PMID:25220459

  4. Sestrin 2 and AMPK Connect Hyperglycemia to Nox4-Dependent Endothelial Nitric Oxide Synthase Uncoupling and Matrix Protein Expression

    PubMed Central

    Eid, Assaad A.; Lee, Doug-Yoon; Roman, Linda J.; Khazim, Khaled

    2013-01-01

    Mesangial matrix accumulation is an early feature of glomerular pathology in diabetes. Oxidative stress plays a critical role in hyperglycemia-induced glomerular injury. Here, we demonstrate that, in glomerular mesangial cells (MCs), endothelial nitric oxide synthase (eNOS) is uncoupled upon exposure to high glucose (HG), with enhanced generation of reactive oxygen species (ROS) and decreased production of nitric oxide. Peroxynitrite mediates the effects of HG on eNOS dysfunction. HG upregulates Nox4 protein, and inhibition of Nox4 abrogates the increase in ROS and peroxynitrite generation, as well as the eNOS uncoupling triggered by HG, demonstrating that Nox4 functions upstream from eNOS. Importantly, this pathway contributes to HG-induced MC fibronectin accumulation. Nox4-mediated eNOS dysfunction was confirmed in glomeruli of a rat model of type 1 diabetes. Sestrin 2-dependent AMP-activated protein kinase (AMPK) activation attenuates HG-induced MC fibronectin synthesis through blockade of Nox4-dependent ROS and peroxynitrite generation, with subsequent eNOS uncoupling. We also find that HG negatively regulates sestrin 2 and AMPK, thereby promoting Nox4-mediated eNOS dysfunction and increased fibronectin. These data identify a protective function for sestrin 2/AMPK and potential targets for intervention to prevent fibrotic injury in diabetes. PMID:23816887

  5. Dolichol phosphate mannose synthase is required in vivo for glycosyl phosphatidylinositol membrane anchoring, O mannosylation, and N glycosylation of protein in Saccharomyces cerevisiae.

    PubMed Central

    Orlean, P

    1990-01-01

    Glycosyl phosphatidylinositol (GPI) anchoring, N glycosylation, and O mannosylation of protein occur in the rough endoplasmic reticulum and involve transfer of precursor structures that contain mannose. Direct genetic evidence is presented that dolichol phosphate mannose (Dol-P-Man) synthase, which transfers mannose from GDPMan to the polyisoprenoid dolichol phosphate, is required in vivo for all three biosynthetic pathways leading to these covalent modifications of protein in yeast cells. Temperature-sensitive yeast mutants were isolated after in vitro mutagenesis of the yeast DPM1 gene. At the nonpermissive temperature of 37 degrees C, the dpm1 mutants were blocked in [2-3H]myo-inositol incorporation into protein and accumulated a lipid that could be radiolabeled with both [2-3H]myo-inositol and [2-3H]glucosamine and met existing criteria for an intermediate in GPI anchor biosynthesis. The likeliest explanation for these results is that Dol-P-Man donates the mannose residues needed for completion of the GPI anchor precursor lipid before it can be transferred to protein. Dol-P-Man synthase is also required in vivo for N glycosylation of protein, because (i) dpm1 cells were unable to make the full-length precursor Dol-PP-GlcNAc2Man9Glc3 and instead accumulated the intermediate Dol-PP-GlcNAc2Man5 in their pool of lipid-linked precursor oligosaccharides and (ii) truncated, endoglycosidase H-resistant oligosaccharides were transferred to the N-glycosylated protein invertase after a shift to 37 degrees C. Dol-P-Man synthase is also required in vivo for O mannosylation of protein, because chitinase, normally a 150-kDa O-mannosylated protein, showed a molecular size of 60 kDa, the size predicted for the unglycosylated protein, after shift of the dpm1 mutant to the nonpermissive temperature. Images PMID:2146492

  6. The Major Antigenic Membrane Protein of “Candidatus Phytoplasma asteris” Selectively Interacts with ATP Synthase and Actin of Leafhopper Vectors

    PubMed Central

    Galetto, Luciana; Bosco, Domenico; Balestrini, Raffaella; Genre, Andrea; Fletcher, Jacqueline; Marzachì, Cristina

    2011-01-01

    Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of “Candidatus Phytoplasma asteris”, the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity. PMID

  7. HMP Binding Protein ThiY and HMP-P Synthase THI5 Are Structural Homologues

    SciTech Connect

    Bale, Shridhar; Rajashankar, Kanagalaghatta R.; Perry, Kay; Begley, Tadhg P.; Ealick, Steven E.

    2010-10-14

    The ATP-binding cassette transporter system ThiXYZ transports N-formyl-4-amino-5-(aminomethyl)-2-methylpyrimidine (FAMP), a thiamin salvage pathway intermediate, into cells. FAMP is then converted to 4-amino-5-(hydroxymethyl)-2-methylpyrimidine (HMP) and recycled into the thiamin biosynthetic pathway. ThiY is the periplasmic substrate binding protein of the ThiXYZ system and delivers the substrate FAMP to the transmembrane domain. We report the crystal structure of Bacillus halodurans ThiY with FAMP bound at 2.4 {angstrom} resolution determined by single-wavelength anomalous diffraction phasing. The crystal structure reveals that ThiY belongs to the group II periplasmic binding protein family. The closest structural homologues of ThiY are periplasmic binding proteins involved in alkanesulfonate/nitrate and bicarbonate transport. ThiY is also structurally homologous to thiamin binding protein (TbpA) and to thiaminase-I. THI5 is responsible for the synthesis of 4-amino-5-(hydroxymethyl)-2-methylpyrimidine phosphate in the thiamin biosynthetic pathway of eukaryotes and is approximately 25% identical in sequence with ThiY. A homology model of Saccharomyces cerevisiae THI5 was generated on the basis of the structure of ThiY. Many features of the thiamin pyrimidine binding site are shared between ThiY and THI5, suggesting a common ancestor.

  8. TATA-binding protein and associated factors in polymerase II and polymerase III transcription.

    PubMed Central

    Meyers, R E; Sharp, P A

    1993-01-01

    Transcription by RNA polymerase I (pol I), pol II, and pol III requires the TATA-binding protein (TBP). This protein functions in association with distinct TBP-associated factors (TAFs) which may specify the nature of the polymerase selected for initiation at a promoter site. In the pol III transcription system, the TBP-TAF complex is a component of the TFIIIB factor. This factor has been resolved into a TBP-TAF complex and another component, both of which are required for reconstitution of transcription by pol III. Neither the TBP-TAF complexes B-TFIID and D-TFIID, which were previously characterized as active for pol II transcription, nor TBP alone can complement pol III transcription reactions that are dependent upon the TBP-TAF subcomponent of TFIIIB. Surprisingly, the TBP-TAF subcomponent of TFIIIB is active in reconstitution of pol II transcription. Images PMID:8247010

  9. Transcriptional activation of Brassica napus β-ketoacyl-ACP synthase II with an engineered zinc finger protein transcription factor.

    PubMed

    Gupta, Manju; DeKelver, Russell C; Palta, Asha; Clifford, Carla; Gopalan, Sunita; Miller, Jeffrey C; Novak, Stephen; Desloover, Daniel; Gachotte, Daniel; Connell, James; Flook, Josh; Patterson, Thomas; Robbins, Kelly; Rebar, Edward J; Gregory, Philip D; Urnov, Fyodor D; Petolino, Joseph F

    2012-09-01

    Targeted gene regulation via designed transcription factors has great potential for precise phenotypic modification and acceleration of novel crop trait development. Canola seed oil composition is dictated largely by the expression of genes encoding enzymes in the fatty acid biosynthetic pathway. In the present study, zinc finger proteins (ZFPs) were designed to bind DNA sequences common to two canola β-ketoacyl-ACP Synthase II (KASII) genes downstream of their transcription start site. Transcriptional activators (ZFP-TFs) were constructed by fusing these ZFP DNA-binding domains to the VP16 transcriptional activation domain. Following transformation using Agrobacterium, transgenic events expressing ZFP-TFs were generated and shown to have elevated KASII transcript levels in the leaves of transgenic T(0) plants when compared to 'selectable marker only' controls as well as of T(1) progeny plants when compared to null segregants. In addition, leaves of ZFP-TF-expressing T(1) plants contained statistically significant decreases in palmitic acid (consistent with increased KASII activity) and increased total C18. Similarly, T(2) seed displayed statistically significant decreases in palmitic acid, increased total C18 and reduced total saturated fatty acid contents. These results demonstrate that designed ZFP-TFs can be used to regulate the expression of endogenous genes to elicit specific phenotypic modifications of agronomically relevant traits in a crop species. PMID:22520333

  10. The innervation of rainbow trout (Oncorhynchus mykiss) liver: protein gene product 9.5 and neuronal nitric oxide synthase immunoreactivities

    PubMed Central

    ESTEBAN, F. J.; JIMÉNEZ, A.; BARROSO, J. B.; PEDROSA, J. A.; DEL MORAL, M. L.; RODRIGO, J.; PEINADO, M. A.

    1998-01-01

    We have explored the innervation of the rainbow trout (O. mykiss) liver using immunohistochemical procedures and light microscopy to detect in situ protein gene product 9.5 and neuronal nitric oxide synthase immunoreactivities (PGP-IR and NOS-IR). The results showed PGP-IR nerve fibres running with the extralobular biliary duct (EBD), hepatic artery (EHA) and portal vein (EPV) that form the hepatic hilum, as well as following the spatial distribution of the intrahepatic blood vessel and biliary channels. These nerve fibres appear as single varicose processes, thin bundles, or thick bundles depending on their diameter and location in the wall of the blood vessel or biliary duct. No PGP-IR fibres were detected in the liver parenchyma. NOS-IR nerve fibres were located only in the vessels and ducts that form the hepatic hilum (EBD, EHA, EPV); in addition, NOS-IR nerve cell bodies were found isolated or forming ganglionated plexuses in the peribiliary fibromuscular tissue of the EBD. No PGP-IR ganglionated plexuses were detected in the EBD. The location of the general (PGP-IR) and nitrergic (nNOS-IR) intrinsic nerves of the trout liver suggest a conserved evolutionary role of the nervous control of hepatic blood flow and hepatobiliary activity. PMID:9827640

  11. Glycogen Synthase Kinase 3β Is Positively Regulated by Protein Kinase Cζ-Mediated Phosphorylation Induced by Wnt Agonists

    PubMed Central

    Tejeda-Muñoz, Nydia; González-Aguilar, Héctor; Santoyo-Ramos, Paula; Castañeda-Patlán, M. Cristina

    2015-01-01

    The molecular events that drive Wnt-induced regulation of glycogen synthase kinase 3β (GSK-3β) activity are poorly defined. In this study, we found that protein kinase Cζ (PKCζ) and GSK-3β interact mainly in colon cancer cells. Wnt stimulation induced a rapid GSK-3β redistribution from the cytoplasm to the nuclei in malignant cells and a transient PKC-mediated phosphorylation of GSK-3β at a different site from serine 9. In addition, while Wnt treatment induced a decrease in PKC-mediated phosphorylation of GSK-3β in nonmalignant cells, in malignant cells, this phosphorylation was increased. Pharmacological inhibition and small interfering RNA (siRNA)-mediated silencing of PKCζ abolished all of these effects, but unexpectedly, it also abolished the constitutive basal activity of GSK-3β. In vitro activity assays demonstrated that GSK-3β phosphorylation mediated by PKCζ enhanced GSK-3β activity. We mapped Ser147 of GSK-3β as the site phosphorylated by PKCζ, i.e., its mutation into alanine abolished GSK-3β activity, resulting in β-catenin stabilization and increased transcriptional activity, whereas phosphomimetic replacement of Ser147 by glutamic acid maintained GSK-3β basal activity. Thus, we found that PKCζ phosphorylates GSK-3β at Ser147 to maintain its constitutive activity in resting cells and that Wnt stimulation modifies the phosphorylation of Ser147 to regulate GSK-3β activity in opposite manners in normal and malignant colon cells. PMID:26711256

  12. Phosphorylation and activation of calcineurin by glycogen synthase (casein) kinase-1 and cyclic AMP-dependent protein kinase

    SciTech Connect

    Singh, T.J.; Wang, J.H.

    1986-05-01

    Calcineurin is a phosphoprotein phosphatase that is activated by divalent cations and further stimulated by calmodulin. In this study calcineurin is shown to be a substrate for both glycogen synthase (casein) kinase-1 (CK-1) and cyclic AMP-dependent protein kinase (A-kinase). Either kinase can catalyze the incorporation of 1.0-1.4 mol /sup 32/P/mol calcineurin. Analysis by SDS-PAGE revealed that only the ..cap alpha.. subunit is phosphorylated. Phosphorylation of calcineurin by either kinase leads to its activation. Using p-nitrophenyl phosphate as a substrate the authors observed a 2-3 fold activation of calcineurin by either Mn/sup 2 +/ or Ni/sup 2 +/ (in the presence or absence of calmodulin) after phosphorylation of calcineurin by either CK-1 or A-kinase. In the absence of Mn/sup 2 +/ or Ni/sup 2 +/ phosphorylated calcineurin, like the nonphosphorylated enzyme, showed very little activity. Ni/sup 2 +/ was a more potent activator of phosphorylated calcineurin compared to Mn/sup 2 +/. Higher levels of activation (5-8 fold) of calcineurin by calmodulin was observed when phosphorylated calcineurin was pretreated with Ni/sup 2 +/ before measurement of phosphatase activity. These results indicate that phosphorylation may be an important mechanism by which calcineurin activity is regulated by Ca/sup 2 +/.

  13. GapIII, a new brain-enriched member of the GTPase-activating protein family.

    PubMed

    Baba, H; Fuss, B; Urano, J; Poullet, P; Watson, J B; Tamanoi, F; Macklin, W B

    1995-08-15

    Ras GTPase-activating proteins (GAPs) are negative regulators of ras, which controls proliferation and differentiation in many cells. Ras GAPs have been found in a variety of species from yeast to mammals. We describe here a newly identified mammalian GAP, GapIII, which was obtained by differential screening of a rat oligodendrocyte cDNA library. GapIII putatively encodes a 834 amino acid protein with a predicted molecular weight of 96 kDa, which contains a consensus GAP-related domain (GRD). The protein encoded by this cDNA has high homology with Gap1m, which was recently identified as a putative mammalian homolog of Drosophila Gap1. These proteins contain three structural domains, an N-terminal calcium-dependent phospholipid binding domain, GRD, and a C-terminal PH/Btk domain. Because of the sequence homology and the structural similarities of this protein with Gap1m, we hypothesize that GapIII and Gap1m may be members of a mammalian GAP gene family, separate from p120GAP, neurofibromin (NF1), and IQGAP. To confirm the GapIII protein activity, constructs containing different GapIII-GRD domains were transformed into iral mutant yeast to determine their relative ability to replace IRA1 functionally. Constructs that contained essentially the full-length protein (all three domains), the GRD alone, or the GRD plus PH/Btk domain suppressed heat shock sensitivity of ira1, whereas constructs that contained the GRD with part of the PH/Btk domain had only a weak ability to suppress heat shock sensitivity. These results suggest that the GapIII GRD itself is sufficient to down-regulate ras proteins in yeast. Expression of GapIII mRNA (4.2 kb) was examined by Northern analysis and in situ hybridization. This mRNA was expressed at highest levels in the brain, where its expression increased with development. Lower levels of the mRNA were expressed in the spleen and lung. Among neural cells, GapIII mRNA was expressed in neurons and oligodendrocytes, but not in astrocytes

  14. Protein signatures correspond to survival outcomes of AJCC stage III melanoma patients

    PubMed Central

    Mactier, Swetlana; Kaufman, Kimberley L; Wang, Penghao; Crossett, Ben; Pupo, Gulietta M; Kohnke, Philippa L; Thompson, John F; Scolyer, Richard A; Yang, Jean Y; Mann, Graham J; Christopherson, Richard I

    2014-01-01

    Summary Outcomes for melanoma patients with stage III disease differ widely even within the same subcategory. Molecular signatures that more accurately predict prognosis are needed to stratify patients according to risk. Proteomic analyses were used to identify differentially abundant proteins in extracts of surgically excised samples from patients with stage IIIc melanoma lymph node metastases. Analysis of samples from patients with poor (n = 14, <1 yr) and good (n = 19, >4 yr) survival outcomes identified 84 proteins that were differentially abundant between prognostic groups. Subsequent selected reaction monitoring analysis verified 21 proteins as potential biomarkers for survival. Poor prognosis patients are characterized by increased levels of proteins involved in protein metabolism, nucleic acid metabolism, angiogenesis, deregulation of cellular energetics and methylation processes, and decreased levels of proteins involved in apoptosis and immune response. These proteins are able to classify stage IIIc patients into prognostic subgroups (P < 0.02). This is the first report of potential prognostic markers from stage III melanoma using proteomic analyses. Validation of these protein markers in larger patient cohorts should define protein signatures that enable better stratification of stage III melanoma patients. PMID:24995518

  15. Protein Kinase A Opposes the Phosphorylation-dependent Recruitment of Glycogen Synthase Kinase 3β to A-kinase Anchoring Protein 220.

    PubMed

    Whiting, Jennifer L; Nygren, Patrick J; Tunquist, Brian J; Langeberg, Lorene K; Seternes, Ole-Morten; Scott, John D

    2015-08-01

    The proximity of an enzyme to its substrate can influence rate and magnitude of catalysis. A-kinase anchoring protein 220 (AKAP220) is a multivalent anchoring protein that can sequester a variety of signal transduction enzymes. These include protein kinase A (PKA) and glycogen synthase kinase 3β (GSK3β). Using a combination of molecular and cellular approaches we show that GSK3β phosphorylation of Thr-1132 on AKAP220 initiates recruitment of this kinase into the enzyme scaffold. We also find that AKAP220 anchors GSK3β and its substrate β-catenin in membrane ruffles. Interestingly, GSK3β can be released from the multienzyme complex in response to PKA phosphorylation on serine 9, which suppresses GSK3β activity. The signaling scaffold may enhance this regulatory mechanism, as AKAP220 has the capacity to anchor two PKA holoenzymes. Site 1 on AKAP220 (residues 610-623) preferentially interacts with RII, whereas site 2 (residues 1633-1646) exhibits a dual specificity for RI and RII. In vitro affinity measurements revealed that site 2 on AKAP220 binds RII with ∼10-fold higher affinity than site 1. Occupancy of both R subunit binding sites on AKAP220 could provide a mechanism to amplify local cAMP responses and enable cross-talk between PKA and GSK3β. PMID:26088133

  16. Structural Characterisation of the Beta-Ketoacyl-Acyl Carrier Protein Synthases, FabF and FabH, of Yersinia pestis

    PubMed Central

    Nanson, Jeffrey D.; Himiari, Zainab; Swarbrick, Crystall M. D.; Forwood, Jade K.

    2015-01-01

    Yersinia pestis, the causative agent of bubonic, pneumonic, and septicaemic plague, remains a major public health threat, with outbreaks of disease occurring in China, Madagascar, and Peru in the last five years. The existence of multidrug resistant Y. pestis and the potential of this bacterium as a bioterrorism agent illustrates the need for new antimicrobials. The β-ketoacyl-acyl carrier protein synthases, FabB, FabF, and FabH, catalyse the elongation of fatty acids as part of the type II fatty acid biosynthesis (FASII) system, to synthesise components of lipoproteins, phospholipids, and lipopolysaccharides essential for bacterial growth and survival. As such, these enzymes are promising targets for the development of novel therapeutic agents. We have determined the crystal structures of the Y. pestis β-ketoacyl-acyl carrier protein synthases FabF and FabH, and compared these with the unpublished, deposited structure of Y. pestis FabB. Comparison of FabB, FabF, and FabH provides insights into the substrate specificities of these enzymes, and investigation of possible interactions with known β-ketoacyl-acyl carrier protein synthase inhibitors suggests FabB, FabF and FabH may be targeted simultaneously to prevent synthesis of the fatty acids necessary for growth and survival. PMID:26469877

  17. Structural Characterisation of the Beta-Ketoacyl-Acyl Carrier Protein Synthases, FabF and FabH, of Yersinia pestis.

    PubMed

    Nanson, Jeffrey D; Himiari, Zainab; Swarbrick, Crystall M D; Forwood, Jade K

    2015-01-01

    Yersinia pestis, the causative agent of bubonic, pneumonic, and septicaemic plague, remains a major public health threat, with outbreaks of disease occurring in China, Madagascar, and Peru in the last five years. The existence of multidrug resistant Y. pestis and the potential of this bacterium as a bioterrorism agent illustrates the need for new antimicrobials. The β-ketoacyl-acyl carrier protein synthases, FabB, FabF, and FabH, catalyse the elongation of fatty acids as part of the type II fatty acid biosynthesis (FASII) system, to synthesise components of lipoproteins, phospholipids, and lipopolysaccharides essential for bacterial growth and survival. As such, these enzymes are promising targets for the development of novel therapeutic agents. We have determined the crystal structures of the Y. pestis β-ketoacyl-acyl carrier protein synthases FabF and FabH, and compared these with the unpublished, deposited structure of Y. pestis FabB. Comparison of FabB, FabF, and FabH provides insights into the substrate specificities of these enzymes, and investigation of possible interactions with known β-ketoacyl-acyl carrier protein synthase inhibitors suggests FabB, FabF and FabH may be targeted simultaneously to prevent synthesis of the fatty acids necessary for growth and survival. PMID:26469877

  18. Crystal structure of the Yersinia type III secretion protein YscE

    SciTech Connect

    Phan, Jason; Austin, Brian P.; Waugh, David S.

    2010-12-06

    The plague-causing bacterium Yersinia pestis utilizes a contact-dependent (type III) secretion system (T3SS) to transport virulence factors from the bacterial cytosol directly into the interior of mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. The type III secretion apparatus is composed of 20-25 different Yersinia secretion (Ysc) proteins. We report here the structure of YscE, the smallest Ysc protein, which is a dimer in solution. The probable mode of oligomerization is discussed.

  19. Regulation of endothelial nitric oxide synthase: involvement of protein kinase G 1 beta, serine 116 phosphorylation and lipid structures.

    PubMed

    John, Theresa A; Ibe, Basil O; Raj, J Usha

    2008-02-01

    1. Endothelial nitric oxide synthase (NOS3) is important for vascular homeostasis. The role of protein kinase G (PKG) in regulation of NOS3 activity was studied in primary cultures of newborn lamb lung microvascular endothelial cells (LMVEC). 2. We determined the presence of PKG in fetal and neonatal LMVEC as well as subcellular localization of PKG isoforms in the neonatal cells by fluorescence immunohistochemistry. We used diaminofluorescein (DAF) fluorophore to measure nitric oxide (NO) production from neonatal LMVEC. We confirmed that NO measured was from constitutive NOS3 by inhibiting it with NOS inhibitors. 3. To identify a role for PKG in basal NO production, we measured NO release from LMVEC cells using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM; 0.5-0.8 micromol/L) with and without prior stimulation with the PKG activator 8-bromo-cGMP (8-Br-cGMP; 0.3 and 3 micromol/L) or prior PKG inhibition with beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothionate (BPC; 0.3 and 3 micromol/L). With the same drugs, we determined the role of PKG on cellular expression of NOS3 and serine 116 phosphorylated NOS (pSer116-NOS) by qualitative and quantitative immunofluorescence assays, as well as western blotting. 4. Because PKG 1 beta was distributed throughout the cytosol in a punctate expression, we used 2 mmol/L cyclodextrin, a cholesterol extractor, to determine a role for lipid vesicles in PKG regulation of NO production. 5. Protein kinase G 1 beta gave a punctate appearance, indicating its presence in intracellular vesicles. Nitric oxide production decreased by approximately 20% with 300 nmol/L and 3 micromol/L 8-Br cGMP (P < 0.05) and increased by 20.8 +/- 3.7% with 3 micromol/L BPC (P < 0.001), indicating that both stimulated and basal PKG activity has inhibitory effects on basal NOS3 function. Nitric oxide synthase immunofluorescence and immunoblot expression were decreased and pSer116-NOS immunofluorescence was increased by 800 nmol

  20. The multiple phenylpropene synthases in both Clarkia breweri and Petunia hybrida represent two distinct protein lineages

    PubMed Central

    Koeduka, Takao; Louie, Gordon V.; Orlova, Irina; Kish, Christine M.; Ibdah, Mwafaq; Wilkerson, Curtis G.; Bowman, Marianne E.; Baiga, Thomas J.; Noel, Joseph P.; Dudareva, Natalia; Pichersky, Eran

    2009-01-01

    Summary Many plants synthesize the volatile phenylpropene compounds eugenol and isoeugenol to serve in defense against herbivores and pathogens and to attract pollinators. Clarkia breweri flowers emit a mixture of eugenol and isoeugenol, while Petunia hybrida flowers emit mostly isoeugenol with small amounts of eugenol. We recently reported the identification of a petunia enzyme, PhIGS1, that catalyzes the formation of isoeugenol, and an Ocimum basilicum (basil) enzyme, ObEGS1, that produces eugenol. ObEGS1 and PhIGS1 both utilize coniferyl acetate, are 52% sequence identical, and belong to a family of NADPH-dependent reductases involved in secondary metabolism. Here we show that C. breweri flowers have two closely related proteins (96% identity), CbIGS1 and CbEGS1, that are similar to ObEGS1 (58% and 59%) and catalyze the formation of isoeugenol and eugenol, respectively. In vitro mutagenesis experiments demonstrate that substitution of only a single residue can substantially affect the product specificity of these enzymes. A third C. breweri enzyme identified, CbEGS2, also catalyzes the formation of eugenol from coniferyl acetate and is only 46% identical to CbIGS1 and CbEGS1 but more similar (>70%) to other types of reductases. We also found that petunia flowers contain an enzyme, PhEGS1, that is highly similar to CbEGS2 (82% identity) and that converts coniferyl acetate to eugenol. Our results indicate that plant enzymes with EGS and IGS activities have arisen multiple times and in different protein lineages. PMID:18208524

  1. A phycocyanin·phellandrene synthase fusion enhances recombinant protein expression and β-phellandrene (monoterpene) hydrocarbons production in Synechocystis (cyanobacteria).

    PubMed

    Formighieri, Cinzia; Melis, Anastasios

    2015-11-01

    Cyanobacteria can be exploited as photosynthetic platforms for heterologous generation of terpene hydrocarbons with industrial applications. Transformation of Synechocystis and heterologous expression of the β-phellandrene synthase (PHLS) gene alone is necessary and sufficient to confer to Synechocystis the ability to divert intermediate terpenoid metabolites and to generate the monoterpene β-phellandrene during photosynthesis. However, terpene synthases, including the PHLS, have a slow Kcat (low Vmax) necessitating high levels of enzyme concentration to enable meaningful rates and yield of product formation. Here, a novel approach was applied to increase the PHLS protein expression alleviating limitations in the rate and yield of β-phellandrene product generation. Different PHLS fusion constructs were generated with the Synechocystis endogenous cpcB sequence, encoding for the abundant in cyanobacteria phycocyanin β-subunit, expressed under the native cpc operon promoter. In one of these constructs, the CpcB·PHLS fusion protein accumulated to levels approaching 20% of the total cellular protein, i.e., substantially higher than expressing the PHLS protein alone under the same endogenous cpc promoter. The CpcB·PHLS fusion protein retained the activity of the PHLS enzyme and catalyzed β-phellandrene synthesis, yielding an average of 3.2 mg product g(-1) dry cell weight (dcw) versus the 0.03 mg g(-1)dcw measured with low-expressing constructs, i.e., a 100-fold yield improvement. In conclusion, the terpene synthase fusion-protein approach is promising, as, in this case, it substantially increased the amount of the PHLS in cyanobacteria, and commensurately improved rates and yield of β-phellandrene hydrocarbons production in these photosynthetic microorganisms. PMID:26410450

  2. The Arabidopsis Protein CONSERVED ONLY IN THE GREEN LINEAGE160 Promotes the Assembly of the Membranous Part of the Chloroplast ATP Synthase[W

    PubMed Central

    Rühle, Thilo; Razeghi, Jafar Angouri; Vamvaka, Evgenia; Viola, Stefania; Gandini, Chiara; Kleine, Tatjana; Schünemann, Danja; Barbato, Roberto; Jahns, Peter; Leister, Dario

    2014-01-01

    The chloroplast F1Fo-ATP synthase/ATPase (cpATPase) couples ATP synthesis to the light-driven electrochemical proton gradient. The cpATPase is a multiprotein complex and consists of a membrane-spanning protein channel (comprising subunit types a, b, b′, and c) and a peripheral domain (subunits α, β, γ, δ, and ε). We report the characterization of the Arabidopsis (Arabidopsis thaliana) CONSERVED ONLY IN THE GREEN LINEAGE160 (AtCGL160) protein (AtCGL160), conserved in green algae and plants. AtCGL160 is an integral thylakoid protein, and its carboxyl-terminal portion is distantly related to prokaryotic ATP SYNTHASE PROTEIN1 (Atp1/UncI) proteins that are thought to function in ATP synthase assembly. Plants without AtCGL160 display an increase in xanthophyll cycle activity and energy-dependent nonphotochemical quenching. These photosynthetic perturbations can be attributed to a severe reduction in cpATPase levels that result in increased acidification of the thylakoid lumen. AtCGL160 is not an integral cpATPase component but is specifically required for the efficient incorporation of the c-subunit into the cpATPase. AtCGL160, as well as a chimeric protein containing the amino-terminal part of AtCGL160 and Synechocystis sp. PCC6803 Atp1, physically interact with the c-subunit. We conclude that AtCGL160 and Atp1 facilitate the assembly of the membranous part of the cpATPase in their hosts, but loss of their functions provokes a unique compensatory response in each organism. PMID:24664203

  3. Trapping of intermediates with substrate analog HBOCoA in the polymerizations catalyzed by class III polyhydroxybutyrate (PHB) synthase from Allochromatium vinosum.

    PubMed

    Chen, Chao; Cao, Ruikai; Shrestha, Ruben; Ward, Christina; Katz, Benjamin B; Fischer, Christopher J; Tomich, John M; Li, Ping

    2015-05-15

    Polyhydroxybutyrate (PHB) synthases (PhaCs) catalyze the formation of biodegradable PHB polymers that are considered as an ideal alternative to petroleum-based plastics. To provide strong evidence for the preferred mechanistic model involving covalent and noncovalent intermediates, a substrate analog HBOCoA was synthesized chemoenzymatically. Substitution of sulfur in the native substrate HBCoA with an oxygen in HBOCoA enabled detection of (HB)nOCoA (n = 2-6) intermediates when the polymerization was catalyzed by wild-type (wt-)PhaECAv at 5.84 h(-1). This extremely slow rate is due to thermodynamically unfavorable steps that involve the formation of enzyme-bound PHB species (thioesters) from corresponding CoA oxoesters. Synthesized standards (HB)nOCoA (n = 2-3) were found to undergo both reacylation and hydrolysis catalyzed by the synthase. Distribution of the hydrolysis products highlights the importance of the penultimate ester group as previously suggested. Importantly, the reaction between primed synthase [(3)H]-sT-PhaECAv and HBOCoA yielded [(3)H]-sTet-O-CoA at a rate constant faster than 17.4 s(-1), which represents the first example that a substrate analog undergoes PHB chain elongation at a rate close to that of the native substrate (65.0 s(-1)). Therefore, for the first time with a wt-synthase, strong evidence was obtained to support our favored PHB chain elongation model. PMID:25686368

  4. Trapping of Intermediates with Substrate Analog HBOCoA in the Polymerizations Catalyzed by Class III Polyhydroxybutyrate (PHB) Synthase from Allochromatium Vinosum

    PubMed Central

    Shrestha, Ruben; Ward, Christina; Katz, Benjamin B.; Fischer, Christopher J.; Tomich, John M.; Li, Ping

    2016-01-01

    Polyhydroxybutyrate (PHB) synthases (PhaCs) catalyze the formation of biodegradable PHB polymers that are considered as an ideal alternative to petroleum-based plastics. To provide strong evidence for the preferred mechanistic model involving covalent and noncovalent intermediates, a substrate analog HBOCoA was synthesized chemoenzymatically. Substitution of sulfur in the native substrate HBCoA with an oxygen in HBOCoA enabled detection of (HB)nOCoA (n = 2–6) intermediates when the polymerization was catalyzed by wild-type (wt-)PhaECAv at 5.84 hr−1. This extremely slow rate is due to thermodynamically unfavorable steps that involve formation of enzyme-bound PHB species (thioesters) from corresponding CoA oxoesters. Synthesized standards (HB)nOCoA (n = 2–3) were found to undergo both reacylation and hydrolysis catalyzed by the synthase. Distribution of the hydrolysis products highlights the importance of the penultimate ester group as previously suggested. Importantly, the reaction between primed synthase [3H]-sT-PhaECAv and HBOCoA yielded [3H]-sTet-O-CoA at a rate constant faster than 17.4 s−1, which represents the first example that a substrate analog undergoes PHB chain elongation at a rate close to that of the native substrate (65.0 s−1). Therefore, for the first time with a wt-synthase, strong evidence was obtained to support our favored PHB chain elongation model. PMID:25686368

  5. Tomato Cutin Deficient 1 (CD1) and Putative Orthologs Comprise an Ancient Family of Cutin Synthase-like (CUS) Proteins that are Conserved among Land Plants

    PubMed Central

    Yeats, Trevor H.; Huang, Wenlin; Chatterjee, Subhasish; Viart, Hélène M-F.; Clausen, Mads H.; Stark, Ruth E.; Rose, Jocelyn K.C.

    2014-01-01

    Summary The aerial epidermis of all land plants is covered with a hydrophobic cuticle that provides essential protection from desiccation, and so its evolution is believed to have been prerequisite for terrestrial colonization. A major structural component of apparently all plant cuticles is cutin, a polyester of hydroxy fatty acids. However, despite its ubiquity, the details of cutin polymeric structure and the mechanisms of its formation and remodeling are not well understood. We recently reported that cutin polymerization in tomato (Solanum lycopersicum) fruit occurs via transesterification of hydroxyacylglycerol precursors, catalyzed by the GDSL-motif lipase/hydrolase family protein (GDSL) Cutin Deficient 1 (CD1). Here we present additional biochemical characterization of CD1 and putative orthologs from Arabidopsis thaliana and the moss Physcomitrella patens, which represent a distinct clade of cutin synthases within the large GDSL super-family. We demonstrate that members of this ancient and conserved family of cutin synthase-like (CUS) proteins act as polyester synthases with negligible hydrolytic activity. Moreover, solution-state NMR analysis indicates that CD1 catalyzes the formation of primarily linear cutin oligomeric products in vitro. These results reveal a conserved mechanism of cutin polyester synthesis in land plants, and suggest that elaborations of the linear polymer, such as branching or cross-linking, may require additional, as yet unknown, factors. PMID:24372802

  6. Biological and protein-binding studies of newly synthesized polymer-cobalt(III) complexes.

    PubMed

    Vignesh, G; Pradeep, I; Arunachalam, S; Vignesh, S; Arthur James, R; Arun, R; Premkumar, K

    2016-03-01

    The polymer-cobalt(III) complexes, [Co(bpy)(dien)BPEI]Cl3 · 4H2O (bpy = 2,2'-bipyridine, dien = diethylentriamine, BPEI = branched polyethyleneimine) were synthesized and characterized. The interaction of these complexes with human serum albumin (HSA) and bovine serum albumin (BSA) was investigated under physiological conditions using various physico-chemical techniques. The results reveal that the fluorescence quenching of serum albumins by polymer-cobalt(III) complexes took place through static quenching. The binding of these complexes changed the molecular conformation of the protein considerably. The polymer-cobalt(III) complex with x = 0.365 shows antimicrobial activity against several human pathogens. This complex also induces cytotoxicity against MCF-7 through apoptotic induction. However, further studies are needed to decipher the molecular mode of action of polymer-cobalt(III) complex and for its possible utilization in anticancer therapy. PMID:26278128

  7. A Polyketide Synthase Acyltransferase Domain Structure Suggests a Recognition Mechanism for Its Hydroxymalonyl-Acyl Carrier Protein Substrate

    PubMed Central

    Park, Hyunjun; Kevany, Brian M.; Dyer, David H.; Thomas, Michael G.; Forest, Katrina T.

    2014-01-01

    We have previously shown that the acyl transferase domain of ZmaA (ZmaA-AT) is involved in the biosynthesis of the aminopolyol polyketide/nonribosomal peptide hybrid molecule zwittermicin A from cereus UW85, and that it specifically recognizes the precursor hydroxymalonyl-acyl carrier protein (ACP) and transfers the hydroxymalonyl extender unit to a downstream second ACP via a transacylated AT domain intermediate. We now present the X-ray crystal structure of ZmaA-AT at a resolution of 1.7 Å. The structure shows a patch of solvent-exposed hydrophobic residues in the area where the AT is proposed to interact with the precursor ACP. We addressed the significance of the AT/ACP interaction in precursor specificity of the AT by testing whether malonyl- or methylmalonyl-ACP can be recognized by ZmaA-AT. We found that the ACP itself biases extender unit selection. Until now, structural information for ATs has been limited to ATs specific for the CoA-linked precursors malonyl-CoA and (2S)-methylmalonyl-CoA. This work contributes to polyketide synthase engineering efforts by expanding our knowledge of AT/substrate interactions with the structure of an AT domain that recognizes an ACP-linked substrate, the rare hydroxymalonate. Our structure suggests a model in which ACP interaction with a hydrophobic motif promotes secondary structure formation at the binding site, and opening of the adjacent substrate pocket lid to allow extender unit binding in the AT active site. PMID:25340352

  8. G-rich, a Drosophila selenoprotein, is a Golgi-resident type III membrane protein

    SciTech Connect

    Chen, Chang Lan; Shim, Myoung Sup; Chung, Jiyeol; Yoo, Hyun-Seung; Ha, Ji Min; Kim, Jin Young; Choi, Jinmi; Zang, Shu Liang; Hou, Xiao; Carlson, Bradley A.; Hatfield, Dolph L.; Lee, Byeong Jae . E-mail: imbglmg@plaza.snu.ac.kr

    2006-10-06

    G-rich is a Drosophila melanogaster selenoprotein, which is a homologue of human and mouse SelK. Subcellular localization analysis using GFP-tagged G-rich showed that G-rich was localized in the Golgi apparatus. The fusion protein was co-localized with the Golgi marker proteins but not with an endoplasmic reticulum (ER) marker protein in Drosophila SL2 cells. Bioinformatic analysis of G-rich suggests that this protein is either type II or type III transmembrane protein. To determine the type of transmembrane protein experimentally, GFP-G-rich in which GFP was tagged at the N-terminus of G-rich, or G-rich-GFP in which GFP was tagged at the C-terminus of G-rich, were expressed in SL2 cells. The tagged proteins were then digested with trypsin, and analyzed by Western blot analysis. The results showed that the C-terminus of the G-rich protein was exposed to the cytoplasm indicating it is a type III microsomal membrane protein. G-rich is First selenoprotein identified in the Golgi apparatus.

  9. Positive modulation of RNA polymerase III transcription by ribosomal proteins

    SciTech Connect

    Dieci, Giorgio; Carpentieri, Andrea; Amoresano, Angela; Ottonello, Simone

    2009-02-06

    A yeast nuclear fraction of unknown composition, named TFIIIE, was reported previously to enhance transcription of tRNA and 5S rRNA genes in vitro. We show that TFIIIE activity co-purifies with a specific subset of ribosomal proteins (RPs) which, as revealed by chromatin immunoprecipitation analysis, generally interact with tRNA and 5S rRNA genes, but not with a Pol II-specific promoter. Only Rpl6Ap and Rpl6Bp, among the tested RPs, were found associated to a TATA-containing tRNA{sup Ile}(TAT) gene. The RPL6A gene also emerged as a strong multicopy suppressor of a conditional mutation in the basal transcription factor TFIIIC, while RPL26A and RPL14A behaved as weak suppressors. The data delineate a novel extra-ribosomal role for one or a few RPs which, by influencing 5S rRNA and tRNA synthesis, could play a key role in the coordinate regulation of the different sub-pathways required for ribosome biogenesis and functionality.

  10. Oxygen effect in the radiolysis of proteins. III. Haemoglobin.

    PubMed

    Puchała, M; Schuessler, H

    1993-08-01

    Radiolysis of haemoglobin was carried out in phosphate buffer under air, N2 or N2O and with and without ethanol. Radiation products were separated by SDS-PAGE. The loss of subunits and simultaneous aggregation and fragmentation of haemoglobin was measured, if OH-radicals were unscavenged. There was no sensitizing effect of oxygen on the degradation process. Radiation-induced fragmentation was not a random process, but produced specific fragments. The estimated molecular weights of these fragments gave further support to the assumption that the aminoacyl-proline peptide group is the preferential breaking site if OH radicals react with proteins in the presence of oxygen. In contrast with lactate dehydrogenase and bovine serum albumin such fragmentation was observed not only after aerobic radiolysis but also under anaerobic conditions. This difference must be caused by the Feporphyrin system which reacts with H2O2 under release of oxygen. If haemoglobin was irradiated under air the yield of aggregates was much lower than under N2O or N2. PMID:8103537

  11. The Role of Cysteine Residues in Redox Regulation and Protein Stability of Arabidopsis thaliana Starch Synthase 1

    PubMed Central

    Skryhan, Katsiaryna; Cuesta-Seijo, Jose A.; Nielsen, Morten M.; Marri, Lucia; Mellor, Silas B.; Glaring, Mikkel A.; Jensen, Poul E.; Palcic, Monica M.; Blennow, Andreas

    2015-01-01

    Starch biosynthesis in Arabidopsis thaliana is strictly regulated. In leaf extracts, starch synthase 1 (AtSS1) responds to the redox potential within a physiologically relevant range. This study presents data testing two main hypotheses: 1) that specific thiol-disulfide exchange in AtSS1 influences its catalytic function 2) that each conserved Cys residue has an impact on AtSS1 catalysis. Recombinant AtSS1 versions carrying combinations of cysteine-to-serine substitutions were generated and characterized in vitro. The results demonstrate that AtSS1 is activated and deactivated by the physiological redox transmitters thioredoxin f1 (Trxf1), thioredoxin m4 (Trxm4) and the bifunctional NADPH-dependent thioredoxin reductase C (NTRC). AtSS1 displayed an activity change within the physiologically relevant redox range, with a midpoint potential equal to -306 mV, suggesting that AtSS1 is in the reduced and active form during the day with active photosynthesis. Cys164 and Cys545 were the key cysteine residues involved in regulatory disulfide formation upon oxidation. A C164S_C545S double mutant had considerably decreased redox sensitivity as compared to wild type AtSS1 (30% vs 77%). Michaelis-Menten kinetics and molecular modeling suggest that both cysteines play important roles in enzyme catalysis, namely, Cys545 is involved in ADP-glucose binding and Cys164 is involved in acceptor binding. All the other single mutants had essentially complete redox sensitivity (98–99%). In addition of being part of a redox directed activity “light switch”, reactivation tests and low heterologous expression levels indicate that specific cysteine residues might play additional roles. Specifically, Cys265 in combination with Cys164 can be involved in proper protein folding or/and stabilization of translated protein prior to its transport into the plastid. Cys442 can play an important role in enzyme stability upon oxidation. The physiological and phylogenetic relevance of these findings

  12. Insights into the C-terminal Peptide Binding Specificity of the PDZ Domain of Neuronal Nitric-oxide Synthase: CHARACTERIZATION OF THE INTERACTION WITH THE TIGHT JUNCTION PROTEIN CLAUDIN-3.

    PubMed

    Merino-Gracia, Javier; Costas-Insua, Carlos; Canales, María Ángeles; Rodríguez-Crespo, Ignacio

    2016-05-27

    Neuronal nitric-oxide synthase, unlike its endothelial and inducible counterparts, displays a PDZ (PSD-95/Dlg/ZO-1) domain located at its N terminus involved in subcellular targeting. The C termini of various cellular proteins insert within the binding groove of this PDZ domain and determine the subcellular distribution of neuronal NOS (nNOS). The molecular mechanisms underlying these interactions are poorly understood because the PDZ domain of nNOS can apparently exhibit class I, class II, and class III binding specificity. In addition, it has been recently suggested that the PDZ domain of nNOS binds with very low affinity to the C termini of target proteins, and a necessary simultaneous lateral interaction must take place for binding to occur. We describe herein that the PDZ domain of nNOS can behave as a bona fide class III PDZ domain and bind to C-terminal sequences with acidic residues at the P-2 position with low micromolar binding constants. Binding to C-terminal sequences with a hydrophobic residue at the P-2 position plus an acidic residue at the P-3 position (class II) can also occur, although interactions involving residues extending up to the P-7 position mediate this type of binding. This promiscuous behavior also extends to its association to class I sequences, which must display a Glu residue at P-3 and a Thr residue at P-2 By means of site-directed mutagenesis and NMR spectroscopy, we have been able to identify the residues involved in each specific type of binding and rationalize the mechanisms used to recognize binding partners. Finally, we have analyzed the high affinity association of the PDZ domain of nNOS to claudin-3 and claudin-14, two tight junction tetraspan membrane proteins that are essential components of the paracellular barrier. PMID:27030110

  13. Metabolism of glucose 1,6-P2--III. Partial purification and characterization of glucose 1,6-P2 synthase from pig skeletal muscle.

    PubMed

    Carreras, M; Carreras, J; Climent, F

    1988-01-01

    1. Glycerate 1,3-P2-dependent glucose, 1,6-P2 synthase has been purified 2000-fold from pig skeletal muscle, with a yield of 75%. 2. The enzyme possesses fructose 1,6-P2-dependent glucose 1,6-P2 synthase and phosphoglucomutase activities, which represent 0.1 and 60% of the main activity, respectively. 3. Both glucose 1-P and glucose 6-P can act as acceptors of the phosphoryl group from glycerate 1,3-P2. 4. The Km values are 19 microM and 67 nM for glucose 1-P and glycerate 1,3-P2, respectively. 5. The enzyme is inhibited by glycerate 2,3-P2, fructose 1,6-P2, glycerate 3-P, phosphoenolpyruvate and lithium, the inhibition pattern varying with the compound. PMID:2854765

  14. Endothelial cytosolic proteins bind to the 3' untranslated region of endothelial nitric oxide synthase mRNA: regulation by tumor necrosis factor alpha.

    PubMed Central

    Alonso, J; Sánchez de Miguel, L; Montón, M; Casado, S; López-Farré, A

    1997-01-01

    Changes in endothelial nitric oxide synthase (eNOS) expression may be involved in the endothelium-dependent vasorelaxation dysfunction associated with several vascular diseases. In the present work, we demonstrate that eNOS mRNA contains a previously undescribed cis element in the 3' untranslated region (3' UTR). A U+C-rich segment in the 3' UTR is critical in complex formation with bovine aortic endothelial cell cytosolic proteins. Tumor necrosis factor alpha (TNF-alpha), which destabilizes eNOS mRNA, increased the binding activity of the cytosolic proteins in a time-dependent manner. These data suggest that endothelial cytosolic proteins bind to the 3' UTR of eNOS mRNA. These proteins may play a role in TNF-alpha-induced eNOS mRNA destabilization. PMID:9315630

  15. Endoplasmic Reticulum-Localized Iridium(III) Complexes as Efficient Photodynamic Therapy Agents via Protein Modifications.

    PubMed

    Nam, Jung Seung; Kang, Myeong-Gyun; Kang, Juhye; Park, Sun-Young; Lee, Shin Jung C; Kim, Hyun-Tak; Seo, Jeong Kon; Kwon, Oh-Hoon; Lim, Mi Hee; Rhee, Hyun-Woo; Kwon, Tae-Hyuk

    2016-08-31

    Protein inactivation by reactive oxygen species (ROS) such as singlet oxygen ((1)O2) and superoxide radical (O2(•-)) is considered to trigger cell death pathways associated with protein dysfunction; however, the detailed mechanisms and direct involvement in photodynamic therapy (PDT) have not been revealed. Herein, we report Ir(III) complexes designed for ROS generation through a rational strategy to investigate protein modifications by ROS. The Ir(III) complexes are effective as PDT agents at low concentrations with low-energy irradiation (≤ 1 J cm(-2)) because of the relatively high (1)O2 quantum yield (> 0.78), even with two-photon activation. Furthermore, two types of protein modifications (protein oxidation and photo-cross-linking) involved in PDT were characterized by mass spectrometry. These modifications were generated primarily in the endoplasmic reticulum and mitochondria, producing a significant effect for cancer cell death. Consequently, we present a plausible biologically applicable PDT modality that utilizes rationally designed photoactivatable Ir(III) complexes. PMID:27494510

  16. Chitin synthase III: Synthetic lethal mutants and “stress related” chitin synthesis that bypasses the CSD3/CHS6 localization pathway

    PubMed Central

    Osmond, Barbara C.; Specht, Charles A.; Robbins, Phillips W.

    1999-01-01

    We screened Saccharomyces strains for mutants that are synthetically lethal with deletion of the major chitin synthase gene CHS3. In addition to finding, not surprisingly, that mutations in major cell wall-related genes such as FKS1 (glucan synthase) and mutations in any of the Golgi glycosylation complex genes (MNN9 family) are lethal in combination with chs3Δ, we found that a mutation in Srv2p, a bifunctional regulatory gene, is notably lethal in the chs3 deletion. In extending studies of fks1-chitin synthase 3 interactions, we made the surprising discovery that deletion of CSD3/CHS6, a gene normally required for Chs3p delivery and activity in vivo, was not lethal with fks1 and, in fact, that lack of Csd3p/Chs6p did not decrease the high level of stress-related chitin made in the fks1 mutant. This finding suggests that “stress response” chitin synthesis proceeds through an alternate Chs3p targeting pathway. PMID:10500155

  17. An Arabidopsis callose synthase.

    PubMed

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole; Mundy, John

    2002-08-01

    Beta-1,3-glucan polymers are major structural components of fungal cell walls, while cellulosic beta-1,4-glucan is the predominant polysaccharide in plant cell walls. Plant beta-1,3-glucan, called callose, is produced in pollen and in response to pathogen attack and wounding, but it has been unclear whether callose synthases can also produce cellulose and whether plant cellulose synthases may also produce beta-1,3-glucans. We describe here an Arabidopsis gene, AtGsl5, encoding a plasma membrane-localized protein homologous to yeast beta-1,3-glucan synthase whose expression partially complements a yeast beta-1,3-glucan synthase mutant. AtGsl5 is developmentally expressed at highest levels in flowers, consistent with flowers having high beta-1,3-glucan synthase activities for deposition of callose in pollen. A role for AtGsl5 in callose synthesis is also indicated by AtGsl5 expression in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while expression of the nahG salicylate hydroxylase reduces AtGsl5 mRNA levels in the mpk4 mutant. These results indicate that AtGsl5 is likely involved in callose synthesis in flowering tissues and in the mpk4 mutant. PMID:12081364

  18. Solution structure of monomeric BsaL, the type III secretion needle protein of Burkholderia pseudomallei.

    PubMed

    Zhang, Lingling; Wang, Yu; Picking, Wendy L; Picking, William D; De Guzman, Roberto N

    2006-06-01

    Many gram-negative bacteria that are important human pathogens possess type III secretion systems as part of their required virulence factor repertoire. During the establishment of infection, these pathogens coordinately assemble greater than 20 different proteins into a macromolecular structure that spans the bacterial inner and outer membranes and, in many respects, resembles and functions like a syringe. This type III secretion apparatus (TTSA) is used to inject proteins into a host cell's membrane and cytoplasm to subvert normal cellular processes. The external portion of the TTSA is a needle that is composed of a single type of protein that is polymerized in a helical fashion to form an elongated tube with a central channel of 2-3 nm in diameter. TTSA needle proteins from a variety of bacterial pathogens share sequence conservation; however, no atomic structure for any TTSA needle protein is yet available. Here, we report the structure of a TTSA needle protein called BsaL from Burkholderia pseudomallei determined by nuclear magnetic resonance (NMR) spectroscopy. The central part of the protein assumes a helix-turn-helix core domain with two well-defined alpha-helices that are joined by an ordered, four-residue linker. This forms a two-helix bundle that is stabilized by interhelix hydrophobic contacts. Residues that flank this presumably exposed core region are not completely disordered, but adopt a partial helical conformation. The atomic structure of BsaL and its sequence homology with other TTSA needle proteins suggest potentially unique structural dynamics that could be linked with a universal mechanism for control of type III secretion in diverse gram-negative bacterial pathogens. PMID:16631790

  19. Response of Purkinje neurons to hypobaric hypoxic exposure as shown by alteration in expression of glutamate receptors, nitric oxide synthases and calcium binding proteins.

    PubMed

    Kaur, C; Sivakumar, V; Singh, G; Singh, J; Ling, E A

    2005-01-01

    Hypobaric hypoxia is known to impair muscular coordination. It is not known whether hypobaric hypoxia causes any damage to the Purkinje neurons which may be responsible for impairment of muscular coordination. Expression of ionotropic glutamate receptors N-methyl-d-aspartate receptor subunit 1, amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid GluR2/3, calcium binding proteins and nitric oxide synthases in the Purkinje neurons was examined in rats exposed to hypobaric hypoxia. The mRNA expression of N-methyl-d-aspartate receptor subunit 1, GluR2, GluR3 and nitric oxide synthases [neuronal, endothelial and inducible] was upregulated at 3 h peaking at 24 h after the exposure. This was sustained up to 3 days; thereafter, it was comparable to the controls. Immunohistochemical analysis confirmed a marked expression of N-methyl-d-aspartate receptor subunit 1 and GluR2/3 at the above time intervals. Immunoexpression of calbindin-D28k (calbindin) and parvalbumin was intense in the soma of Purkinje neurons in the control rats. It was, however, drastically downregulated up to 3 days after exposure. At 3 days the neuronal dendrites showed intense expression of calbindin which returned to control levels at 7 days. Expression of neuronal nitric oxide synthase and inducible nitric oxide synthase was markedly upregulated from 3 h to 3 days whereas endothelial nitric oxide synthase expression, localized in the blood vessels and Purkinje neurons, remained elevated up to 24 h after the exposure. A progressive darkening of the Purkinje neuron cell bodies was observed at ultrastructural level up to 3 days but degenerating cells were not observed. A salient alteration was the dilation and stacking of smooth endoplasmic reticulum in the dendrites up to 14 days after the exposure. The present results suggest that hypobaric hypoxia leads to overexpression of N-methyl-d-aspartate receptor subunit 1 and GluR2/3 in Purkinje neurons that may be responsive to altered calcium levels as

  20. Cutting edge: Mouse NAIP1 detects the type III secretion system needle protein.

    PubMed

    Rayamajhi, Manira; Zak, Daniel E; Chavarria-Smith, Joseph; Vance, Russell E; Miao, Edward A

    2013-10-15

    The NAIP/NLRC4 inflammasomes activate caspase-1 in response to bacterial type III secretion systems (T3SSs). Inadvertent injection of the T3SS rod protein and flagellin into the cytosol is detected through murine NAIP2 and NAIP5/6, respectively. In this study, we identify the agonist for the orphan murine NAIP1 receptor as the T3SS needle protein. NAIP1 is poorly expressed in resting mouse bone marrow-derived macrophages; however, priming with polyinosinic-polycytidylic acid induces it and confers needle protein sensitivity. Further, overexpression of NAIP1 in immortalized bone marrow-derived macrophages by retroviral transduction enabled needle detection. In contrast, peritoneal cavity macrophages basally express NAIP1 and respond to needle protein robustly, independent of priming. Human macrophages are known to express only one NAIP gene, which detects the needle protein, but not rod or flagellin. Thus, murine NAIP1 is functionally analogous to human NAIP. PMID:24043898

  1. Inhibitory activity to protein prenylation and antifungal activity of zaragozic acid D3, a potent inhibitor of squalene synthase produced by the fungus, Mollisia sp. SANK 10294.

    PubMed

    Tanimoto, T; Ohya, S; Tsujita, Y

    1998-04-01

    Recently we found novel zaragozic acids (ZAs), F-10863A (zaragozic acid D3, ZAD3), B, C and D in the culture broth of the fungus Mollisia sp. SANK 10294 as potent inhibitors of squalene synthase. There are several other enzymes that use farnesylpyrophosphate as their substrate. Among them we chose farnesyl-protein transferase and examined whether ZAD3 and F-10863B inhibit this enzyme's activity. ZAD3 and F-10863B inhibited farnesyl-protein transferase with IC50 values of 0.60 and 3.7 microM, respectively. They also inhibited geranylgeranyl-protein transferase at similar concentrations. In addition, they exhibited potent antifungal activity. PMID:9630865

  2. Connective tissue-activating peptide III (CTAP-III): cloning the synthetic gene and characterization of the protein expressed in E. coli

    SciTech Connect

    Johnson, P.H.; Castor, C.W.; Walz, D.A.

    1986-05-01

    CTAP-III, an ..cap alpha..-granule protein secreted by human platelets, is known to stimulate mitogenesis, extracellular matrix synthesis, and plasminogen activator synthesis in human fibroblast cultures. From its primary sequence, a synthetic gene was constructed to code for a methionine-free derivative (Leu substituted for Met-21), then cloned and expressed in E. coli using a new expression vector containing regulatory elements of the colicin E1 operon. Partially purified recombinant CTAP-III showed a line of identity with CTAP-III by immunodiffusion against rabbit antibody to platelet-derived CTAP-III. Immunodetection of the reduced protein after SDS-PAGE electrophoresis showed a molecular weight (mobility) in agreement with the natural form. Biologic activity of rCTAP-III eluted from an antiCTAP-III immunoaffinity column was measured in human synovial cell bioassay systems. rCTAP-III stimulated synovial cell synthesis of /sup 14/C-hyaluronic acid approximately 13-fold; significant (P < 0.001) mitogenesis was also observed. These studies indicate that a sufficient quantity of bioactive peptide can be obtained for a more comprehensive study of its biologic properties.

  3. Proteomic Upregulation of Fatty Acid Synthase and Fatty Acid Binding Protein 5 and Identification of Cancer- and Race-Specific Pathway Associations in Human Prostate Cancer Tissues

    PubMed Central

    Myers, Jennifer S.; von Lersner, Ariana K.; Sang, Qing-Xiang Amy

    2016-01-01

    Protein profiling studies of prostate cancer have been widely used to characterize molecular differences between diseased and non-diseased tissues. When combined with pathway analysis, profiling approaches are able to identify molecular mechanisms of prostate cancer, group patients by cancer subtype, and predict prognosis. This strategy can also be implemented to study prostate cancer in very specific populations, such as African Americans who have higher rates of prostate cancer incidence and mortality than other racial groups in the United States. In this study, age-, stage-, and Gleason score-matched prostate tumor specimen from African American and Caucasian American men, along with non-malignant adjacent prostate tissue from these same patients, were compared. Protein expression changes and altered pathway associations were identified in prostate cancer generally and in African American prostate cancer specifically. In comparing tumor to non-malignant samples, 45 proteins were significantly cancer-associated and 3 proteins were significantly downregulated in tumor samples. Notably, fatty acid synthase (FASN) and epidermal fatty acid-binding protein (FABP5) were upregulated in human prostate cancer tissues, consistent with their known functions in prostate cancer progression. Aldehyde dehydrogenase family 1 member A3 (ALDH1A3) was also upregulated in tumor samples. The Metastasis Associated Protein 3 (MTA3) pathway was significantly enriched in tumor samples compared to non-malignant samples. While the current experiment was unable to detect statistically significant differences in protein expression between African American and Caucasian American samples, differences in overrepresentation and pathway enrichment were found. Structural components (Cytoskeletal Proteins and Extracellular Matrix Protein protein classes, and Biological Adhesion Gene Ontology (GO) annotation) were overrepresented in African American but not Caucasian American tumors. Additionally, 5

  4. Protein kinase C and tyrosine kinase pathways regulate lipopolysaccharide-induced nitric oxide synthase activity in RAW 264.7 murine macrophages.

    PubMed Central

    Paul, A; Pendreigh, R H; Plevin, R

    1995-01-01

    1. In RAW 264.7 macrophages, lipopolysaccharide (LPS) and gamma-interferon (IFN gamma) alone or in combination stimulated the induction of nitric oxide synthase (iNOS) activity and increased the expression of the 130 kDa isoform of NOS. 2. LPS-induced NOS activity was reduced by incubation with CD14 neutralising antibodies and abolished in macrophages deprived of serum. 3. LPS stimulated a small increase in protein kinase C (PKC) activity in RAW 264.7 macrophages which was dependent on the presence of serum. However, IFN gamma did not potentiate LPS-stimulated PKC activity. 4. The protein kinase C inhibitor, Ro-318220, abolished both LPS- and IFN gamma-stimulated protein kinase C activity and the induction of NOS activity. 5. LPS- and IFN gamma-induced NOS activity was reduced by the tyrosine kinase inhibitor genestein. Genestein also reduced LPS-stimulated protein kinase C activity but did not affect the response to the protein kinase C activator, tetradecanoylphorbol acetate (TPA). 6. Nicotinamide, an inhibitor of poly-ADP ribosylation, abolished LPS- and IFN gamma-induced NOS activity. 7. Brefeldin A, an inhibitor of a factor which stimulates nucleotide exchange activity on the 21 kDa ADP-ribosylation factor, ARF, reduced LPS- and IFN gamma-induced NOS activity by approximately 80%. 8. These results suggest the involvement of protein kinase C, tyrosine kinase and poly-ADP ribosylation pathways in the regulation of the induction of nitric oxide synthase in RAW 264.7 macrophages by LPS and IFN gamma. Images Figure 2 PMID:7533621

  5. Biophysical Characterization of the Type III Secretion System Translocator Proteins and the Translocator Proteins Attached to Bacterium-Like Particles.

    PubMed

    Chen, Xiaotong; Choudhari, Shyamal P; Kumar, Prashant; Toth, Ronald T; Kim, Jae Hyun; Van Roosmalen, Maarten L; Leenhouts, Kees; Middaugh, C Russell; Picking, Wendy L; Picking, William D

    2015-12-01

    Diarrhea caused by Shigella, Salmonella, and Yersinia is an important public health problem, but development of safe and effective vaccines against such diseases is challenging. A new antigen delivery platform called bacterium-like particles (BLPs) was explored as a means for delivering protective antigens from the type III secretion systems (T3SS) of these pathogens. BLPs are peptidoglycan skeletons derived from Lactococcus lactis that are safe for newborns and can carry multiple antigens. Hydrophobic T3SS translocator proteins were fused to a peptidoglycan anchor (PA) for BLP attachment. The proteins and protein-BLP complexes associated with BLPs were characterized and the resulting data used to create three-index empirical phase diagrams (EPDs). On the basis of these EPDs, IpaB (Shigella) and SipB (Salmonella) behave distinctly from YopB (Yersinia) under different environmental stresses. Adding the PA domain appears to enhance the stability of both the PA and translocator proteins, which was confirmed using differential scanning calorimetry, and although the particles dominated the spectroscopic signals in the protein-loaded BLPs, structural changes in the proteins were still detected. The protein-BLPs were most stable near neutral pH, but these proteins' hydrophobicity made them sensitive to environmental stresses. PMID:26422758

  6. A bacterial type III secretion-based protein delivery tool for broad applications in cell biology

    PubMed Central

    Ittig, Simon J.; Schmutz, Christoph; Kasper, Christoph A.; Amstutz, Marlise; Schmidt, Alexander; Sauteur, Loïc; Vigano, M. Alessandra; Low, Shyan Huey; Affolter, Markus; Cornelis, Guy R.; Nigg, Erich A.

    2015-01-01

    Methods enabling the delivery of proteins into eukaryotic cells are essential to address protein functions. Here we propose broad applications to cell biology for a protein delivery tool based on bacterial type III secretion (T3S). We show that bacterial, viral, and human proteins, fused to the N-terminal fragment of the Yersinia enterocolitica T3S substrate YopE, are effectively delivered into target cells in a fast and controllable manner via the injectisome of extracellular bacteria. This method enables functional interaction studies by the simultaneous injection of multiple proteins and allows the targeting of proteins to different subcellular locations by use of nanobody-fusion proteins. After delivery, proteins can be freed from the YopE fragment by a T3S-translocated viral protease or fusion to ubiquitin and cleavage by endogenous ubiquitin proteases. Finally, we show that this delivery tool is suitable to inject proteins in living animals and combine it with phosphoproteomics to characterize the systems-level impact of proapoptotic human truncated BID on the cellular network. PMID:26598622

  7. Electrostatic Interactions between Elongated Monomers Drive Filamentation of Drosophila Shrub, a Metazoan ESCRT-III Protein.

    PubMed

    McMillan, Brian J; Tibbe, Christine; Jeon, Hyesung; Drabek, Andrew A; Klein, Thomas; Blacklow, Stephen C

    2016-08-01

    The endosomal sorting complex required for transport (ESCRT) is a conserved protein complex that facilitates budding and fission of membranes. It executes a key step in many cellular events, including cytokinesis and multi-vesicular body formation. The ESCRT-III protein Shrub in flies, or its homologs in yeast (Snf7) or humans (CHMP4B), is a critical polymerizing component of ESCRT-III needed to effect membrane fission. We report the structural basis for polymerization of Shrub and define a minimal region required for filament formation. The X-ray structure of the Shrub core shows that individual monomers in the lattice interact in a staggered arrangement using complementary electrostatic surfaces. Mutations that disrupt interface salt bridges interfere with Shrub polymerization and function. Despite substantial sequence divergence and differences in packing interactions, the arrangement of Shrub subunits in the polymer resembles that of Snf7 and other family homologs, suggesting that this intermolecular packing mechanism is shared among ESCRT-III proteins. PMID:27452459

  8. Inhibition of the p53/hDM2 protein-protein interaction by cyclometallated iridium(III) compounds

    PubMed Central

    Liu, Li-Juan; He, Bingyong; Miles, Jennifer A.; Wang, Wanhe; Mao, Zhifeng; Che, Weng Ian; Lu, Jin-Jian; Chen, Xiu-Ping; Wilson, Andrew J.; Ma, Dik-Lung; Leung, Chung-Hang

    2016-01-01

    Inactivation of the p53 transcription factor by mutation or other mechanisms is a frequent event in tumorigenesis. One of the major endogenous negative regulators of p53 in humans is hDM2, a ubiquitin E3 ligase that binds to p53 causing proteasomal p53 degradation. In this work, a library of organometallic iridium(III) compounds were synthesized and evaluated for their ability to disrupt the p53/hDM2 protein-protein interaction. The novel cyclometallated iridium(III) compound 1 [Ir(eppy)2(dcphen)](PF6) (where eppy = 2-(4-ethylphenyl)pyridine and dcphen = 4, 7-dichloro-1, 10-phenanthroline) blocked the interaction of p53/hDM2 in human amelanotic melanoma cells. Finally, 1 exhibited anti-proliferative activity and induced apoptosis in cancer cell lines consistent with inhibition of the p53/hDM2 interaction. Compound 1 represents the first reported organometallic p53/hDM2 protein-protein interaction inhibitor. PMID:26883110

  9. Methylmercury Alters the Activities of Hsp90 Client Proteins, Prostaglandin E Synthase/p23 (PGES/23) and nNOS

    PubMed Central

    Caito, Samuel; Zeng, Heng; Aschner, Judy L.; Aschner, Michael

    2014-01-01

    Methylmercury (MeHg) is a persistent pollutant with known neurotoxic effects. We have previously shown that astrocytes accumulate MeHg and play a prominent role in mediating MeHg toxicity in the central nervous system (CNS) by altering glutamate signaling, generating oxidative stress, depleting glutathione (GSH) and initiating lipid peroxidation. Interestingly, all of these pathways can be regulated by the constitutively expressed, 90-kDa heat shock protein, Hsp90. As Hsp90 function is regulated by oxidative stress, we hypothesized that MeHg disrupts Hsp90-client protein functions. Astrocytes were treated with MeHg and expression of Hsp90, as well as the abundance of complexes of Hsp90-neuronal nitric oxide synthase (nNOS) and Hsp90-prostaglandin E synthase/p23 (PGES/p23) were assessed. MeHg exposure decreased Hsp90 protein expression following 12 h of treatment while shorter exposures had no effect on Hsp90 protein expression. Interestingly, following 1 or 6 h of MeHg exposure, Hsp90 binding to PGES/p23 or nNOS was significantly increased, resulting in increased prostaglandin E2 (PGE2) synthesis from MeHg-treated astrocytes. These effects were attenuated by the Hsp90 antagonist, geldanmycin. NOS activity was increased following MeHg treatment while cGMP formation was decreased. This was accompanied by an increase in •O2− and H2O2 levels, suggesting that MeHg uncouples NO formation from NO-dependent signaling and increases oxidative stress. Altogether, our data demonstrates that Hsp90 interactions with client proteins are increased following MeHg exposure, but over time Hsp90 levels decline, contributing to oxidative stress and MeHg-dependent excitotoxicity. PMID:24852575

  10. Human NAIP and mouse NAIP1 recognize bacterial type III secretion needle protein for inflammasome activation.

    PubMed

    Yang, Jieling; Zhao, Yue; Shi, Jianjin; Shao, Feng

    2013-08-27

    Inflammasome mediated by central nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) protein is critical for defense against bacterial infection. Here we show that type III secretion system (T3SS) needle proteins from several bacterial pathogens, including Salmonella typhimurium, enterohemorrhagic Escherichia coli, Shigella flexneri, and Burkholderia spp., can induce robust inflammasome activation in both human monocyte-derived and mouse bone marrow macrophages. Needle protein activation of human NRL family CARD domain containing 4 (NLRC4) inflammasome requires the sole human neuronal apoptosis inhibitory protein (hNAIP). Among the seven mouse NAIPs, NAIP1 functions as the mouse counterpart of hNAIP. We found that NAIP1 recognition of T3SS needle proteins was more robust in mouse dendritic cells than in bone marrow macrophages. Needle proteins, as well as flagellin and rod proteins from five different bacteria, exhibited differential and cell type-dependent inflammasome-stimulating activity. Comprehensive profiling of the three types of NAIP ligands revealed that NAIP1 sensing of the needle protein dominated S. flexneri-induced inflammasome activation, particularly in dendritic cells. hNAIP/NAIP1 and NAIP2/5 formed a large oligomeric complex with NLRC4 in the presence of corresponding bacterial ligands, and could support reconstitution of the NLRC4 inflammasome in a ligand-specific manner. PMID:23940371

  11. Using Transcriptional Control To Increase Titers of Secreted Heterologous Proteins by the Type III Secretion System

    PubMed Central

    Metcalf, Kevin J.; Finnerty, Casey; Azam, Anum; Valdivia, Elias

    2014-01-01

    The type III secretion system (T3SS) encoded at the Salmonella pathogenicity island 1 (SPI-1) locus secretes protein directly from the cytosol to the culture media in a concerted, one-step process, bypassing the periplasm. While this approach is attractive for heterologous protein production, product titers are too low for many applications. In addition, the expression of the SPI-1 gene cluster is subject to native regulation, which requires culturing conditions that are not ideal for high-density growth. We used transcriptional control to increase the amount of protein that is secreted into the extracellular space by the T3SS of Salmonella enterica. The controlled expression of the gene encoding SPI-1 transcription factor HilA circumvents the requirement of endogenous induction conditions and allows for synthetic induction of the secretion system. This strategy increases the number of cells that express SPI-1 genes, as measured by promoter activity. In addition, protein secretion titer is sensitive to the time of addition and the concentration of inducer for the protein to be secreted and SPI-1 gene cluster. Overexpression of hilA increases secreted protein titer by >10-fold and enables recovery of up to 28 ± 9 mg/liter of secreted protein from an 8-h culture. We also demonstrate that the protein beta-lactamase is able to adopt an active conformation after secretion, and the increase in secreted titer from hilA overexpression also correlates to increased enzyme activity in the culture supernatant. PMID:25038096

  12. Evaluation of Salmonella enterica Type III Secretion System Effector Proteins as Carriers for Heterologous Vaccine Antigens

    PubMed Central

    Hegazy, Wael Abdel Halim; Xu, Xin; Metelitsa, Leonid

    2012-01-01

    Live attenuated strains of Salmonella enterica have a high potential as carriers of recombinant vaccines. The type III secretion system (T3SS)-dependent translocation of S. enterica can be deployed for delivery of heterologous antigens to antigen-presenting cells. Here we investigated the efficacy of various effector proteins of the Salmonella pathogenicity island (SPI2)-encoded T3SS for the translocation of model antigens and elicitation of immune responses. The SPI2 T3SS effector proteins SifA, SteC, SseL, SseJ, and SseF share an endosomal membrane-associated subcellular localization after translocation. We observed that all effector proteins could be used to translocate fusion proteins with the model antigens ovalbumin and listeriolysin into the cytosol of host cells. Under in vitro conditions, fusion proteins with SseJ and SteC stimulated T-cell responses that were superior to those triggered by fusion proteins with SseF. However, in mice vaccinated with Salmonella carrier strains, only fusion proteins based on SseJ or SifA elicited potent T-cell responses. These data demonstrate that the selection of an optimal SPI2 effector protein for T3SS-mediated translocation is a critical parameter for the rational design of effective Salmonella-based recombinant vaccines. PMID:22252866

  13. Maize yellow stripe1 encodes a membrane protein directly involved in Fe(III) uptake.

    PubMed

    Curie, C; Panaviene, Z; Loulergue, C; Dellaporta, S L; Briat, J F; Walker, E L

    2001-01-18

    Frequently, crop plants do not take up adequate amounts of iron from the soil, leading to chlorosis, poor yield and decreased nutritional quality. Extremely limited soil bioavailability of iron has led plants to evolve two distinct uptake strategies: chelation, which is used by the world's principal grain crops; and reduction, which is used by other plant groups. The chelation strategy involves extrusion of low-molecular-mass secondary amino acids (mugineic acids) known as 'phytosiderophores' which chelate sparingly soluble iron. The Fe(III)-phytosiderophore complex is then taken up by an unknown transporter at the root surface. The maize yellow stripe1 (ys1) mutant is deficient in Fe(III)-phytosiderophore uptake, therefore YS1 has been suggested to be the Fe(III)-phytosiderophore transporter. Here we show that ys1 is a membrane protein that mediates iron uptake. Expression of YS1 in a yeast iron uptake mutant restores growth specifically on Fe(III)-phytosiderophore media. Under iron-deficient conditions, ys1 messenger RNA levels increase in both roots and shoots. Cloning of ys1 is an important step in understanding iron uptake in grasses, and has implications for mechanisms controlling iron homeostasis in all plants. PMID:11201743

  14. Mumps Virus Induces Protein-Kinase-R-Dependent Stress Granules, Partly Suppressing Type III Interferon Production.

    PubMed

    Hashimoto, Shin; Yamamoto, Soh; Ogasawara, Noriko; Sato, Toyotaka; Yamamoto, Keisuke; Katoh, Hiroshi; Kubota, Toru; Shiraishi, Tsukasa; Kojima, Takashi; Himi, Tetsuo; Tsutsumi, Hiroyuki; Yokota, Shin-Ichi

    2016-01-01

    Stress granules (SGs) are cytoplasmic granular aggregations that are induced by cellular stress, including viral infection. SGs have opposing antiviral and proviral roles, which depend on virus species. The exact function of SGs during viral infection is not fully understood. Here, we showed that mumps virus (MuV) induced SGs depending on activation of protein kinase R (PKR). MuV infection strongly induced interferon (IFN)-λ1, 2 and 3, and IFN-β through activation of IFN regulatory factor 3 (IRF3) via retinoic acid inducible gene-I (RIG-I) and the mitochondrial antiviral signaling (MAVS) pathway. MuV-induced IFNs were strongly upregulated in PKR-knockdown cells. MuV-induced SG formation was suppressed by knockdown of PKR and SG marker proteins, Ras-GTPase-activating protein SH3-domain-binding protein 1 and T-cell-restricted intracellular antigen-1, and significantly increased the levels of MuV-induced IFN-λ1. However, viral titer was not altered by suppression of SG formation. PKR was required for induction of SGs by MuV infection and regulated type III IFN (IFN-λ1) mRNA stability. MuV-induced SGs partly suppressed type III IFN production by MuV; however, the limited suppression was not sufficient to inhibit MuV replication in cell culture. Our results provide insight into the relationship between SGs and IFN production induced by MuV infection. PMID:27560627

  15. Mumps Virus Induces Protein-Kinase-R-Dependent Stress Granules, Partly Suppressing Type III Interferon Production

    PubMed Central

    Hashimoto, Shin; Yamamoto, Soh; Ogasawara, Noriko; Sato, Toyotaka; Yamamoto, Keisuke; Katoh, Hiroshi; Kubota, Toru; Shiraishi, Tsukasa; Kojima, Takashi; Himi, Tetsuo; Tsutsumi, Hiroyuki; Yokota, Shin-ichi

    2016-01-01

    Stress granules (SGs) are cytoplasmic granular aggregations that are induced by cellular stress, including viral infection. SGs have opposing antiviral and proviral roles, which depend on virus species. The exact function of SGs during viral infection is not fully understood. Here, we showed that mumps virus (MuV) induced SGs depending on activation of protein kinase R (PKR). MuV infection strongly induced interferon (IFN)-λ1, 2 and 3, and IFN-β through activation of IFN regulatory factor 3 (IRF3) via retinoic acid inducible gene-I (RIG-I) and the mitochondrial antiviral signaling (MAVS) pathway. MuV-induced IFNs were strongly upregulated in PKR-knockdown cells. MuV-induced SG formation was suppressed by knockdown of PKR and SG marker proteins, Ras-GTPase-activating protein SH3-domain-binding protein 1 and T-cell-restricted intracellular antigen-1, and significantly increased the levels of MuV-induced IFN-λ1. However, viral titer was not altered by suppression of SG formation. PKR was required for induction of SGs by MuV infection and regulated type III IFN (IFN-λ1) mRNA stability. MuV-induced SGs partly suppressed type III IFN production by MuV; however, the limited suppression was not sufficient to inhibit MuV replication in cell culture. Our results provide insight into the relationship between SGs and IFN production induced by MuV infection. PMID:27560627

  16. Development of a biomarker for Geobacter activity and strain composition: Proteogenomic analysis of the citrate synthase protein during bioremediation of U(VI)

    SciTech Connect

    Wilkins, M.J.; Callister, S.J.; Miletto, M.; Williams, K.H.; Nicora, C.D.; Lovley, D.R.; Long, P.E.; Lipton, M.S.

    2010-02-15

    Monitoring the activity of target microorganisms during stimulated bioremediation is a key problem for the development of effective remediation strategies. At the US Department of Energy's Integrated Field Research Challenge (IFRC) site in Rifle, CO, the stimulation of Geobacter growth and activity via subsurface acetate addition leads to precipitation of U(VI) from groundwater as U(IV). Citrate synthase (gltA) is a key enzyme in Geobacter central metabolism that controls flux into the TCA cycle. Here, we utilize shotgun proteomic methods to demonstrate that the measurement of gltA peptides can be used to track Geobacter activity and strain evolution during in situ biostimulation. Abundances of conserved gltA peptides tracked Fe(III) reduction and changes in U(VI) concentrations during biostimulation, whereas changing patterns of unique peptide abundances between samples suggested sample-specific strain shifts within the Geobacter population. Abundances of unique peptides indicated potential differences at the strain level between Fe(III)-reducing populations stimulated during in situ biostimulation experiments conducted a year apart at the Rifle IFRC. These results offer a novel technique for the rapid screening of large numbers of proteomic samples for Geobacter species and will aid monitoring of subsurface bioremediation efforts that rely on metal reduction for desired outcomes.

  17. Development of a biomarker for Geobacter activity and strain composition; Proteogenomic analysis of the citrate synthase protein during bioremediation of U(VI).

    SciTech Connect

    Wilkins, Michael J.; Callister, Stephen J.; Miletto, Marzia; Williams, Kenneth H.; Nicora, Carrie D.; Lovely, Derek R.; Long, Philip E.; Lipton, Mary S.

    2011-01-01

    Monitoring the activity of target microorganisms during stimulated bioremediation is a key problem for the development of effective remediation strategies. At the U.S. Department of Energy’s Integrated Field Research Challenge (IFRC) site in Rifle, CO, the stimulation of Geobacter growth and activity via subsurface acetate addition leads to precipitation of U(VI) from groundwater as U(IV). Citrate synthase (gltA) is a key enzyme in Geobacter central metabolism that controls flux into the TCA cycle. Here, we utilize shotgun proteomic methods to demonstrate that the measurement of gltA peptides can be used to track Geobacter activity and strain evolution during in situ biostimulation. Abundances of conserved gltA peptides tracked Fe(III) reduction and changes in U(VI) concentrations during biostimulation, whereas changing patterns of unique peptide abundances between samples suggested sample-specific strain shifts within the Geobacter population. Abundances of unique peptides indicated potential differences at the strain level between Fe(III)-reducing populations stimulated during in situ biostimulation experiments conducted a year apart at the Rifle IFRC. These results offer a novel technique for the rapid screening of large numbers of proteomic samples for Geobacter species and will aid monitoring of subsurface bioremediation efforts that rely on metal reduction for desired outcomes.

  18. Use of proline mutants to help solve the NMR solution structure of type III antifreeze protein.

    PubMed Central

    Chao, H.; Davies, P. L.; Sykes, B. D.; Sönnichsen, F. D.

    1993-01-01

    To help understand the structure/function relationships in antifreeze proteins (AFP), and to define the motifs required for ice binding, a Type III AFP suitable for two-dimensional (2D) NMR studies was produced in Escherichia coli. A synthetic gene for one of the Type III AFP isoforms was assembled in a T7 polymerase-directed expression vector. The 67-amino acid-long gene product differed from the natural AFP by inclusion of an N-terminal methionine but was indistinguishable in activity. The NMR spectra of this AFP were complicated by cis-trans proline isomerization from the C-terminal sequence YPPA. Substitution of this sequence by YAA eliminated isomer signals without altering the activity or structure of the mutant AFP. This variant (rQAE m1.1) was selected for sequential assignment and the secondary structure determination using 2D 1H NMR spectroscopy. Nine beta-strands are paired to form two triple-stranded antiparallel sheets and one double-stranded antiparallel sheet. Two further proline replacements, P29A and P33A, were made to delineate the role of conserved prolines in Type III AFP. These mutants were valuable in clarifying ambiguous NMR spectral assignments amongst the remaining six prolines of rQAE m1.1. In contrast to the replacement of the C-terminal prolyl residues, the exchange of P29 and P33 caused some structural changes and significantly decreased protein solubility and antifreeze activity. PMID:8401227

  19. Invertebrate and Vertebrate Class III Myosins Interact with MORN Repeat-Containing Adaptor Proteins

    PubMed Central

    Mecklenburg, Kirk L.; Freed, Stephanie A.; Raval, Manmeet; Quintero, Omar A.; Yengo, Christopher M.; O'Tousa, Joseph. E.

    2015-01-01

    In Drosophila photoreceptors, the NINAC-encoded myosin III is found in a complex with a small, MORN-repeat containing, protein Retinophilin (RTP). Expression of these two proteins in other cell types showed NINAC myosin III behavior is altered by RTP. NINAC deletion constructs were used to map the RTP binding site within the proximal tail domain of NINAC. In vertebrates, the RTP ortholog is MORN4. Co-precipitation experiments demonstrated that human MORN4 binds to human myosin IIIA (MYO3A). In COS7 cells, MORN4 and MYO3A, but not MORN4 and MYO3B, co-localize to actin rich filopodia extensions. Deletion analysis mapped the MORN4 binding to the proximal region of the MYO3A tail domain. MYO3A dependent MORN4 tip localization suggests that MYO3A functions as a motor that transports MORN4 to the filopodia tips and MORN4 may enhance MYO3A tip localization by tethering it to the plasma membrane at the protrusion tips. These results establish conserved features of the RTP/MORN4 family: they bind within the tail domain of myosin IIIs to control their behavior. PMID:25822849

  20. a Computational Approach to Explore Protein Translocation Through Type III Secretion Apparatus

    NASA Astrophysics Data System (ADS)

    Rathinavelan, Thenmalarchelvi; Im, Wonpil

    2010-01-01

    Many Gram-negative bacteria initiate infections by injecting effector proteins into host cells through the type III secretion apparatus (TTSA) that is comprised of a basal body, a needle, and a tip. The needle channel is formed by the assembly of a single needle protein. To explore the export mechanisms of MxiH needle protein through the needle of Shigella flexneri, an essential step during needle assembly, we have performed steered molecular dynamics simulations in implicit solvent. Interestingly, the electronegative channel interior creates an energy barrier for MxiH to enter the channel, while the same may facilitate the ejection of the effectors into host cells. Structurally-known basal regions and ATPase underneath the basal region have also such electronegative interior, while effector proteins have considerable electronegative patches on their surfaces. Based on these observations, we propose a repulsive electrostatic mechanism for protein translocation through the TTSA. This mechanism is supported by the suggestion that an ATPase is required for protein translocation through these nanomachines, which may provide the energy to overcome the initial electrostatic energy barrier. A similar mechanism may be applicable to macromolecular channels in other secretion systems or viruses through which proteins or nucleic acids are transported.

  1. Zaragozic acids D and D2: potent inhibitors of squalene synthase and of Ras farnesyl-protein transferase.

    PubMed

    Dufresne, C; Wilson, K E; Singh, S B; Zink, D L; Bergstrom, J D; Rew, D; Polishook, J D; Meinz, M; Huang, L; Silverman, K C

    1993-11-01

    Two new zaragozic acids, D and D2, have been isolated from the keratinophilic fungus Amauroascus niger. Zaragozic acids D [4] and D2 [5] are related to the previously described zaragozic acids A [1], B [2], and C [3] and are potent inhibitors of squalene synthase. Furthermore, all the zaragozic acids (A, B, C, D, and D2) are also active against farnesyl transferase. Zaragozic acids D and D2 inhibit farnesyl transferase with IC50 values of 100 nM, while zaragozic acids A and B are less potent. PMID:8289063

  2. LAP5 and LAP6 encode anther-specific proteins with similarity to chalcone synthase essential for pollen exine development in Arabidopsis.

    PubMed

    Dobritsa, Anna A; Lei, Zhentian; Nishikawa, Shuh-Ichi; Urbanczyk-Wochniak, Ewa; Huhman, David V; Preuss, Daphne; Sumner, Lloyd W

    2010-07-01

    Pollen grains of land plants have evolved remarkably strong outer walls referred to as exine that protect pollen and interact with female stigma cells. Exine is composed of sporopollenin, and while the composition and synthesis of this biopolymer are not well understood, both fatty acids and phenolics are likely components. Here, we describe mutations in the Arabidopsis (Arabidopsis thaliana) LESS ADHESIVE POLLEN (LAP5) and LAP6 that affect exine development. Mutation of either gene results in abnormal exine patterning, whereas pollen of double mutants lacked exine deposition and subsequently collapsed, causing male sterility. LAP5 and LAP6 encode anther-specific proteins with homology to chalcone synthase, a key flavonoid biosynthesis enzyme. lap5 and lap6 mutations reduced the accumulation of flavonoid precursors and flavonoids in developing anthers, suggesting a role in the synthesis of phenolic constituents of sporopollenin. Our in vitro functional analysis of LAP5 and LAP6 using 4-coumaroyl-coenzyme A yielded bis-noryangonin (a commonly reported derailment product of chalcone synthase), while similar in vitro analyses using fatty acyl-coenzyme A as the substrate yielded medium-chain alkyl pyrones. Thus, in vitro assays indicate that LAP5 and LAP6 are multifunctional enzymes and may play a role in both the synthesis of pollen fatty acids and phenolics found in exine. Finally, the genetic interaction between LAP5 and an anther gene involved in fatty acid hydroxylation (CYP703A2) demonstrated that they act synergistically in exine production. PMID:20442277

  3. Optimized green fluorescent protein fused to FoF1-ATP synthase for single-molecule FRET using a fast anti-Brownian electrokinetic trap

    NASA Astrophysics Data System (ADS)

    Dienerowitz, Maria; Ilchenko, Mykhailo; Su, Bertram; Deckers-Hebestreit, Gabriele; Mayer, Günter; Henkel, Thomas; Heitkamp, Thomas; Börsch, Michael

    2016-02-01

    Observation times of freely diffusing single molecules in solution are limited by the photophysics of the attached fluorescence markers and by a small observation volume in the femtolitre range that is required for a sufficient signal-to-background ratio. To extend diffusion-limited observation times through a confocal detection volume, A. E. Cohen and W. E. Moerner have invented and built the ABELtrap -- a microfluidic device to actively counteract Brownian motion of single nanoparticles with an electrokinetic trap. Here we present a version of an ABELtrap with a laser focus pattern generated by electro-optical beam deflectors and controlled by a programmable FPGA chip. This ABELtrap holds single fluorescent nanoparticles for more than 100 seconds, increasing the observation time of fluorescent nanoparticles compared to free diffusion by a factor of 10000. To monitor conformational changes of individual membrane proteins in real time, we record sequential distance changes between two specifically attached dyes using Förster resonance energy transfer (smFRET). Fusing the a-subunit of the FoF1-ATP synthase with mNeonGreen results in an improved signal-to-background ratio at lower laser excitation powers. This increases our measured trap duration of proteoliposomes beyond 2 s. Additionally, we observe different smFRET levels attributed to varying distances between the FRET donor (mNeonGreen) and acceptor (Alexa568) fluorophore attached at the a- and c-subunit of the FoF1-ATP synthase respectively.

  4. THI1, a Thiamine Thiazole Synthase, Interacts with Ca2+-Dependent Protein Kinase CPK33 and Modulates the S-Type Anion Channels and Stomatal Closure in Arabidopsis.

    PubMed

    Li, Chun-Long; Wang, Mei; Wu, Xiao-Meng; Chen, Dong-Hua; Lv, Hong-Jun; Shen, Jian-Lin; Qiao, Zhu; Zhang, Wei

    2016-02-01

    Thiamine is required for both plant growth and development. Here, the involvement of a thiamine thiazole synthase, THI1, has been demonstrated in both guard cell abscisic acid (ABA) signaling and the drought response in Arabidopsis (Arabidopsis thaliana). THI1 overexpressors proved to be more sensitive to ABA than the wild type with respect to both the activation of guard cell slow type anion channels and stomatal closure; this effectively reduced the rate of water loss from the plant and thereby enhanced its level of drought tolerance. A yeast two-hybrid strategy was used to screen a cDNA library from epidermal strips of leaves for THI1 regulatory factors, and identified CPK33, a Ca(2+)-dependent protein kinase, as interactor with THI1 in a plasma membrane-delimited manner. Loss-of-function cpk33 mutants were hypersensitive to ABA activation of slow type anion channels and ABA-induced stomatal closure, while the CPK33 overexpression lines showed opposite phenotypes. CPK33 kinase activity was essential for ABA-induced stomatal closure. Consistent with their contrasting regulatory role over stomatal closure, THI1 suppressed CPK33 kinase activity in vitro. Together, our data reveal a novel regulatory role of thiamine thiazole synthase to kinase activity in guard cell signaling. PMID:26662273

  5. Differential Expression of 1-Aminocyclopropane-1-Carboxylate Synthase Genes during Orchid Flower Senescence Induced by the Protein Phosphatase Inhibitor Okadaic Acid1

    PubMed Central

    Wang, Ning Ning; Yang, Shang Fa; Charng, Yee-yung

    2001-01-01

    Applying 10 pmol of okadaic acid (OA), a specific inhibitor of type 1 or type 2A serine/threonine protein phosphatases, to the orchid (Phalaenopsis species) stigma induced a dramatic increase in ethylene production and an accelerated senescence of the whole flower. Aminoethoxyvinylglycine or silver thiosulfate, inhibitors of ethylene biosynthesis or action, respectively, effectively inhibited the OA-induced ethylene production and retarded flower senescence, suggesting that the protein phosphatase inhibitor induced orchid flower senescence through an ethylene-mediated signaling pathway. OA treatment induced a differential expression pattern for the 1-aminocyclopropane-1-carboxylic acid synthase multigene family. Accumulation of Phal-ACS1 transcript in the stigma, labelum, and ovary induced by OA were higher than those induced by pollination as determined by “semiquantitative” reverse transcriptase-polymerase chain reaction. In contrast, the transcript levels of Phal-ACS2 and Phal-ACS3 induced by OA were much lower than those induced by pollination. Staurosporine, a protein kinase inhibitor, on the other hand, inhibited the OA-induced Phal-ACS1 expression in the stigma and delayed flower senescence. Our results suggest that a hyper-phosphorylation status of an unidentified protein(s) is involved in up-regulating the expression of Phal-ACS1 gene resulting in increased ethylene production and accelerated the senescence process of orchid flower. PMID:11351088

  6. A Medicago truncatula Cystathionine-β-Synthase-like Domain-Containing Protein Is Required for Rhizobial Infection and Symbiotic Nitrogen Fixation.

    PubMed

    Sinharoy, Senjuti; Liu, Chengwu; Breakspear, Andrew; Guan, Dian; Shailes, Sarah; Nakashima, Jin; Zhang, Shulan; Wen, Jiangqi; Torres-Jerez, Ivone; Oldroyd, Giles; Murray, Jeremy D; Udvardi, Michael K

    2016-04-01

    The symbiosis between leguminous plants and soil rhizobia culminates in the formation of nitrogen-fixing organs called nodules that support plant growth. Two Medicago truncatula Tnt1-insertion mutants were identified that produced small nodules, which were unable to fix nitrogen effectively due to ineffective rhizobial colonization. The gene underlying this phenotype was found to encode a protein containing a putative membrane-localized domain of unknown function (DUF21) and a cystathionine-β-synthase domain. The cbs1 mutants had defective infection threads that were sometimes devoid of rhizobia and formed small nodules with greatly reduced numbers of symbiosomes. We studied the expression of the gene, designated M truncatula Cystathionine-β-Synthase-like1 (MtCBS1), using a promoter-β-glucuronidase gene fusion, which revealed expression in infected root hair cells, developing nodules, and in the invasion zone of mature nodules. An MtCBS1-GFP fusion protein localized itself to the infection thread and symbiosomes. Nodulation factor-induced Ca(2+) responses were observed in the cbs1 mutant, indicating that MtCBS1 acts downstream of nodulation factor signaling. MtCBS1 expression occurred exclusively during Medicago-rhizobium symbiosis. Induction of MtCBS1 expression during symbiosis was found to be dependent on Nodule Inception (NIN), a key transcription factor that controls both rhizobial infection and nodule organogenesis. Interestingly, the closest homolog of MtCBS1, MtCBS2, was specifically induced in mycorrhizal roots, suggesting common infection mechanisms in nodulation and mycorrhization. Related proteins in Arabidopsis have been implicated in cell wall maturation, suggesting a potential role for CBS1 in the formation of the infection thread wall. PMID:26884486

  7. A Medicago truncatula Cystathionine-β-Synthase-like Domain-Containing Protein Is Required for Rhizobial Infection and Symbiotic Nitrogen Fixation1[OPEN

    PubMed Central

    Breakspear, Andrew; Guan, Dian; Nakashima, Jin; Zhang, Shulan; Torres-Jerez, Ivone; Oldroyd, Giles; Murray, Jeremy D.; Udvardi, Michael K.

    2016-01-01

    The symbiosis between leguminous plants and soil rhizobia culminates in the formation of nitrogen-fixing organs called nodules that support plant growth. Two Medicago truncatula Tnt1-insertion mutants were identified that produced small nodules, which were unable to fix nitrogen effectively due to ineffective rhizobial colonization. The gene underlying this phenotype was found to encode a protein containing a putative membrane-localized domain of unknown function (DUF21) and a cystathionine-β-synthase domain. The cbs1 mutants had defective infection threads that were sometimes devoid of rhizobia and formed small nodules with greatly reduced numbers of symbiosomes. We studied the expression of the gene, designated M. truncatula Cystathionine-β-Synthase-like1 (MtCBS1), using a promoter-β-glucuronidase gene fusion, which revealed expression in infected root hair cells, developing nodules, and in the invasion zone of mature nodules. An MtCBS1-GFP fusion protein localized itself to the infection thread and symbiosomes. Nodulation factor-induced Ca2+ responses were observed in the cbs1 mutant, indicating that MtCBS1 acts downstream of nodulation factor signaling. MtCBS1 expression occurred exclusively during Medicago-rhizobium symbiosis. Induction of MtCBS1 expression during symbiosis was found to be dependent on Nodule Inception (NIN), a key transcription factor that controls both rhizobial infection and nodule organogenesis. Interestingly, the closest homolog of MtCBS1, MtCBS2, was specifically induced in mycorrhizal roots, suggesting common infection mechanisms in nodulation and mycorrhization. Related proteins in Arabidopsis have been implicated in cell wall maturation, suggesting a potential role for CBS1 in the formation of the infection thread wall. PMID:26884486

  8. Molecular evolution and sequence divergence of plant chalcone synthase and chalcone synthase-Like genes.

    PubMed

    Han, Yingying; Zhao, Wenwen; Wang, Zhicui; Zhu, Jingying; Liu, Qisong

    2014-06-01

    Plant chalcone synthase (CHS) and CHS-Like (CHSL) proteins are polyketide synthases. In this study, we evaluated the molecular evolution of this gene family using representative types of CHSL genes, including stilbene synthase (STS), 2-pyrone synthase (2-PS), bibenzyl synthase (BBS), acridone synthase (ACS), biphenyl synthase (BIS), benzalacetone synthase, coumaroyl triacetic acid synthase (CTAS), and benzophenone synthase (BPS), along with their CHS homologs from the same species of both angiosperms and gymnosperms. A cDNA-based phylogeny indicated that CHSLs had diverse evolutionary patterns. STS, ACS, and 2-PS clustered with CHSs from the same species (late diverged pattern), while CTAS, BBS, BPS, and BIS were distant from their CHS homologs (early diverged pattern). The amino-acid phylogeny suggested that CHS and CHSL proteins formed clades according to enzyme function. The CHSs and CHSLs from Polygonaceae and Arachis had unique evolutionary histories. Synonymous mutation rates were lower in late diverged CHSLs than in early diverged ones, indicating that gene duplications occurred more recently in late diverged CHSLs than in early diverged ones. Relative rate tests proved that late diverged CHSLs had unequal rates to CHSs from the same species when using fatty acid synthase, which evolved from the common ancestor with the CHS superfamily, as the outgroup, while the early diverged lineages had equal rates. This indicated that late diverged CHSLs experienced more frequent mutation than early diverged CHSLs after gene duplication, allowing obtaining new functions in relatively short period of time. PMID:24849013

  9. Luminescent Alkyne-Bearing Terbium(III) Complexes and Their Application to Bioorthogonal Protein Labeling.

    PubMed

    O'Malley, William I; Abdelkader, Elwy H; Aulsebrook, Margaret L; Rubbiani, Riccardo; Loh, Choy-Theng; Grace, Michael R; Spiccia, Leone; Gasser, Gilles; Otting, Gottfried; Tuck, Kellie L; Graham, Bim

    2016-02-15

    Two new bifunctional macrocyclic chelate ligands that form luminescent terbium(III) complexes featuring an alkyne group for conjugation to (bio)molecules via the Cu(I)-catalyzed "click" reaction were synthesized. Upon ligation, the complexes exhibit a significant luminescent enhancement when excited at the λ(max) of the "clicked" products. To demonstrate the utility of the complexes for luminescent labeling, they were conjugated in vitro to E. coli aspartate/glutamate-binding protein incorporating a genetically encoded p-azido-L-phenylalanine or p-(azidomethyl)-L-phenylalanine residue. The complexes may prove useful for time-gated assay applications. PMID:26821062

  10. Specificity of a protein phosphatase inhibitor from rabbit skeletal muscle.

    PubMed Central

    Cohen, P; Nimmo, G A; Antoniw, J F

    1977-01-01

    A hear-stable protein, which is a specific inhibitor of protein phosphatase-III, was purified 700-fold from skeletal muscle by a procedure that involved heat-treatment at 95 degrees C, chromatography on DEAE-cellulose and gel filtration on Sephadex G-100. The final step completely resolved the protein phosphatase inhibitor from the protein inhibitor of cyclic AMP-dependent protein kinase. The phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities of protein phosphatase-III [Antoniw, J. F., Nimmo, H. G., Yeaman, S. J. & Cohen, P.(1977) Biochem.J. 162, 423-433] were inhibited in a very similar manner by the protein phosphatase inhibitor and at least 95% inhibition was observed at high concentrations of inhibitor. The two forms of protein phosphatase-III, termed IIIA and IIIB, were equally susceptible to the protein phosphatase inhibitor. The protein phosphatase inhibitor was at least 200 times less effective in inhibiting the activity of protein phosphatase-I and protein phosphatase-II. The high degree of specificity of the inhibitor for protein phosphatase-III was used to show that 90% of the phosphorylase phosphatase and glycogen synthase phosphatase activities measured in muscle extracts are catalysed by protein phosphatase-III. Protein phosphatase-III was tightly associated with the protein-glycogen complex that can be isolated from skeletal muscle, whereas the protein phosphatase inhibitor and protein phosphatase-II were not. The results provide further evidence that the enzyme that catalyses the dephosphorylation of the alpha-subunit of phosphorylase kinase (protein phosphatase-II) and the enzyme that catalyses the dephosphorylation of the beta-subunit of phosphorylase kinase (protein phosphatase-III) are distinct. The results suggest that the protein phosphatase inhibitor may be a useful probe for differentiating different classes of protein phosphatases in mammalian

  11. A Ser/Thr protein kinase phosphorylates MA-ACS1 (Musa acuminata 1-aminocyclopropane-1-carboxylic acid synthase 1) during banana fruit ripening.

    PubMed

    Choudhury, Swarup Roy; Roy, Sujit; Sengupta, Dibyendu N

    2012-08-01

    1-Aminocyclopropane-1-carboxylic acid synthase (ACS) catalyzes the rate-limiting step in ethylene biosynthesis during ripening. ACS isozymes are regulated both transcriptionally and post-translationally. However, in banana, an important climacteric fruit, little is known about post-translational regulation of ACS. Here, we report the post-translational modification of MA-ACS1 (Musa acuminata ACS1), a ripening inducible isozyme in the ACS family, which plays a key role in ethylene biosynthesis during banana fruit ripening. Immunoprecipitation analyses of phospholabeled protein extracts from banana fruit using affinity-purified anti-MA-ACS1 antibody have revealed phosphorylation of MA-ACS1, particularly in ripe fruit tissue. We have identified the induction of a 41-kDa protein kinase activity in pulp at the onset of ripening. The 41-kDa protein kinase has been identified as a putative protein kinase by MALDI-TOF/MS analysis. Biochemical analyses using partially purified protein kinase fraction from banana fruit have identified the protein kinase as a Ser/Thr family of protein kinase and its possible involvement in MA-ACS1 phosphorylation during ripening. In vitro phosphorylation analyses using synthetic peptides and site-directed mutagenized recombinant MA-ACS1 have revealed that serine 476 and 479 residues at the C-terminal region of MA-ACS1 are phosphorylated. Overall, this study provides important novel evidence for in vivo phosphorylation of MA-ACS1 at the molecular level as a possible mechanism of post-translational regulation of this key regulatory protein in ethylene signaling pathway in banana fruit during ripening. PMID:22419220

  12. Deletion of the N- or C-Terminal Helix of Apolipophorin III To Create a Four-Helix Bundle Protein.

    PubMed

    Dwivedi, Pankaj; Rodriguez, Johana; Ibe, Nnejiuwa U; Weers, Paul M M

    2016-07-01

    Apolipophorin III (apoLp-III) is an exchangeable apolipoprotein found in insects and plays an important function in lipid transport. The protein has an unusual five-helix bundle architecture, deviating from the common four-helix bundle motif. To understand the role of the additional helix in apoLp-III, the N-terminal or C-terminal helix was deleted to create a putative four-helix bundle protein. While the protein lacking helix-1 could be expressed in bacteria albeit at reduced yields, apoLp-III lacking helix-5 could not be produced. Mutational analysis by truncating helix-5 showed that a minimum segment of approximately one-third of the C-terminal helix is required for protein expression. The variant lacking helix-5 was produced by inserting a methionine residue between helix-4 and -5; subsequent cyanogenbromide cleavage generated the four-helix variant. Both N- and C-terminal helix deletion variants displayed significantly reduced helical content, protein stability, and tertiary structure. Despite the significantly altered structure, the variants were still fully functional. The rate of dimyristoylphosphatidylcholine vesicle solubilization was enhanced 4-5-fold compared to the wild-type protein, and the deletion variants were effective in binding to lipolyzed low density lipoprotein thereby preventing lipoprotein aggregation. These results show that the additional helix of apoLp-III is not essential for lipid binding but is required for proper folding to keep the protein into a stable conformation. PMID:27280697

  13. ESCRT-III drives the final stages of CUPS maturation for unconventional protein secretion.

    PubMed

    Curwin, Amy J; Brouwers, Nathalie; Alonso Y Adell, Manuel; Teis, David; Turacchio, Gabriele; Parashuraman, Seetharaman; Ronchi, Paolo; Malhotra, Vivek

    2016-01-01

    The unconventional secretory pathway exports proteins that bypass the endoplasmic reticulum. In Saccharomyces cerevisiae, conditions that trigger Acb1 secretion via this pathway generate a Grh1 containing compartment composed of vesicles and tubules surrounded by a cup-shaped membrane and collectively called CUPS. Here we report a quantitative assay for Acb1 secretion that reveals requirements for ESCRT-I, -II, and -III but, surprisingly, without the involvement of the Vps4 AAA-ATPase. The major ESCRT-III subunit Snf7 localizes transiently to CUPS and this was accelerated in vps4Δ cells, correlating with increased Acb1 secretion. Microscopic analysis suggests that, instead of forming intraluminal vesicles with the help of Vps4, ESCRT-III/Snf7 promotes direct engulfment of preexisting Grh1 containing vesicles and tubules into a saccule to generate a mature Acb1 containing compartment. This novel multivesicular / multilamellar compartment, we suggest represents the stable secretory form of CUPS that is competent for the release of Acb1 to cells exterior. PMID:27115345

  14. ESCRT-III drives the final stages of CUPS maturation for unconventional protein secretion

    PubMed Central

    Curwin, Amy J; Brouwers, Nathalie; Alonso Y Adell, Manuel; Teis, David; Turacchio, Gabriele; Parashuraman, Seetharaman; Ronchi, Paolo; Malhotra, Vivek

    2016-01-01

    The unconventional secretory pathway exports proteins that bypass the endoplasmic reticulum. In Saccharomyces cerevisiae, conditions that trigger Acb1 secretion via this pathway generate a Grh1 containing compartment composed of vesicles and tubules surrounded by a cup-shaped membrane and collectively called CUPS. Here we report a quantitative assay for Acb1 secretion that reveals requirements for ESCRT-I, -II, and -III but, surprisingly, without the involvement of the Vps4 AAA-ATPase. The major ESCRT-III subunit Snf7 localizes transiently to CUPS and this was accelerated in vps4Δ cells, correlating with increased Acb1 secretion. Microscopic analysis suggests that, instead of forming intraluminal vesicles with the help of Vps4, ESCRT-III/Snf7 promotes direct engulfment of preexisting Grh1 containing vesicles and tubules into a saccule to generate a mature Acb1 containing compartment. This novel multivesicular / multilamellar compartment, we suggest represents the stable secretory form of CUPS that is competent for the release of Acb1 to cells exterior. DOI: http://dx.doi.org/10.7554/eLife.16299.001 PMID:27115345

  15. Multivalency in the Inhibition of Oxidative Protein Folding by Arsenic(III) Species

    PubMed Central

    2015-01-01

    The renewed use of arsenicals as chemotherapeutics has rekindled interest in the biochemistry of As(III) species. In this work, simple bis- and tris-arsenical derivatives were synthesized with the aim of exploiting the chelate effect in the inhibition of thiol-disulfide oxidoreductases (here, Quiescin sulfhydryl oxidase, QSOX, and protein disulfide isomerase, PDI) that utilize two or more CxxC motifs in the catalysis of oxidative protein folding. Coupling 4-aminophenylarsenoxide (APAO) to acid chloride or anhydride derivatives yielded two bis-arsenical prototypes, BA-1 and BA-2, and a tris-arsenical, TA-1. Unlike the monoarsenical, APAO, these new reagents proved to be strong inhibitors of oxidative protein folding in the presence of a realistic intracellular concentration of competing monothiol (here, 5 mM reduced glutathione, GSH). However, this inhibition does not reflect direct inactivation of QSOX or PDI, but avid binding of MVAs to the reduced unfolded protein substrates themselves. Titrations of reduced riboflavin-binding protein with MVAs show that all 18 protein −SH groups can be captured by these arsenicals. With reduced RNase, addition of substoichiometric levels of MVAs is accompanied by the formation of Congo Red- and Thioflavin T-positive fibrillar aggregates. Even with Kd values of ∼50 nM, MVAs are ineffective inhibitors of PDI in the presence of millimolar levels of competing GSH. These results underscore the difficulties of designing effective and specific arsenical inhibitors for folded enzymes and proteins. Some of the cellular effects of arsenicals likely reflect their propensity to associate very tightly and nonspecifically to conformationally mobile cysteine-rich regions of proteins, thereby interfering with folding and/or function. PMID:25506675

  16. A bacterial type III secretion assay for delivery of fungal effector proteins into wheat.

    PubMed

    Upadhyaya, Narayana M; Mago, Rohit; Staskawicz, Brian J; Ayliffe, Michael A; Ellis, Jeffrey G; Dodds, Peter N

    2014-03-01

    Large numbers of candidate effectors from fungal pathogens are being identified through whole-genome sequencing and in planta expression studies. Although Agrobacterium-mediated transient expression has enabled high-throughput functional analysis of effectors in dicot plants, this assay is not effective in cereal leaves. Here, we show that a nonpathogenic Pseudomonas fluorescens engineered to express the type III secretion system (T3SS) of P. syringae and the wheat pathogen Xanthomonas translucens can deliver fusion proteins containing T3SS signals from P. syringae (AvrRpm1) and X. campestris (AvrBs2) avirulence (Avr) proteins, respectively, into wheat leaf cells. A calmodulin-dependent adenylate cyclase reporter protein was delivered effectively into wheat and barley by both bacteria. Absence of any disease symptoms with P. fluorescens makes it more suitable than X. translucens for detecting a hypersensitive response (HR) induced by an effector protein with avirulence activity. We further modified the delivery system by removal of the myristoylation site from the AvrRpm1 fusion to prevent its localization to the plasma membrane which could inhibit recognition of an Avr protein. Delivery of the flax rust AvrM protein by the modified delivery system into transgenic tobacco leaves expressing the corresponding M resistance protein induced a strong HR, indicating that the system is capable of delivering a functional rust Avr protein. In a preliminary screen of effectors from the stem rust fungus Puccinia graminis f. sp. tritici, we identified one effector that induced a host genotype-specific HR in wheat. Thus, the modified AvrRpm1:effector-Pseudomonas fluorescens system is an effective tool for large-scale screening of pathogen effectors for recognition in wheat. PMID:24156769

  17. Type III secretion needle proteins induce cell signaling and cytokine secretion via Toll-like receptors.

    PubMed

    Jessen, Danielle L; Osei-Owusu, Patrick; Toosky, Melody; Roughead, William; Bradley, David S; Nilles, Matthew L

    2014-06-01

    Pathogens are recognized by hosts by use of various receptors, including the Toll-like receptor (TLR) and Nod-like receptor (NLR) families. Ligands for these varied receptors, including bacterial products, are identified by the immune system, resulting in development of innate immune responses. Only a couple of components from type III secretion (T3S) systems are known to be recognized by TLR or NLR family members. Known T3S components that are detected by pattern recognition receptors (PRRs) are (i) flagellin, detected by TLR5 and NLRC4 (Ipaf); and (ii) T3S rod proteins (PrgJ and homologs) and needle proteins (PrgI and homologs), detected by NAIP and the NLRC4 inflammasome. In this report, we characterize the induction of proinflammatory responses through TLRs by the Yersinia pestis T3S needle protein, YscF, the Salmonella enterica needle proteins PrgI and SsaG, and the Shigella needle protein, MxiH. More specifically, we determine that the proinflammatory responses occur through TLR2 and -4. These data support the hypothesis that T3S needles have an unrecognized role in bacterial pathogenesis by modulating immune responses. PMID:24643544

  18. Diffusible signal factor (DSF) synthase RpfF of Xylella fastidiosa is a multifunction protein also required for response to DSF.

    PubMed

    Ionescu, Michael; Baccari, Clelia; Da Silva, Aline Maria; Garcia, Angelica; Yokota, Kenji; Lindow, Steven E

    2013-12-01

    Xylella fastidiosa, like related Xanthomonas species, employs an Rpf cell-cell communication system consisting of a diffusible signal factor (DSF) synthase, RpfF, and a DSF sensor, RpfC, to coordinate expression of virulence genes. While phenotypes of a ΔrpfF strain in Xanthomonas campestris could be complemented by its own DSF, the DSF produced by X. fastidiosa (XfDSF) did not restore expression of the XfDSF-dependent genes hxfA and hxfB to a ΔrpfF strain of X. fastidiosa, suggesting that RpfF is involved in XfDSF sensing or XfDSF-dependent signaling. To test this conjecture, rpfC and rpfF of X. campestris were replaced by those of X. fastidiosa, and the contribution of each gene to the induction of a X. campestris DSF-dependent gene was assessed. As in X. fastidiosa, XfDSF-dependent signaling required both X. fastidiosa proteins RpfF and RpfC. RpfF repressed RpfC signaling activity, which in turn was derepressed by XfDSF. A mutated X. fastidiosa RpfF protein with two substitutions of glutamate to alanine in its active site was incapable of XfDSF production yet enabled a response to XfDSF, indicating that XfDSF production and the response to XfDSF are two separate functions in which RpfF is involved. This mutant was also hypervirulent to grape, demonstrating the antivirulence effects of XfDSF itself in X. fastidiosa. The Rpf system of X. fastidiosa is thus a novel example of a quorum-sensing signal synthase that is also involved in the response to the signal molecule that it synthesizes. PMID:24056101

  19. Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II.

    PubMed

    Davis, Keersten M; Bitting, Anna L; Markwalter, Christine F; Bauer, Westley S; Wright, David W

    2015-01-01

    This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2(+) (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 µm magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging. PMID:26273845

  20. Detergent Isolation Stabilizes and Activates the Shigella Type III Secretion System Translocator Protein IpaC.

    PubMed

    Bernard, Abram R; Duarte, Shari M; Kumar, Prashant; Dickenson, Nicholas E

    2016-07-01

    Shigella rely on a type III secretion system as the primary virulence factor for invasion and colonization of human hosts. Although there are an estimated 90 million Shigella infections, annually responsible for more than 100,000 deaths worldwide, challenges isolating and stabilizing many type III secretion system proteins have prevented a full understanding of the Shigella invasion mechanism and additionally slowed progress toward a much needed Shigella vaccine. Here, we show that the non-denaturing zwitterionic detergent N, N-dimethyldodecylamine N-oxide (LDAO) and non-ionic detergent n-octyl-oligo-oxyethylene efficiently isolated the hydrophobic Shigella translocator protein IpaC from the co-purified IpaC/IpgC chaperone-bound complex. Both detergents resulted in monomeric IpaC that exhibits strong membrane binding and lysis characteristics while the chaperone-bound complex does not, suggesting that the stabilizing detergents provide a means of following IpaC "activation" in vitro. Additionally, biophysical characterization found that LDAO provides significant thermal and temporal stability to IpaC, protecting it for several days at room temperature and brief exposure to temperatures reaching 90°C. In summary, this work identified and characterized conditions that provide stable, membrane active IpaC, providing insight into key interactions with membranes and laying a strong foundation for future vaccine formulation studies taking advantage of the native immunogenicity of IpaC and the stability provided by LDAO. PMID:27297397

  1. PROTEIN TARGETING TO STARCH Is Required for Localising GRANULE-BOUND STARCH SYNTHASE to Starch Granules and for Normal Amylose Synthesis in Arabidopsis

    PubMed Central

    Seung, David; Soyk, Sebastian; Coiro, Mario; Maier, Benjamin A.; Eicke, Simona; Zeeman, Samuel C.

    2015-01-01

    The domestication of starch crops underpinned the development of human civilisation, yet we still do not fully understand how plants make starch. Starch is composed of glucose polymers that are branched (amylopectin) or linear (amylose). The amount of amylose strongly influences the physico-chemical behaviour of starchy foods during cooking and of starch mixtures in non-food manufacturing processes. The GRANULE-BOUND STARCH SYNTHASE (GBSS) is the glucosyltransferase specifically responsible for elongating amylose polymers and was the only protein known to be required for its biosynthesis. Here, we demonstrate that PROTEIN TARGETING TO STARCH (PTST) is also specifically required for amylose synthesis in Arabidopsis. PTST is a plastidial protein possessing an N-terminal coiled coil domain and a C-terminal carbohydrate binding module (CBM). We discovered that Arabidopsis ptst mutants synthesise amylose-free starch and are phenotypically similar to mutants lacking GBSS. Analysis of granule-bound proteins showed a dramatic reduction of GBSS protein in ptst mutant starch granules. Pull-down assays with recombinant proteins in vitro, as well as immunoprecipitation assays in planta, revealed that GBSS physically interacts with PTST via a coiled coil. Furthermore, we show that the CBM domain of PTST, which mediates its interaction with starch granules, is also required for correct GBSS localisation. Fluorescently tagged Arabidopsis GBSS, expressed either in tobacco or Arabidopsis leaves, required the presence of Arabidopsis PTST to localise to starch granules. Mutation of the CBM of PTST caused GBSS to remain in the plastid stroma. PTST fulfils a previously unknown function in targeting GBSS to starch. This sheds new light on the importance of targeting biosynthetic enzymes to sub-cellular sites where their action is required. Importantly, PTST represents a promising new gene target for the biotechnological modification of starch composition, as it is exclusively involved

  2. In vivo footprinting of the mouse inducible nitric oxide synthase gene: inducible protein occupation of numerous sites including Oct and NF-IL6.

    PubMed Central

    Goldring, C E; Reveneau, S; Algarté, M; Jeannin, J F

    1996-01-01

    A wide variety of cells usefully but sometimes destructively produce nitric oxide via inducible nitric oxide synthase (iNOS). Data obtained by gel shift analysis and reporter assays have linked murine iNOS gene induction by cytokines and bacterial products with the binding of a number of proteins to a proximal promoter, as well as to a distal enhancer of the iNOS gene. Nevertheless, these techniques do not necessarily reflect protein occupation of sites in vivo. To address this, we have used dimethyl sulphate in vivo footprinting to determine binding events in the two murine iNOS transcription control regions, using a classical lipopolysaccharide induction of RAW 264.7 macrophages. Protein-DNA interactions are absent before activation. Exposure to lipopolysaccharide induces protection at a NF-kappaB site and hypersensitivity at a shared gamma-activated site/interferon-stimulated response element within the enhancer. Protections are seen at a NF-IL6, and an Oct site within the promoter. We also observe modulations in guanine methylation at two regions which do not correspond to any known putative binding elements. Furthermore, we confirm the probable involvement of interferon regulatory factor-1 (binding to its -901 to -913 site) and the binding of NF-kappaB to its proximal site. Our data demonstrate an abundance of hitherto-unrecognised protein-DNA binding events upon simple lipopolysaccharide activation of the iNOS gene and suggests a role for protein-protein interactions in its transcriptional induction. PMID:8649986

  3. Bacterial type III secretion systems: specialized nanomachines for protein delivery into target cells

    PubMed Central

    Galán, Jorge E.; Lara-Tejero, Maria; Marlovits, Thomas C.; Wagner, Samuel

    2015-01-01

    One of the most exciting developments in the field of bacterial pathogenesis in recent years is the discovery that many pathogens utilized complex nanomachines to deliver bacterially encoded effector proteins into target eukaryotic cells. These effector proteins modulate a variety of cellular functions for the pathogen’s benefit. One of these protein-delivery machines is the type III secretion system (T3SS). T3SSs are widespread in nature and are encoded not only by bacteria pathogenic to vertebrates or plants, but also by bacteria that are symbiotic to plants or insects. A central component of T3SSs is the needle complex, a supramolecular structure that mediates the passage of the secreted proteins across the bacterial envelope. Working in conjunction with several cytoplasmic components, the needle complex engages specific substrates in sequential order, moves them across the bacterial envelope, and ultimately delivers them into eukaryotic cells. The central role of T3SSs in pathogenesis makes them great targets for novel antimicrobial strategies. PMID:25002086

  4. Enrichment of Functional Redox Reactive Proteins and Identification by Mass Spectrometry Results in Several Terminal Fe(III)-reducing Candidate Proteins in Shewanella oneidensis MR-1.

    SciTech Connect

    Elias, Dwayne A.; Yang, Feng; Mottaz, Heather M.; Beliaev, Alex S.; Lipton, Mary S.

    2007-02-01

    Identification of the proteins directly involved in microbial metal-reduction is important to understanding the biochemistry involved in heavy metal reduction/immobilization and the ultimate cleanup of DOE contaminated sites. Although previous strategies for the identification of these proteins have traditionally required laborious protein purification/characterization of metal-reducing capability, activity is often lost before the final purification step, thus creating a significant knowledge gap. In the current study, subcellular fractions of S. oneidensis MR-1 were enriched for Fe(III)-NTA reducing proteins in a single step using several orthogonal column matrices. The protein content of eluted fractions that demonstrated activity were determined by ultra high pressure liquid chromatography coupled with tandem mass spectrometry (LCMS/ MS). A comparison of the proteins identified from active fractions in all separations produced 30 proteins that may act as the terminal electron-accepting protein for Fe(III)-reduction. These include MtrA, MtrB, MtrC and OmcA as well as a number of other proteins not previously associated with Fe(III)-reduction. This is the first report of such an approach where the laborious procedures for protein purification are not required for identification of metal-reducing proteins. Such work provides the basis for a similar approach with other cultured organisms as well as analysis of sediment and groundwater samples from biostimulation efforts at contaminated sites.

  5. Overexpression of interleukin-18 protein reduces viability and induces apoptosis of tongue squamous cell carcinoma cells by activation of glycogen synthase kinase-3β signaling

    PubMed Central

    LIU, WEIWEI; HU, MIN; WANG, YUMEI; SUN, BAOZHEN; GUO, YU; XU, ZHIMIN; LI, JIA; HAN, BING

    2015-01-01

    The aim of this study was to investigate the effects of interleukin-18 (IL-18) expression on regulating the viability and apoptosis of tongue squamous cell carcinoma (TSCC) cells in vitro and examine the underlying molecular events. Human IL-18 cDNA was cloned into the vector pcDNA3.1 (+) and transfected into CRL-1623™ cells. Quantitative reverse transcription-PCR (RT-qPCR), western blot analysis, immunofluorescence, cell viability MTT assay, flow cytometric Annexin V/propidium iodide (PI), Giemsa staining, and caspase-3 activity assay were performed. The data showed that overexpression of IL-18 protein reduced TSCC cell viability by inducing apoptosis. Compared with cells transfected with the control vector, IL-18 expression activated caspase-3, -7, and -9 by inducing their cleavage and increased the expression of interferon (IFN)-γ and cytochrome c mRNA, but reduced cyclin D1 and A1 expression in TSCC cells. IL-18 expression upregulated the expression and phosphorylation of glycogen synthase kinase (GSK)-3β protein in CRL1623 cells, whereas the selective GSK-3β inhibitor kenpaullone antagonized the effects of IL-18 protein on TSCC cells in vitro. The results indicated that IL-18 played an important role in the inhibition of TSCC cell growth and may be further investigated as a novel therapeutic target against TSCC. PMID:25591548

  6. The TWD40-2 protein and the AP2 complex cooperate in the clathrin-mediated endocytosis of cellulose synthase to regulate cellulose biosynthesis

    PubMed Central

    Bashline, Logan; Li, Shundai; Zhu, Xiaoyu; Gu, Ying

    2015-01-01

    Cellulose biosynthesis is performed exclusively by plasma membrane-localized cellulose synthases (CESAs). Therefore, the trafficking of CESAs to and from the plasma membrane is an important mechanism for regulating cellulose biosynthesis. CESAs were recently identified as cargo proteins of the classic adaptor protein 2 (AP2) complex of the clathrin-mediated endocytosis (CME) pathway. The AP2 complex of the CME pathway is conserved in yeast, animals, and plants, and has been well-characterized in many systems. In contrast, the recently discovered TPLATE complex (TPC), which is proposed to function as a CME adaptor complex, is only conserved in plants and a few other eukaryotes. In this study, we discovered that the TWD40-2 protein, a putative member of the TPC, is also important for the endocytosis of CESAs. Genetic analysis between TWD40-2 and AP2M of the AP2 complex revealed that the roles of TWD40-2 in CME are both distinct from and cooperative with the AP2 complex. Loss of efficient CME in twd40-2-3 resulted in the unregulated overaccumulation of CESAs at the plasma membrane. In seedlings of twd40-2-3 and other CME-deficient mutants, a direct correlation was revealed between endocytic deficiency and cellulose content deficiency, highlighting the importance of controlled CESA endocytosis in regulating cellulose biosynthesis. PMID:26417106

  7. Arabidopsis protein phosphatase 2C ABI1 interacts with type I ACC synthases and is involved in the regulation of ozone-induced ethylene biosynthesis.

    PubMed

    Ludwików, Agnieszka; Cieśla, Agata; Kasprowicz-Maluśki, Anna; Mituła, Filip; Tajdel, Małgorzata; Gałgański, Łukasz; Ziółkowski, Piotr A; Kubiak, Piotr; Małecka, Arleta; Piechalak, Aneta; Szabat, Marta; Górska, Alicja; Dąbrowski, Maciej; Ibragimow, Izabela; Sadowski, Jan

    2014-06-01

    Ethylene plays a crucial role in various biological processes and therefore its biosynthesis is strictly regulated by multiple mechanisms. Posttranslational regulation, which is pivotal in controlling ethylene biosynthesis, impacts 1-aminocyclopropane 1-carboxylate synthase (ACS) protein stability via the complex interplay of specific factors. Here, we show that the Arabidopsis thaliana protein phosphatase type 2C, ABI1, a negative regulator of abscisic acid signaling, is involved in the regulation of ethylene biosynthesis under oxidative stress conditions. We found that ABI1 interacts with ACS6 and dephosphorylates its C-terminal fragment, a target of the stress-responsive mitogen-activated protein kinase, MPK6. In addition, ABI1 controls MPK6 activity directly and by this means also affects the ACS6 phosphorylation level. Consistently with this, ozone-induced ethylene production was significantly higher in an ABI1 knockout strain (abi1td) than in wild-type plants. Importantly, an increase in stress-induced ethylene production in the abi1td mutant was compensated by a higher ascorbate redox state and elevated antioxidant activities. Overall, the results of this study provide evidence that ABI1 restricts ethylene synthesis by affecting the activity of ACS6. The ABI1 contribution to stress phenotype underpins its role in the interplay between the abscisic acid (ABA) and ethylene signaling pathways. PMID:24637173

  8. Protein kinase R-like endoplasmic reticulum kinase and glycogen synthase kinase-3α/β regulate foam cell formation[S

    PubMed Central

    McAlpine, Cameron S.; Werstuck, Geoff H.

    2014-01-01

    Evidence suggests a causative role for endoplasmic reticulum (ER) stress in the development of atherosclerosis. This study investigated the potential role of glycogen synthase kinase (GSK)-3α/β in proatherogenic ER stress signaling. Thp1-derived macrophages were treated with the ER stress-inducing agents, glucosamine, thapsigargin, or palmitate. Using small-molecule inhibitors of specific unfolded protein response (UPR) signaling pathways, we found that protein kinase R-like ER kinase (PERK), but not inositol requiring enzyme 1 or activating transcription factor 6, is required for the activation of GSK3α/β by ER stress. GSK3α/β inhibition or siRNA-directed knockdown attenuated ER stress-induced expression of distal components of the PERK pathway. Macrophage foam cells within atherosclerotic plaques and isolated macrophages from ApoE−/− mice fed a diet supplemented with the GSK3α/β inhibitor valproate had reduced levels of C/EBP homologous protein (CHOP). GSK3α/β inhibition blocked ER stress-induced lipid accumulation and the upregulation of genes associated with lipid metabolism. In primary mouse macrophages, PERK inhibition blocked ER stress-induced lipid accumulation, whereas constitutively active S9A-GSK3β promoted foam cell formation and CHOP expression, even in cells treated with a PERK inhibitor. These findings suggest that ER stress-PERK-GSK3α/β signaling promotes proatherogenic macrophage lipid accumulation. PMID:25183803

  9. Steady state protein levels in Geobacter metallireducens grown with Iron (III) citrate or nitrate as terminal electron acceptor.

    SciTech Connect

    Ahrendt, A. J.; Tollaksen, S. L.; Lindberg, C.; Zhu, W.; Yates, J. R., III; Nevin, K. P.; Lovley, D.; Giometti, C. S.; Biosciences Division; The Scripps Research Inst.; Univ. of Massachusetts

    2007-01-01

    Geobacter species predominate in aquatic sediments and submerged soils where organic carbon sources are oxidized with the reduction of Fe(III). The natural occurrence of Geobacter in some waste sites suggests this microorganism could be useful for bioremediation if growth and metabolic activity can be regulated. 2-DE was used to monitor the steady state protein levels of Geobacter metallireducens grown with either Fe(III) citrate or nitrate to elucidate metabolic differences in response to different terminal electron acceptors present in natural environments populated by Geobacter. Forty-six protein spots varied significantly in abundance (p<0.05) between the two growth conditions; proteins were identified by tryptic peptide mass and peptide sequence determined by MS/MS. Enzymes involved in pyruvate metabolism and the tricarboxylic acid (TCA) cycle were more abundant in cells grown with Fe(III) citrate, while proteins associated with nitrate metabolism and sensing cellular redox status along with several proteins of unknown function were more abundant in cells grown with nitrate. These results indicate a higher level of flux through the TCA cycle in the presence of Fe(III) compared to nitrate. The oxidative stress response observed in previous studies of Geobacter sulfurreducens grown with Fe(III) citrate was not seen in G. metallireducens.

  10. Three-dimensional (3D) structure prediction of the American and African oil-palms β-ketoacyl-[ACP] synthase-II protein by comparative modelling

    PubMed Central

    Wang, Edina; Chinni, Suresh; Bhore, Subhash Janardhan

    2014-01-01

    Background: The fatty-acid profile of the vegetable oils determines its properties and nutritional value. Palm-oil obtained from the African oil-palm [Elaeis guineensis Jacq. (Tenera)] contains 44% palmitic acid (C16:0), but, palm-oil obtained from the American oilpalm [Elaeis oleifera] contains only 25% C16:0. In part, the b-ketoacyl-[ACP] synthase II (KASII) [EC: 2.3.1.179] protein is responsible for the high level of C16:0 in palm-oil derived from the African oil-palm. To understand more about E. guineensis KASII (EgKASII) and E. oleifera KASII (EoKASII) proteins, it is essential to know its structures. Hence, this study was undertaken. Objective: The objective of this study was to predict three-dimensional (3D) structure of EgKASII and EoKASII proteins using molecular modelling tools. Materials and Methods: The amino-acid sequences for KASII proteins were retrieved from the protein database of National Center for Biotechnology Information (NCBI), USA. The 3D structures were predicted for both proteins using homology modelling and ab-initio technique approach of protein structure prediction. The molecular dynamics (MD) simulation was performed to refine the predicted structures. The predicted structure models were evaluated and root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values were calculated. Results: The homology modelling showed that EgKASII and EoKASII proteins are 78% and 74% similar with Streptococcus pneumonia KASII and Brucella melitensis KASII, respectively. The EgKASII and EoKASII structures predicted by using ab-initio technique approach shows 6% and 9% deviation to its structures predicted by homology modelling, respectively. The structure refinement and validation confirmed that the predicted structures are accurate. Conclusion: The 3D structures for EgKASII and EoKASII proteins were predicted. However, further research is essential to understand the interaction of EgKASII and EoKASII proteins with its substrates. PMID

  11. Purification and biochemical characterization of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthases KasA and KasB.

    PubMed

    Schaeffer, M L; Agnihotri, G; Volker, C; Kallender, H; Brennan, P J; Lonsdale, J T

    2001-12-14

    Mycolic acids are vital components of the Mycobacterium tuberculosis cell wall, and enzymes involved in their formation represent attractive targets for the discovery of novel anti-tuberculosis agents. Biosynthesis of the fatty acyl chains of mycolic acids involves two fatty acid synthetic systems, the multifunctional polypeptide fatty acid synthase I (FASI), which performs de novo fatty acid synthesis, and the dissociated FASII system, which consists of monofunctional enzymes, and acyl carrier protein (ACP) and elongates FASI products to long chain mycolic acid precursors. In this study, we present the initial characterization of purified KasA and KasB, two beta-ketoacyl-ACP synthase (KAS) enzymes of the M. tuberculosis FASII system. KasA and KasB were expressed in E. coli and purified by affinity chromatography. Both enzymes showed activity typical of bacterial KASs, condensing an acyl-ACP with malonyl-ACP. Consistent with the proposed role of FASII in mycolic acid synthesis, analysis of various acyl-ACP substrates indicated KasA and KasB had higher specificity for long chain acyl-ACPs containing at least 16 carbons. Activity of KasA and KasB increased with use of M. tuberculosis AcpM, suggesting that structural differences between AcpM and E. coli ACP may affect their recognition by the enzymes. Both enzymes were sensitive to KAS inhibitors cerulenin and thiolactomycin. These results represent important steps in characterizing KasA and KasB as targets for antimycobacterial drug discovery. PMID:11600501

  12. Glycogen synthase and phosphofructokinase protein and mRNA levels in skeletal muscle from insulin-resistant patients with non-insulin-dependent diabetes mellitus.

    PubMed Central

    Vestergaard, H; Lund, S; Larsen, F S; Bjerrum, O J; Pedersen, O

    1993-01-01

    In patients with non-insulin-dependent diabetes mellitus (NIDDM) and matched control subjects we examined the interrelationships between in vivo nonoxidative glucose metabolism and glucose oxidation and the muscle activities, as well as the immunoreactive protein and mRNA levels of the rate-limiting enzymes in glycogen synthesis and glycolysis, glycogen synthase (GS) and phosphofructokinase (PFK), respectively. Analysis of biopsies of quadriceps muscle from 19 NIDDM patients and 19 control subjects showed in the basal state a 30% decrease (P < 0.005) in total GS activity and a 38% decrease (P < 0.001) in GS mRNA/microgram DNA in NIDDM patients, whereas the GS protein level was normal. The enzymatic activity and protein and mRNA levels of PFK were all normal in diabetic patients. In subgroups of NIDDM patients and control subjects an insulin-glucose clamp in combination with indirect calorimetry was performed. The rate of insulin-stimulated nonoxidative glucose metabolism was decreased by 47% (P < 0.005) in NIDDM patients, whereas the glucose oxidation rate was normal. The PFK activity, protein level, and mRNA/microgram DNA remained unchanged. The relative activation of GS by glucose-6-phosphate was 33% lower (P < 0.02), whereas GS mRNA/micrograms DNA was 37% lower (P < 0.05) in the diabetic patients after 4 h of hyperinsulinemia. Total GS immunoreactive mass remained normal. In conclusion, qualitative but not quantitative posttranslational abnormalities of the GS protein in muscle determine the reduced insulin-stimulated nonoxidative glucose metabolism in NIDDM. Images PMID:8514849

  13. A functional cellulose synthase from ascidian epidermis

    PubMed Central

    Matthysse, Ann G.; Deschet, Karine; Williams, Melanie; Marry, Mazz; White, Alan R.; Smith, William C.

    2004-01-01

    Among animals, urochordates (e.g., ascidians) are unique in their ability to biosynthesize cellulose. In ascidians cellulose is synthesized in the epidermis and incorporated into a protective coat know as the tunic. A putative cellulose synthase-like gene was first identified in the genome sequences of the ascidian Ciona intestinalis. We describe here a cellulose synthase gene from the ascidian Ciona savignyi that is expressed in the epidermis. The predicted C. savignyi cellulose synthase amino acid sequence showed conserved features found in all cellulose synthases, including plants, but was most similar to cellulose synthases from bacteria, fungi, and Dictyostelium discoidium. However, unlike other known cellulose synthases, the predicted C. savignyi polypeptide has a degenerate cellulase-like region near the carboxyl-terminal end. An expression construct carrying the C. savignyi cDNA was found to restore cellulose biosynthesis to a cellulose synthase (CelA) minus mutant of Agrobacterium tumefaciens, showing that the predicted protein has cellulose synthase activity. The lack of cellulose biosynthesis in all other groups of metazoans and the similarity of the C. savignyi cellulose synthase to enzymes from cellulose-producing organisms support the hypothesis that the urochordates acquired the cellulose biosynthetic pathway by horizontal transfer. PMID:14722352

  14. Presence of unique glyoxalase III proteins in plants indicates the existence of shorter route for methylglyoxal detoxification

    PubMed Central

    Ghosh, Ajit; Kushwaha, Hemant R; Hasan, Mohammad R; Pareek, Ashwani; Sopory, Sudhir K; Singla-Pareek, Sneh L

    2016-01-01

    Glyoxalase pathway, comprising glyoxalase I (GLY I) and glyoxalase II (GLY II) enzymes, is the major pathway for detoxification of methylglyoxal (MG) into D-lactate involving reduced glutathione (GSH). However, in bacteria, glyoxalase III (GLY III) with DJ-1/PfpI domain(s) can do the same conversion in a single step without GSH. Our investigations for the presence of DJ-1/PfpI domain containing proteins in plants have indicated the existence of GLY III-like proteins in monocots, dicots, lycopods, gymnosperm and bryophytes. A deeper in silico analysis of rice genome identified twelve DJ-1 proteins encoded by six genes. Detailed analysis has been carried out including their chromosomal distribution, genomic architecture and localization. Transcript profiling under multiple stress conditions indicated strong induction of OsDJ-1 in response to exogenous MG. A member of OsDJ-1 family, OsDJ-1C, showed high constitutive expression at all developmental stages and tissues of rice. MG depletion study complemented by simultaneous formation of D-lactate proved OsDJ-1C to be a GLY III enzyme that converts MG directly into D-lactate in a GSH-independent manner. Site directed mutagenesis of Cys-119 to Alanine significantly reduces its GLY III activity indicating towards the existence of functional GLY III enzyme in rice—a shorter route for MG detoxification. PMID:26732528

  15. Presence of unique glyoxalase III proteins in plants indicates the existence of shorter route for methylglyoxal detoxification.

    PubMed

    Ghosh, Ajit; Kushwaha, Hemant R; Hasan, Mohammad R; Pareek, Ashwani; Sopory, Sudhir K; Singla-Pareek, Sneh L

    2016-01-01

    Glyoxalase pathway, comprising glyoxalase I (GLY I) and glyoxalase II (GLY II) enzymes, is the major pathway for detoxification of methylglyoxal (MG) into D-lactate involving reduced glutathione (GSH). However, in bacteria, glyoxalase III (GLY III) with DJ-1/PfpI domain(s) can do the same conversion in a single step without GSH. Our investigations for the presence of DJ-1/PfpI domain containing proteins in plants have indicated the existence of GLY III-like proteins in monocots, dicots, lycopods, gymnosperm and bryophytes. A deeper in silico analysis of rice genome identified twelve DJ-1 proteins encoded by six genes. Detailed analysis has been carried out including their chromosomal distribution, genomic architecture and localization. Transcript profiling under multiple stress conditions indicated strong induction of OsDJ-1 in response to exogenous MG. A member of OsDJ-1 family, OsDJ-1C, showed high constitutive expression at all developmental stages and tissues of rice. MG depletion study complemented by simultaneous formation of D-lactate proved OsDJ-1C to be a GLY III enzyme that converts MG directly into D-lactate in a GSH-independent manner. Site directed mutagenesis of Cys-119 to Alanine significantly reduces its GLY III activity indicating towards the existence of functional GLY III enzyme in rice-a shorter route for MG detoxification. PMID:26732528

  16. Geranyl diphosphate synthase molecules, and nucleic acid molecules encoding same

    SciTech Connect

    Croteau, Rodney Bruce; Burke, Charles Cullen

    2008-06-24

    In one aspect, the present invention provides isolated nucleic acid molecules that each encode a geranyl diphosphate synthase protein, wherein each isolated nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:1 under conditions of 5.times.SSC at 45.degree. C. for one hour. The present invention also provides isolated geranyl diphosphate synthase proteins, and methods for altering the level of expression of geranyl diphosphate synthase protein in a host cell.

  17. Geranyl diphosphate synthase large subunit, and methods of use

    DOEpatents

    Croteau, Rodney B.; Burke, Charles C.; Wildung, Mark R.

    2001-10-16

    A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

  18. PscI is a type III secretion needle anchoring protein with in vitro polymerization capacities.

    PubMed

    Monlezun, Laura; Liebl, David; Fenel, Daphna; Grandjean, Teddy; Berry, Alice; Schoehn, Guy; Dessein, Rodrigue; Faudry, Eric; Attree, Ina

    2015-04-01

    The export of bacterial toxins across the bacterial envelope requires the assembly of complex, membrane-embedded protein architectures. Pseudomonas aeruginosa employs type III secretion (T3S) injectisome to translocate exotoxins directly into the cytoplasm of a target eukaryotic cell. This multi-protein channel crosses two bacterial membranes and extends further as a needle through which the proteins travel. We show in this work that PscI, proposed to form the T3S system (T3SS) inner rod, possesses intrinsic properties to polymerize into flexible and regularly twisted fibrils and activates IL-1β production in mouse bone marrow macrophages in vitro. We also found that point mutations within C-terminal amphipathic helix of PscI alter needle assembly in vitro and T3SS function in cell infection assays, suggesting that this region is essential for an efficient needle assembly. The overexpression of PscF partially compensates for the absence of the inner rod in PscI-deficient mutant by forming a secretion-proficient injectisome. All together, we propose that the polymerized PscI in P. aeruginosa optimizes the injectisome function by anchoring the needle within the envelope-embedded complex of the T3S secretome and - contrary to its counterpart in Salmonella - is not involved in substrate switching. PMID:25614137

  19. ESCRT-III binding protein MITD1 is involved in cytokinesis and has an unanticipated PLD fold that binds membranes

    PubMed Central

    Hadders, Michael A.; Agromayor, Monica; Obita, Takayuki; Perisic, Olga; Caballe, Anna; Kloc, Magdalena; Lamers, Meindert H.; Williams, Roger L.; Martin-Serrano, Juan

    2012-01-01

    The endosomal sorting complexes required for transport (ESCRT) proteins have a critical function in abscission, the final separation of the daughter cells during cytokinesis. Here, we describe the structure and function of a previously uncharacterized ESCRT-III interacting protein, MIT-domain containing protein 1 (MITD1). Crystal structures of MITD1 reveal a dimer, with a microtubule-interacting and trafficking (MIT) domain at the N terminus and a unique, unanticipated phospholipase D-like (PLD) domain at the C terminus that binds membranes. We show that the MIT domain binds to a subset of ESCRT-III subunits and that this interaction mediates MITD1 recruitment to the midbody during cytokinesis. Depletion of MITD1 causes a distinct cytokinetic phenotype consistent with destabilization of the midbody and abscission failure. These results suggest a model whereby MITD1 coordinates the activity of ESCRT-III during abscission with earlier events in the final stages of cell division. PMID:23045692

  20. Immunogenicity of a novel tetravalent vaccine formulation with four recombinant lipidated dengue envelope protein domain IIIs in mice

    PubMed Central

    Chiang, Chen-Yi; Pan, Chien-Hsiung; Chen, Mei-Yu; Hsieh, Chun-Hsiang; Tsai, Jy-Ping; Liu, Hsueh-Hung; Liu, Shih-Jen; Chong, Pele; Leng, Chih-Hsiang; Chen, Hsin-Wei

    2016-01-01

    We developed a novel platform to express high levels of recombinant lipoproteins with intrinsic adjuvant properties. Based on this technology, our group developed recombinant lipidated dengue envelope protein domain IIIs as vaccine candidates against dengue virus. This work aims to evaluate the immune responses in mice to the tetravalent formulation. We demonstrate that 4 serotypes of recombinant lipidated dengue envelope protein domain III induced both humoral and cellular immunity against all 4 serotypes of dengue virus on the mixture that formed the tetravalent formulation. Importantly, the immune responses induced by the tetravalent formulation in the absence of the exogenous adjuvant were functional in clearing the 4 serotypes of dengue virus in vivo. We affirm that the tetravalent formulation of recombinant lipidated dengue envelope protein domain III is a potential vaccine candidate against dengue virus and suggest further detailed studies of this formulation in nonhuman primates. PMID:27470096

  1. Immunogenicity of a novel tetravalent vaccine formulation with four recombinant lipidated dengue envelope protein domain IIIs in mice.

    PubMed

    Chiang, Chen-Yi; Pan, Chien-Hsiung; Chen, Mei-Yu; Hsieh, Chun-Hsiang; Tsai, Jy-Ping; Liu, Hsueh-Hung; Liu, Shih-Jen; Chong, Pele; Leng, Chih-Hsiang; Chen, Hsin-Wei

    2016-01-01

    We developed a novel platform to express high levels of recombinant lipoproteins with intrinsic adjuvant properties. Based on this technology, our group developed recombinant lipidated dengue envelope protein domain IIIs as vaccine candidates against dengue virus. This work aims to evaluate the immune responses in mice to the tetravalent formulation. We demonstrate that 4 serotypes of recombinant lipidated dengue envelope protein domain III induced both humoral and cellular immunity against all 4 serotypes of dengue virus on the mixture that formed the tetravalent formulation. Importantly, the immune responses induced by the tetravalent formulation in the absence of the exogenous adjuvant were functional in clearing the 4 serotypes of dengue virus in vivo. We affirm that the tetravalent formulation of recombinant lipidated dengue envelope protein domain III is a potential vaccine candidate against dengue virus and suggest further detailed studies of this formulation in nonhuman primates. PMID:27470096

  2. Post-synaptic density-95 promotes calcium/calmodulin-dependent protein kinase II-mediated Ser847 phosphorylation of neuronal nitric oxide synthase.

    PubMed Central

    Watanabe, Yasuo; Song, Tao; Sugimoto, Katsuyoshi; Horii, Mariko; Araki, Nobukazu; Tokumitsu, Hiroshi; Tezuka, Tohru; Yamamoto, Tadashi; Tokuda, Masaaki

    2003-01-01

    Post-synaptic density-95 (PSD-95) is a neuronal scaffolding protein that associates with N -methyl-D-aspartate (NMDA) receptors and links them to intracellular signalling molecules. In neurons, neuronal nitric oxide synthase (nNOS) binds selectively to the second PDZ domain (PDZ2) of PSD-95, thereby exhibiting physiological activation triggered via NMDA receptors. We have demonstrated previously that Ca(2+)/calmodulin-dependent protein kinase IIalpha (CaM-K IIalpha) directly phosphorylates nNOS at residue Ser(847), and can attenuate the catalytic activity of the enzyme in neuronal cells [Komeima, Hayashi, Naito and Watanabe (2000) J. Biol. Chem. 275, 28139-28143]. In the present study, we examined how CaM-K II participates in the phosphorylation by analysing the functional interaction between nNOS and PSD-95 in cells. The results showed that PSD-95 directly promotes the nNOS phosphorylation at Ser(847) induced by endogenous CaM-K II. In transfected cells, this effect of PSD-95 required its dual palmitoylation and the PDZ2 domain, but did not rely on its guanylate kinase domain. CaM-K Ialpha and CaM-K IV failed to phosphorylate nNOS at Ser(847) in transfected cells. Thus PSD-95 mediates cellular trafficking of nNOS, and may be required for the efficient phosphorylation of nNOS at Ser(847) by CaM-K II in neuronal cells. PMID:12630910

  3. Inhibition of G-protein-coupled Receptor Kinase 2 Prevents the Dysfunctional Cardiac Substrate Metabolism in Fatty Acid Synthase Transgenic Mice.

    PubMed

    Abd Alla, Joshua; Graemer, Muriel; Fu, Xuebin; Quitterer, Ursula

    2016-02-01

    Impairment of myocardial fatty acid substrate metabolism is characteristic of late-stage heart failure and has limited treatment options. Here, we investigated whether inhibition of G-protein-coupled receptor kinase 2 (GRK2) could counteract the disturbed substrate metabolism of late-stage heart failure. The heart failure-like substrate metabolism was reproduced in a novel transgenic model of myocardium-specific expression of fatty acid synthase (FASN), the major palmitate-synthesizing enzyme. The increased fatty acid utilization of FASN transgenic neonatal cardiomyocytes rapidly switched to a heart failure phenotype in an adult-like lipogenic milieu. Similarly, adult FASN transgenic mice developed signs of heart failure. The development of disturbed substrate utilization of FASN transgenic cardiomyocytes and signs of heart failure were retarded by the transgenic expression of GRKInh, a peptide inhibitor of GRK2. Cardioprotective GRK2 inhibition required an intact ERK axis, which blunted the induction of cardiotoxic transcripts, in part by enhanced serine 273 phosphorylation of Pparg (peroxisome proliferator-activated receptor γ). Conversely, the dual-specific GRK2 and ERK cascade inhibitor, RKIP (Raf kinase inhibitor protein), triggered dysfunctional cardiomyocyte energetics and the expression of heart failure-promoting Pparg-regulated genes. Thus, GRK2 inhibition is a novel approach that targets the dysfunctional substrate metabolism of the failing heart. PMID:26670611

  4. Allicin protects spinal cord neurons from glutamate-induced oxidative stress through regulating the heat shock protein 70/inducible nitric oxide synthase pathway.

    PubMed

    Liu, Shu-Guang; Ren, Peng-Yu; Wang, Guo-Yu; Yao, Shu-Xin; He, Xi-Jing

    2015-01-01

    Allicin, the main biologically active compound derived from garlic, exerts a broad spectrum of pharmacological activities and is considered to have therapeutic potential in many neurological disorders. Using an in vitro spinal cord injury model induced by glutamate treatment, we sought to investigate the neuroprotective effects of allicin in primary cultured spinal cord neurons. We found that allicin treatment significantly attenuated glutamate-induced lactate dehydrogenase (LDH) release, loss of cell viability and apoptotic neuronal death. This protection was associated with reduced oxidative stress, as evidenced by decreased reactive oxygen species (ROS) generation, reduced lipid peroxidation and preservation of antioxidant enzyme activities. The results of western blot analysis showed that allicin decreased the expression of inducible nitric oxide synthase (iNOS), but had no effects on the expression of neuronal NOS (nNOS) following glutamate exposure. Moreover, allicin treatment significantly increased the expression of heat shock protein 70 (HSP70) at both mRNA and protein levels. Knockdown of HSP70 by specific targeted small interfere RNA (siRNA) not only mitigated allicin-induced protective activity, but also partially nullified its effects on the regulation of iNOS. Collectively, these data demonstrate that allicin treatment may be an effective therapeutic strategy for spinal cord injury, and that the potential underlying mechanism involves HSP70/iNOS pathway-mediated inhibition of oxidative stress. PMID:25473931

  5. Quantitative Phosphoproteomic Study Reveals that Protein Kinase A Regulates Neural Stem Cell Differentiation Through Phosphorylation of Catenin Beta-1 and Glycogen Synthase Kinase 3β.

    PubMed

    Wang, Shuxin; Li, Zheyi; Shen, Hongyan; Zhang, Zhong; Yin, Yuxin; Wang, Qingsong; Zhao, Xuyang; Ji, Jianguo

    2016-08-01

    Protein phosphorylation is central to the understanding of multiple cellular signaling pathways responsible for regulating the self-renewal and differentiation of neural stem cells (NSCs). Here we performed a large-scale phosphoproteomic analysis of rat fetal NSCs using strong cation exchange chromatography prefractionation and citric acid-assisted two-step enrichment with TiO2 strategy followed by nanoLC-MS/MS analysis. Totally we identified 32,546 phosphosites on 5,091 phosphoproteins, among which 23,945 were class I phosphosites, and quantified 16,000 sites during NSC differentiation. More than 65% of class I phosphosites were novel when compared with PhosphoSitePlus database. Quantification results showed that the early and late stage of NSC differentiation differ greatly. We mapped 69 changed phosphosites on 20 proteins involved in Wnt signaling pathway, including S552 on catenin beta-1 (Ctnnb1) and S9 on glycogen synthase kinase 3β (Gsk3β). Western blotting and real-time PCR results proved that Wnt signaling pathway plays critical roles in NSC fate determination. Furthermore, inhibition and activation of PKA dramatically affected the phosphorylation state of Ctnnb1 and Gsk3β, which regulates the differentiation of NSCs. Our data provides a valuable resource for studying the self-renewal and differentiation of NSCs. Stem Cells 2016;34:2090-2101. PMID:27097102

  6. Broadly protective Shigella vaccine based on type III secretion apparatus proteins.

    PubMed

    Martinez-Becerra, Francisco J; Kissmann, Julian M; Diaz-McNair, Jovita; Choudhari, Shyamal P; Quick, Amy M; Mellado-Sanchez, Gabriela; Clements, John D; Pasetti, Marcela F; Picking, Wendy L

    2012-03-01

    Shigella spp. are food- and waterborne pathogens that cause severe diarrheal and dysenteric disease associated with high morbidity and mortality. Individuals most often affected are children under 5 years of age in the developing world. The existence of multiple Shigella serotypes and the heterogenic distribution of pathogenic strains, as well as emerging antibiotic resistance, require the development of a broadly protective vaccine. All Shigella spp. utilize a type III secretion system (TTSS) to initiate infection. The type III secretion apparatus (TTSA) is the molecular needle and syringe that form the energized conduit between the bacterial cytoplasm and the host cell to transport effector proteins that manipulate cellular processes to benefit the pathogen. IpaB and IpaD form a tip complex atop the TTSA needle and are required for pathogenesis. Because they are common to all virulent Shigella spp., they are ideal candidate antigens for a subunit-based, broad-spectrum vaccine. We examined the immunogenicity and protective efficacy of IpaB and IpaD, alone or combined, coadministered with a double mutant heat-labile toxin (dmLT) from Escherichia coli, used as a mucosal adjuvant, in a mouse model of intranasal immunization and pulmonary challenge. Robust systemic and mucosal antibody- and T cell-mediated immunities were induced against both proteins, particularly IpaB. Mice immunized in the presence of dmLT with IpaB alone or IpaB combined with IpaD were fully protected against lethal pulmonary infection with Shigella flexneri and Shigella sonnei. We provide the first demonstration that the Shigella TTSAs IpaB and IpaD are promising antigens for the development of a cross-protective Shigella vaccine. PMID:22202122

  7. In vivo inhibition of nitric oxide synthase impairs upregulation of contractile protein mRNA in overloaded plantaris muscle.

    PubMed

    Sellman, Jeff E; DeRuisseau, Keith C; Betters, Jenna L; Lira, Vitor A; Soltow, Quinlyn A; Selsby, Joshua T; Criswell, David S

    2006-01-01

    Inhibition of nitric oxide synthase (NOS) activity in vivo impedes hypertrophy in the overloaded rat plantaris. We investigated the mechanism for this effect by examining early events leading to muscle growth following 5 or 12 days of functional overload. Male Sprague-Dawley rats (approximately 350 g) were randomly divided into three treatment groups: control, N(G)-nitro-L-arginine methyl ester (L-NAME; 90 mg.kg(-1).day(-1)), and 1-(2-trifluoromethyl-phenyl)-imidazole (TRIM; 10 mg.kg(-1).day(-1)). Unilateral removal of synergists induced chronic overload (OL) of the right plantaris. Sham surgery performed on the left hindlimb served as a normally loaded control. L-NAME and TRIM treatments prevented OL-induced skeletal alpha-actin and type I (slow) myosin heavy chain mRNA expression at 5 days. Conversely, neither L-NAME nor TRIM affected hepatocyte growth factor or VEGF mRNA responses to OL at 5 days. However, OL induction of IGF-I and mechanogrowth factor mRNA was greater (P < 0.05) in the TRIM group compared with the controls. Furthermore, the phosphorylated-to-total p70 S6 kinase ratio was higher in OL muscle from NOS-inhibited groups, compared with control OL. At 12 days of OL, the cumulative proliferation of plantaris satellite cells was assessed by subcutaneous implantation of time release 5'-bromo-2'-deoxyuridine pellets during the OL-inducing surgeries. Although OL caused a fivefold increase in the number of mitotically active (5'-bromo-2'-deoxyuridine positive) sublaminar nuclei, this was unaffected by concurrent NOS inhibition. Therefore, NOS activity may provide negative feedback control of IGF-I/p70 S6 kinase signaling during muscle growth. Moreover, NOS activity may be involved in transcriptional regulation of skeletal alpha-actin and type I (slow) myosin heavy chain during functional overload. PMID:16166235

  8. Post-transcriptional regulation of the human inducible nitric oxide synthase (iNOS) expression by the cytosolic poly(A)-binding protein (PABP).

    PubMed

    Casper, Ingrid; Nowag, Sebastian; Koch, Kathrin; Hubrich, Thomas; Bollmann, Franziska; Henke, Jenny; Schmitz, Katja; Kleinert, Hartmut; Pautz, Andrea

    2013-09-01

    Affinity purification using the 3'-untranslated region (3'-UTR) of the human inducible nitric oxide synthase (iNOS) mRNA identified the cytosolic poly(A)-binding protein (PABP) as a protein interacting with the human iNOS 3'-UTR. Downregulation of PABP expression by RNA interference resulted in a marked reduction of cytokine-induced iNOS mRNA expression without changes in the expression of mRNAs coding for the major subunit of the RNA polymerase II (Pol 2A) or β2-microglobuline (β2M). Along with the mRNA also iNOS protein expression was reduced by siPABP-treatment, whereas in the same cells protein expression of STAT-1α, NF-κB p65, or GAPDH was not altered. Reporter gene analyses showed no change of the inducibility of the human 16kb iNOS promoter in siPABP cells. In contrast, the siPABP-mediated decline of iNOS expression correlated with a reduction in the stability of the iNOS mRNA. As the stability of the Pol 2A and β2M mRNA was not changed, siPABP-treatment seems to have a specific effect on iNOS mRNA decay. UV-crosslinking experiments revealed that PABP interacts with one binding site in the 5'-UTR and two different binding sites in the 3'-UTR of the human iNOS mRNA. Mutation or deletion of the binding site in the 5'-UTR but not in the 3'-UTR reduced luciferase expression in DLD-1 cells transfected with iNOS-5'-UTR or iNOS-3'-UTR luciferase reporter constructs. In summary, our data demonstrate that PABP by binding to specific sequence elements in the 5'-UTR post-transcriptionally enhances human iNOS mRNA stability and thereby iNOS expression. PMID:23711718

  9. Tandem repeats of the extracellular domain of Matrix 2 influenza protein exposed in Brucella lumazine synthase decameric carrier molecule induce protection in mice.

    PubMed

    Alvarez, Paula; Zylberman, Vanesa; Ghersi, Giselle; Boado, Lorena; Palacios, Carlos; Goldbaum, Fernando; Mattion, Nora

    2013-01-21

    The antigenic variation of influenza virus represents a major prevention problem. However, the ectodomain of the protein Matrix 2 (M2e) is nearly invariant in all human influenza A strains and has been considered as a promising candidate for a broadly protective vaccine because antibodies to M2e are protective in animal models. In this work we evaluated the possible use of Brucella abortus lumazine synthase protein (BLS), a highly immunogenic decameric protein, as a carrier of the M2e peptide. Chimeric proteins generated by the fusion of one or four in tandem copies of M2e to BLS were efficiently expressed in Escherichia coli and assembled in decameric subunits similarly to the wild type BLS enzyme, as demonstrated by the comparative circular dichroism spectra and size exclusion chromatography and static light scattering analysis. The M2e peptides were stably exposed at the ten N-terminal ends of each BLS molecule. Immunization of mice with purified chimeras carrying only one M2e (BLS-M2e) copy elicited a significant humoral immune response with the addition of different adjuvants. The fusion of four in tandem copies of the M2e peptide (BLS-4M2e) resulted in similar levels of humoral immune response but in the absence of adjuvant. Survival of mice challenged with live influenza virus was 100% after vaccination with BLS-4M2e adjuvanted with Iscomatrix(®) (P<0.001) and 80% when adjuvanted with alum (P<0.01), while the chimera alone protected 60% of the animals (P<0.05). The approach described in this study is intended as a contribution to the generation of universal influenza immunogens, through a simple production and purification process and using safe carriers that might eventually avoid the use of strong adjuvants. PMID:23246552

  10. Overexpression of Isoforms of Nitric Oxide Synthase 1 Adaptor Protein, Encoded by a Risk Gene for Schizophrenia, Alters Actin Dynamics and Synaptic Function

    PubMed Central

    Hernandez, Kristina; Swiatkowski, Przemyslaw; Patel, Mihir V.; Liang, Chen; Dudzinski, Natasha R.; Brzustowicz, Linda M.; Firestein, Bonnie L.

    2016-01-01

    Proper communication between neurons depends upon appropriate patterning of dendrites and correct distribution and structure of spines. Schizophrenia is a neuropsychiatric disorder characterized by alterations in dendrite branching and spine density. Nitric oxide synthase 1 adaptor protein (NOS1AP), a risk gene for schizophrenia, encodes proteins that are upregulated in the dorsolateral prefrontal cortex (DLPFC) of individuals with schizophrenia. To elucidate the effects of NOS1AP overexpression observed in individuals with schizophrenia, we investigated changes in actin dynamics and spine development when a long (NOS1AP-L) or short (NOS1AP-S) isoform of NOS1AP is overexpressed. Increased NOS1AP-L protein promotes the formation of immature spines when overexpressed in rat cortical neurons from day in vitro (DIV) 14 to DIV 17 and reduces the amplitude of miniature excitatory postsynaptic currents (mEPSCs). In contrast, increased NOS1AP-S protein increases the rate of actin polymerization and the number of immature and mature spines, which may be attributed to a decrease in total Rac1 expression and a reduction in the levels of active cofilin. The increase in the number of mature spines by overexpression of NOS1AP-S is accompanied by an increase in the frequency of mEPSCs. Our findings show that overexpression of NOS1AP-L or NOS1AP-S alters the actin cytoskeleton and synaptic function. However, the mechanisms by which these isoforms induce these changes are distinct. These results are important for understanding how increased expression of NOS1AP isoforms can influence spine development and synaptic function. PMID:26869880

  11. Overexpression of Isoforms of Nitric Oxide Synthase 1 Adaptor Protein, Encoded by a Risk Gene for Schizophrenia, Alters Actin Dynamics and Synaptic Function.

    PubMed

    Hernandez, Kristina; Swiatkowski, Przemyslaw; Patel, Mihir V; Liang, Chen; Dudzinski, Natasha R; Brzustowicz, Linda M; Firestein, Bonnie L

    2016-01-01

    Proper communication between neurons depends upon appropriate patterning of dendrites and correct distribution and structure of spines. Schizophrenia is a neuropsychiatric disorder characterized by alterations in dendrite branching and spine density. Nitric oxide synthase 1 adaptor protein (NOS1AP), a risk gene for schizophrenia, encodes proteins that are upregulated in the dorsolateral prefrontal cortex (DLPFC) of individuals with schizophrenia. To elucidate the effects of NOS1AP overexpression observed in individuals with schizophrenia, we investigated changes in actin dynamics and spine development when a long (NOS1AP-L) or short (NOS1AP-S) isoform of NOS1AP is overexpressed. Increased NOS1AP-L protein promotes the formation of immature spines when overexpressed in rat cortical neurons from day in vitro (DIV) 14 to DIV 17 and reduces the amplitude of miniature excitatory postsynaptic currents (mEPSCs). In contrast, increased NOS1AP-S protein increases the rate of actin polymerization and the number of immature and mature spines, which may be attributed to a decrease in total Rac1 expression and a reduction in the levels of active cofilin. The increase in the number of mature spines by overexpression of NOS1AP-S is accompanied by an increase in the frequency of mEPSCs. Our findings show that overexpression of NOS1AP-L or NOS1AP-S alters the actin cytoskeleton and synaptic function. However, the mechanisms by which these isoforms induce these changes are distinct. These results are important for understanding how increased expression of NOS1AP isoforms can influence spine development and synaptic function. PMID:26869880

  12. Lack of immunogenicity of ice structuring protein type III HPLC12 preparation administered by the oral route to human volunteers.

    PubMed

    Crevel, R W R; Cooper, K J; Poulsen, L K; Hummelshoj, L; Bindslev-Jensen, C; Burks, A W; Sampson, H A

    2007-01-01

    Before a novel protein can be used in foods, its potential allergenicity must be assessed. In this study, healthy volunteers consumed ice structuring protein (ISP) Type III preparation or a control material 5 days a week for a total of 8 weeks. General measures of health were recorded during the study, and the immunogenicity of the protein was assessed by monitoring the levels of IgG and IgE antibodies specific for ISP Type III. The participants remained in good health throughout the study and during the 4 week follow-up period. No IgG or IgE antibodies specific for ISP Type III were detected in the blood of the participants. Investigations of immunogenicity in man have not been previously applied in the context of safety evaluation and they do not form part of the regimens proposed for the evaluation of protein allergenicity. Consequently no standardised protocols exist for such studies, nor any background against which to interpret the results. Nevertheless, the absence of an immune response using a protocol which could have been expected to result in a response with a strongly immunogenic protein, confirms the conclusions of earlier published work, and attests to the lack of allergenicity of ISP Type III preparation. PMID:17027137

  13. [Preparation and crystallization of Polygonum cuspidatum benzalacetone synthase].

    PubMed

    Ma, Wenrui; Liu, Chunmei; Yang, Mingfeng; Xue, Feiyan; Chen, Qing; Ma, Lanqing; Lü, Heshu

    2016-02-01

    The chalcone synthase (CHS) superfamily of the type III polyketide synthases (PKSs) generates backbones of a variety of plant secondary metabolites. Benzalacetone synthase (BAS) catalyzes a condensation reaction of decarboxylation between the substrates of 4-coumaric coenzyme A and malonyl coenzyme A to generate benzylidene acetone, whose derivatives are series of compounds with various biological activities. A BAS gene Pcpks2 and a bifunctional CHS/BAS PcPKSI were isolated from medicinal plant P. cuspidatum. Crystallographic and structure-based mutagenesis studies indicate that the functional diversity of the CHS-superfamily enzymes is principally derived from small modifications of the active site architecture. In order to obtain an understanding of the biosynthesis of polyketides in P. cuspidatum, which has been poorly described, as well as of its activation mechanism, PcPKS2 was overexpressed in Escherichia coli as a C-terminally poly-His-tagged fusion protein, purified to homogeneity and crystallized, which is helpful for the clarification of the catalytic mechanism of the enzyme and lays the foundation for its genetic engineering manipulation. PMID:27382775

  14. Photosynthetic activity and protein overexpression found in Cr(III)-tolerant cells of the green algae Dictyosphaerium chlorelloides.

    PubMed

    Pereira, M; Bartolomé, C M; Sánchez-Fortún, S

    2014-08-01

    Chromium is an important constituent in effluents obtained from chromium plating industries. Due to the highly toxic nature of Cr(VI), attention has been shifted to less hazardous Cr(III) electroplating processes. This study evaluated aquatic toxicity of Cr(III)-containing laboratory samples representative of effluents from chromium electroplating industries, on the photosynthetic activity exhibited by both Cr(III)-sensitive (Dc1M(wt)) and tolerant (Dc1M(Cr(III)R30)) Dictyosphaerium chlorelloides strains. Additionally, selected de novo-determined peptide sequences, obtained from Dc1M(Cr(III)R30), have been analyzed to evidence the possible Cr(III) toxic mechanism involved in the resistance of these cells to high Cr(III) levels in aquatic environments. Dc1M(Cr(III)R30) strain exhibited a gross photosynthetic balance of about five times lower than that exhibited by Dc1M(wt) strain, demonstrating that Dc1M(Cr(III)R30) has a photosynthetic yield significantly lower than Dc1M(wt). SDS-PAGE of Dc1M(Cr(III)R30) samples showed the presence of at least two protein bands (23.05 and 153.46 KDa, respectively) absent in wild-type strain samples. Although it has achieved a low coincidence between the lower molecular weight band and a GTPase identified from genome of the green alga Chlamydomonas reinhardtii, none of de novo peptide sequences obtained showed a significant MS-BLAST score, so that further studies will be required. PMID:24556547

  15. Differences in protein binding and excretion of Triapine and its Fe(III) complex.

    PubMed

    Pelivan, Karla; Miklos, Walter; van Schoonhoven, Sushilla; Koellensperger, Gunda; Gille, Lars; Berger, Walter; Heffeter, Petra; Kowol, Christian R; Keppler, Bernhard K

    2016-07-01

    Triapine has been investigated as anticancer drug in multiple clinical phase I/II trials. Although promising anti-leukemic activity was observed, Triapine was ineffective against solid tumors. The reasons are currently widely unknown. The biological activity of Triapine is strongly connected to its iron complex (Fe-Triapine) which is pharmacologically not investigated. Here, novel analytical tools for Triapine and Fe-Triapine were developed and applied for cell extracts and body fluids of treated mice. Triapine and its iron complex showed a completely different behavior: for Triapine, low protein binding was observed in contrast to fast protein adduct formation of Fe-Triapine. Notably, both drugs were rapidly cleared from the body (serum half-life time <1h). Remarkably, in contrast to Triapine, where (in accordance to clinical data) basically no renal excretion was found, the iron complex was effectively excreted via urine. Moreover, no Fe-Triapine was detected in serum or cytosolic extracts after Triapine treatment. Taken together, our study will help to further understand the biological behavior of Triapine and its Fe-complex and allow the development of novel thiosemicarbazones with pronounced activity against solid tumor types. PMID:26507768

  16. The poly(A)-binding protein Nab2 functions in RNA polymerase III transcription

    PubMed Central

    Reuter, L. Maximilian; Meinel, Dominik M.; Sträßer, Katja

    2015-01-01

    RNA polymerase III (RNAPIII) synthesizes most small RNAs, the most prominent being tRNAs. Although the basic mechanism of RNAPIII transcription is well understood, recent evidence suggests that additional proteins play a role in RNAPIII transcription. Here, we discovered by a genome-wide approach that Nab2, a poly(A)-binding protein important for correct poly(A) tail length and nuclear mRNA export, is present at all RNAPIII transcribed genes. The occupancy of Nab2 at RNAPIII transcribed genes is dependent on transcription. Using a novel temperature-sensitive allele of NAB2, nab2-34, we show that Nab2 is required for the occupancy of RNAPIII and TFIIIB at target genes. Furthermore, Nab2 interacts with RNAPIII, TFIIIB, and RNAPIII transcripts. Importantly, impairment of Nab2 function causes an RNAPIII transcription defect in vivo and in vitro. Taken together, we establish Nab2, an important mRNA biogenesis factor, as a novel player required for RNAPIII transcription by stabilizing TFIIIB and RNAPIII at promoters. PMID:26220998

  17. Light-Activated Protein Inhibition through Photoinduced Electron Transfer of a Ruthenium(II)–Cobalt(III) Bimetallic Complex

    PubMed Central

    Holbrook, Robert J.; Weinberg, David J.; Peterson, Mark D.; Weiss, Emily A.; Meade, Thomas J.

    2015-01-01

    We describe a mechanism of light activation that initiates protein inhibitory action of a biologically inert Co(III) Schiff base (Co(III)-sb) complex. Photoinduced electron transfer (PET) occurs from a Ru(II) bipyridal complex to a covalently attached Co(III) complex and is gated by conformational changes that occur in tens of nanoseconds. Reduction of the Co(III)-sb by PET initiates displacement of the inert axial imidazole ligands, promoting coordination to active site histidines of α-thrombin. Upon exposure to 455 nm light, the rate of ligand exchange with 4-methylimidazole, a histidine mimic, increases by approximately 5-fold, as observed by NMR spectroscopy. Similarly, the rate of α-thrombin inhibition increases over 5-fold upon irradiation. These results convey a strategy for light activation of inorganic therapeutic agents through PET utilizing redox-active metal centers. PMID:25671465

  18. Polyester synthases: natural catalysts for plastics.

    PubMed Central

    Rehm, Bernd H A

    2003-01-01

    Polyhydroxyalkanoates (PHAs) are biopolyesters composed of hydroxy fatty acids, which represent a complex class of storage polyesters. They are synthesized by a wide range of different Gram-positive and Gram-negative bacteria, as well as by some Archaea, and are deposited as insoluble cytoplasmic inclusions. Polyester synthases are the key enzymes of polyester biosynthesis and catalyse the conversion of (R)-hydroxyacyl-CoA thioesters to polyesters with the concomitant release of CoA. These soluble enzymes turn into amphipathic enzymes upon covalent catalysis of polyester-chain formation. A self-assembly process is initiated resulting in the formation of insoluble cytoplasmic inclusions with a phospholipid monolayer and covalently attached polyester synthases at the surface. Surface-attached polyester synthases show a marked increase in enzyme activity. These polyester synthases have only recently been biochemically characterized. An overview of these recent findings is provided. At present, 59 polyester synthase structural genes from 45 different bacteria have been cloned and the nucleotide sequences have been obtained. The multiple alignment of the primary structures of these polyester synthases show an overall identity of 8-96% with only eight strictly conserved amino acid residues. Polyester synthases can been assigned to four classes based on their substrate specificity and subunit composition. The current knowledge on the organization of the polyester synthase genes, and other genes encoding proteins related to PHA metabolism, is compiled. In addition, the primary structures of the 59 PHA synthases are aligned and analysed with respect to highly conserved amino acids, and biochemical features of polyester synthases are described. The proposed catalytic mechanism based on similarities to alpha/beta-hydrolases and mutational analysis is discussed. Different threading algorithms suggest that polyester synthases belong to the alpha/beta-hydrolase superfamily, with

  19. Endothelial nitric-oxide synthase antisense (NOS3AS) gene encodes an autophagy-related protein (APG9-like2) highly expressed in trophoblast.

    PubMed

    Yamada, Takahiro; Carson, Andrew R; Caniggia, Isabella; Umebayashi, Kyohei; Yoshimori, Tamotsu; Nakabayashi, Kazuhiko; Scherer, Stephen W

    2005-05-01

    Macroautophagy is an intracellular degradation system for the majority of proteins and some organelles that is conserved in all eukaryotic species. The precise role of autophagy in mammalian development and potential involvement in disease remain to be discerned. Yeast Atg9p is the first integral membrane protein shown to be essential for the cytoplasm to vacuole targeting (Cvt) pathway and autophagy, whereas its mammalian functional orthologue has yet to be identified. We have identified two human genes homologous to yeast Atg9p and designated these as APG9L1 and APG9L2. We have previously identified APG9L2 as NOS3AS, which participates in the post-transcriptional regulation of the endothelial nitric-oxide synthase (NOS3) gene on chromosome 7 through its antisense overlap. In human adult tissues, APG9L1 was ubiquitously expressed, whereas APG9L2 was highly expressed in placenta (trophoblast cells) and pituitary gland. In transient transfection assays we found that both proteins were primarily localized to the perinuclear region and also scattered throughout the cytosol as dots, a subset of which colocalized with an autophagosome-specific marker LC3 under starvation conditions. Finally, by the small interfering RNA-mediated knockdown of APG9L1 in HeLa cells, we demonstrated that APG9L1 is essential for starvation-induced autophagosome formation. In addition, APG9L2 can functionally complement APG9L1 in this process. These results, taken together with those of phylogenetic and sequence analyses, suggest that both APG9L1 and APG9L2 are functionally orthologous to the yATG9 in autophagosome formation. Moreover, APG9L2 is a vertebrate-specific gene that may have gained critical roles in mammalian-specific developmental events, such as placentation, through rapid evolution. PMID:15755735

  20. Functional and evolutionary analysis of DXL1, a non-essential gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase like protein in Arabidopsis thaliana.

    PubMed

    Carretero-Paulet, Lorenzo; Cairó, Albert; Talavera, David; Saura, Andreu; Imperial, Santiago; Rodríguez-Concepción, Manuel; Campos, Narciso; Boronat, Albert

    2013-07-15

    The synthesis of 1-deoxy-D-xylulose 5-phosphate (DXP), catalyzed by the enzyme DXP synthase (DXS), represents a key regulatory step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis. In plants DXS is encoded by small multigene families that can be classified into, at least, three specialized subfamilies. Arabidopsis thaliana contains three genes encoding proteins with similarity to DXS, including the well-known DXS1/CLA1 gene, which clusters within subfamily I. The remaining proteins, initially named DXS2 and DXS3, have not yet been characterized. Here we report the expression and functional analysis of A. thaliana DXS2. Unexpectedly, the expression of DXS2 failed to rescue Escherichia coli and A. thaliana mutants defective in DXS activity. Coherently, we found that DXS activity was negligible in vitro, being renamed as DXL1 following recent nomenclature recommendation. DXL1 is targeted to plastids as DXS1, but shows a distinct expression pattern. The phenotypic analysis of a DXL1 defective mutant revealed that the function of the encoded protein is not essential for growth and development. Evolutionary analyses indicated that DXL1 emerged from DXS1 through a recent duplication apparently specific of the Brassicaceae lineage. Divergent selective constraints would have affected a significant fraction of sites after diversification of the paralogues. Furthermore, amino acids subjected to divergent selection and likely critical for functional divergence through the acquisition of a novel, although not yet known, biochemical function, were identified. Our results provide with the first evidences of functional specialization at both the regulatory and biochemical level within the plant DXS family. PMID:23154062

  1. Alterations in Fibronectin Type III Domain Containing 1 Protein Gene Are Associated with Hypertension

    PubMed Central

    Deng, Alan Y.; Chauvet, Cristina; Ménard, Annie

    2016-01-01

    Multiple quantitative trait loci (QTLs) for blood pressure (BP) have been detected in rat models of human polygenic hypertension. Great challenges confronting us include molecular identifications of individual QTLs. We first defined the chromosome region harboring C1QTL1 to a segment of 1.9 megabases that carries 9 genes. Among them, we identified the gene encoding the fibronectin type III domain containing 1 protein (Fndc1)/activator of G protein signaling 8 (Ags8) to be the strongest candidate for C1QTL1, since numerous non-synonymous mutations are found. Moreover, the 5’ Fndc1/Ags8 putative promoter contains numerous mutations that can account for its differential expression in kidneys and the heart, prominent organs in modulating BP, although the Fndc1/Ags8 protein was not detectable in these organs under our experimental conditions. This work has provided the premier evidence that Fndc1/Ags8 is a novel and strongest candidate gene for C1QTL1 without completely excluding other 8 genes in the C1QTL1-residing interval. If proven true by future in vivo function studies such as single-gene Fndc1/Ags8 congenics, transgenesis or targeted-gene modifications, it might represent a part of the BP genetic architecture that operates in the upstream position distant from the end-phase physiology of BP control, since it activates a Gbetagamma component in a signaling pathway. Its functional role could validate the concept that a QTL in itself can influence BP ‘indirectly’ by regulating other genes downstream in a pathway. The elucidation of the mechanisms initiated by Fndc/Ags8 variations will reveal novel insights into the BP modulation via a regulatory hierarchy. PMID:27064407

  2. Identification of an abundant 56 kDa protein implicated in food allergy as granule-bound starch synthase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rice, the staple food of South and East Asian counties, is considered to be hypoallergenic. However, several clinical studies have documented rice-induced allergy in sensitive patients. Rice proteins with molecular weights of 14-16 kDa, 26 kDa, 33 kDa and 56 kDa have been identified as allergens. Re...

  3. Die another day: molecular mechanisms of effector-triggered immunity elicited by type III secreted effector proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial pathogens inject type III secreted effector (T3SE) proteins into their hosts where they display dual roles depending on the host genotype. T3SEs promote bacterial virulence in susceptible hosts, and elicit immunity in resistant hosts. T3SEs are typically recognized when they modify a host ...

  4. In silico analysis of an envelope domain III-based multivalent fusion protein as a potential dengue vaccine candidate

    PubMed Central

    2016-01-01

    Purpose Dengue virus infection is now a global problem. Currently, there is no licensed vaccine or proven antiviral treatment against this virus. All four serotypes (1-4) of dengue virus can infect human. An effective dengue vaccine should be tetravalent to induce protective immune responses against all four serotypes. Most of dengue vaccine candidates are monovalent, or in the form of physically mixed multivalent formulations. Recently envelope protein domain III of virus is considered as a vaccine candidate, which plays critical roles in the most important viral activities. Development of a tetravalent protein subunit vaccine is very important for equal induction of immune system and prevention of unbalanced immunity. Here, we have presented and used a rational approach to design a tetravalent dengue vaccine candidate. Materials and Methods We designed a multi domain antigen by fusing four consensus domain III sequences together with appropriate hydrophobic linkers and used several types of bioinformatics software and neural networks to predict structural and immunological properties of the designed tetravalent antigen. Results We designed a tetravalent protein (EDIIIF) based on domain III of dengue virus envelope protein. According to the results of the bioinformatics analysis, the constructed models for EDIIIF protein were structurally stable and potentially immunogenic. Conclusion The designed tetravalent protein can be considered as a potential dengue vaccine candidate. The presented approach can be used for rational design and in silico evaluation of chimeric dengue vaccine candidates. PMID:26866023

  5. Effects of type III antifreeze protein on sperm and embryo cryopreservation in rabbit.

    PubMed

    Nishijima, Kazutoshi; Tanaka, Mai; Sakai, Yusuke; Koshimoto, Chihiro; Morimoto, Masatoshi; Watanabe, Teruo; Fan, Jianglin; Kitajima, Shuji

    2014-08-01

    We investigated the effects of antifreeze protein (AFP) III supplementation on the cryopreservation of rabbit sperm cells and embryos. Ejaculated semen was collected from male Japanese white (JW) rabbits and divided into four AFP-supplemented groups (0.1 μg/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml) and one control group with no AFP-supplementation. The semen samples were treated with egg-yolk HEPES extender containing 6% acetamide before the sperm was cooled from room temperature to 5 °C, then packed into sperm straws. The straws were frozen in steam of liquid nitrogen (LN2) and then preserved in the LN2. The motility of the sperm after thawing in 37 °C water was analyzed. The percentage of rapidly motile sperm in the 1 μg/ml AFP group was significantly higher than in the control group. Morulae were collected from female JW rabbits and divided into three AFP-supplemented groups (100 ng/ml, 500 ng/ml, 1000 ng/ml) and one control group. The morulae, immersed in an embryo-freezing solution (M199-HEPES containing 20% ethylene glycol, 20% dimethylsulfoxide, 10% fetal bovine serum and 0.25 M sucrose), were packed into open pulled embryo straws and vitrified in LN2. The frozen embryos were thawed in the embryo-freezing solution, and the rates of embryo survival and development to blastocyte stage were analyzed after incubation for 72 h. The development rate of the embryos in the 500 ng/ml AFP group was significantly higher than in the control group, but that in the 1000 ng/ml AFP group was significantly lower. In conclusion, the appropriate dose of AFP III increased the number of rapidly motile sperm and embryo survival following freezing and thawing. The results suggest that supplementation with AFP III can increase the efficiency of cryopreservation of rabbit sperm cells and embryos. PMID:24809634

  6. The role of surface electrostatics on the stability, function and regulation of human cystathionine β-synthase, a complex multidomain and oligomeric protein

    PubMed Central

    Pey, Angel L.; Majtan, Tomas; Kraus, Jan P.

    2014-01-01

    Human cystathionine β-synthase (hCBS) is a key enzyme of sulfur amino acid metabolism, controlling the commitment of homocysteine to the transsulfuration pathway and antioxidant defense. Mutations in hCBS cause inherited homocystinuria (HCU), a rare inborn error of metabolism characterized by accumulation of toxic homocysteine in blood and urine. hCBS is a complex multidomain and oligomeric protein whose activity and stability is independently regulated by the binding of S-adenosyl-methionine (SAM) to two different type of sites at its C-terminal regulatory domain. Here we study the role of surface electrostatics on the complex regulation and stability of hCBS using biophysical and biochemical procedures. We show that the kinetic stability of the catalytic and regulatory domains is significantly affected by the modulation of surface electrostatics through noticeable structural and energetic changes along their denaturation pathways. We also show that surface electrostatics strongly affect SAM binding properties to those sites responsible for either enzyme activation or kinetic stabilization. Our results provide new insight into the regulation of hCBS activity and stability in vivo with implications for understanding HCU as a conformational disease. We also lend experimental support to the role of electrostatic interactions in the recently proposed binding modes of SAM leading to hCBS activation and kinetic stabilization. PMID:24780582

  7. Statins and the squalene synthase inhibitor zaragozic acid stimulate the non-amyloidogenic pathway of amyloid-beta protein precursor processing by suppression of cholesterol synthesis.

    PubMed

    Kojro, Elzbieta; Füger, Petra; Prinzen, Claudia; Kanarek, Anna Maria; Rat, Dorothea; Endres, Kristina; Fahrenholz, Falk; Postina, Rolf

    2010-01-01

    Cholesterol-lowering drugs such as statins influence the proteolytic processing of the amyloid-beta protein precursor (AbetaPP) and are reported to stimulate the activity of alpha-secretase, the major preventive secretase of Alzheimer's disease. Statins can increase the alpha-secretase activity by their cholesterol-lowering properties as well as by impairment of isoprenoids synthesis. In the present study, we elucidate the contribution of these pathways in alpha-secretase activation. We demonstrate that zaragozic acid, a potent inhibitor of squalene synthase which blocks cholesterol synthesis but allows synthesis of isoprenoids, also stimulates alpha-secretase activity. Treatment of human neuroblastoma cells with 50 microM zaragozic acid resulted in a approximately 3 fold increase of alpha-secretase activity and reduced cellular cholesterol by approximately 30%. These effects were comparable to results obtained from cells treated with a low lovastatin concentration (2 microM). Zaragozic acid-stimulated secretion of alpha-secretase-cleaved soluble AbetaPP was dose dependent and saturable. Lovastatin- or zaragozic acid-stimulated increase of alpha-secretase activity was completely abolished by a selective ADAM10 inhibitor. By targeting the alpha-secretase ADAM10 to lipid raft domains via a glycosylphosphatidylinositol anchor, we demonstrate that ADAM10 is unable to cleave AbetaPP in a cholesterol-rich environment. Our results indicate that inhibition of cholesterol biosynthesis by a low lovastatin concentration is sufficient for alpha-secretase activation. PMID:20413873

  8. Association of Cholesteryl Ester Transfer Protein and Endothelial Nitric Oxide Synthase Gene Polymorphisms With Coronary Artery Disease in the Multi-Ethnic Malaysian Population.

    PubMed

    Chu, Wern Cui; Aziz, Ahmad Fazli Abdul; Nordin, Abdul Jalil; Cheah, Yoke Kqueen

    2016-09-01

    Genetic variants of cholesteryl ester transfer protein (CETP) and endothelial nitric oxide synthase (eNOS) influence high-density lipoprotein cholesterol (HDL-C) metabolism and nitric oxide (NO) synthesis, respectively, and might increase the risk of coronary artery disease (CAD). This study is to investigate the relationship between genetic polymorphisms and the risk of CAD and to evaluate their potential interactions. A total of 237 patients with CAD and 101 controls were genotyped. The association of the polymorphism with the risk of CAD varied among the ethnic groups. Moreover, the concomitant presence of both CETP B1 and eNOS 4a alleles significantly increased the risk of CAD in the Malay group (OR = 33.8, P < .001) and the Indian group (OR = 10.9, P = .031) but not in the Chinese group. This study has identified a novel ethnic-specific gene-gene interaction and suggested that the combination of CETP B1 allele and eNOS 4a allele significantly increases the risk of CAD in Malays and Indians. PMID:25667236

  9. β-Ketoacyl-acyl Carrier Protein Synthase I (KASI) Plays Crucial Roles in the Plant Growth and Fatty Acids Synthesis in Tobacco

    PubMed Central

    Yang, Tianquan; Xu, Ronghua; Chen, Jianghua; Liu, Aizhong

    2016-01-01

    Fatty acids serve many functions in plants, but the effects of some key genes involved in fatty acids biosynthesis on plants growth and development are not well understood yet. To understand the functions of 3-ketoacyl-acyl-carrier protein synthase I (KASI) in tobacco, we isolated two KASI homologs, which we have designated NtKASI-1 and NtKASI-2. Expression analysis showed that these two KASI genes were transcribed constitutively in all tissues examined. Over-expression of NtKASI-1 in tobacco changed the fatty acid content in leaves, whereas over-expressed lines of NtKASI-2 exhibited distinct phenotypic features such as slightly variegated leaves and reduction of the fatty acid content in leaves, similar to the silencing plants of NtKASI-1 gene. Interestingly, the silencing of NtKASI-2 gene had no discernibly altered phenotypes compared to wild type. The double silencing plants of these two genes enhanced the phenotypic changes during vegetative and reproductive growth compared to wild type. These results uncovered that these two KASI genes had the partially functional redundancy, and that the KASI genes played a key role in regulating fatty acids synthesis and in mediating plant growth and development in tobacco. PMID:27509494

  10. Crocin Suppresses LPS-Stimulated Expression of Inducible Nitric Oxide Synthase by Upregulation of Heme Oxygenase-1 via Calcium/Calmodulin-Dependent Protein Kinase 4

    PubMed Central

    Kim, Ji-Hee; Park, Ga-Young; Bang, Soo Young; Park, Sun Young; Bae, Soo-Kyung; Kim, YoungHee

    2014-01-01

    Crocin is a water-soluble carotenoid pigment that is primarily used in various cuisines as a seasoning and coloring agent, as well as in traditional medicines for the treatment of edema, fever, and hepatic disorder. In this study, we demonstrated that crocin markedly induces the expression of heme oxygenase-1 (HO-1) which leads to an anti-inflammatory response. Crocin inhibited inducible nitric oxide synthase (iNOS) expression and nitric oxide production via downregulation of nuclear factor kappa B activity in lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophages. These effects were abrogated by blocking of HO-1 expression or activity. Crocin also induced Ca2+ mobilization from intracellular pools and phosphorylation of Ca2+/calmodulin-dependent protein kinase 4 (CAMK4). CAMK4 knockdown and kinase-dead mutant inhibited crocin-mediated HO-1 expression, Nrf2 activation, and phosphorylation of Akt, indicating that HO-1 expression is mediated by CAMK4 and that Akt is a downstream mediator of CAMK4 in crocin signaling. Moreover, crocin-mediated suppression of iNOS expression was blocked by CAMK4 inhibition. Overall, these results suggest that crocin suppresses LPS-stimulated expression of iNOS by inducing HO-1 expression via Ca2+/calmodulin-CAMK4-PI3K/Akt-Nrf2 signaling cascades. Our findings provide a novel molecular mechanism for the inhibitory effects of crocin against endotoxin-mediated inflammation. PMID:24839356

  11. A Nucleus-Encoded Chloroplast Protein YL1 Is Involved in Chloroplast Development and Efficient Biogenesis of Chloroplast ATP Synthase in Rice

    PubMed Central

    Chen, Fei; Dong, Guojun; Wu, Limin; Wang, Fang; Yang, Xingzheng; Ma, Xiaohui; Wang, Haili; Wu, Jiahuan; Zhang, Yanli; Wang, Huizhong; Qian, Qian; Yu, Yanchun

    2016-01-01

    Chloroplast ATP synthase (cpATPase) is an importance thylakoid membrane-associated photosynthetic complex involved in the light-dependent reactions of photosynthesis. In this study, we isolated and characterized a rice (Oryza sativa) mutant yellow leaf 1 (yl1), which exhibits chlorotic leaves throughout developmental stages. The YL1 mutation showed reduced chlorophyll contents, abnormal chloroplast morphology, and decreased photochemical efficiency. Moreover, YL1 deficiency disrupts the expression of genes associated with chloroplast development and photosynthesis. Molecular and genetic analyses revealed that YL1 is a nucleus-encoded protein with a predicted transmembrane domain in its carboxyl-terminus that is conserved in the higher plant kingdom. YL1 localizes to chloroplasts and is preferentially expressed in green tissues containing chloroplasts. Immunoblot analyses showed that inactivation of YL1 leads to drastically reduced accumulation of AtpA (α) and AtpB (β), two core subunits of CF1αβ subcomplex of cpATPase, meanwhile, a severe decrease (ca. 41.7%) in cpATPase activity was observed in the yl1-1 mutant compared with the wild type. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation assays revealed a specific interaction between YL1 and AtpB subunit of cpATPase. Taken together, our results suggest that YL1 is a plant lineage-specific auxiliary factor involved in the biogenesis of the cpATPase complex, possibly via interacting with the β-subunit. PMID:27585744

  12. Nitric Oxide Synthase in the Central Nervous System and Peripheral Organs of Stramonita haemastoma: Protein Distribution and Gene Expression in Response to Thermal Stress

    PubMed Central

    Toni, Mattia; De Angelis, Federica; Bonaccorsi di Patti, Maria Carmela; Cioni, Carla

    2015-01-01

    Nitric oxide (NO) is generated via the oxidation of l-arginine by the enzyme NO synthase (NOS) both in vertebrates and invertebrates. Three NOS isoforms, nNOS, iNOS and eNOS, are known in vertebrates, whereas a single NOS isoform is usually expressed in invertebrates, sharing structural and functional characteristics with nNOS or iNOS depending on the species. The present paper is focused on the constitutive Ca2+/calmodulin-dependent nNOS recently sequenced by our group in the neogastropod Stramonita haemastoma (ShNOS). In this paper we provide new data on cellular distribution of ShNOS in the CNS (pedal ganglion) and peripheral organs (osphradium, tentacle, eye and foot) obtained by WB, IF, CM and NADPHd. Results demonstrated that NOS-like proteins are widely expressed in sensory receptor elements, neurons and epithelial cells. The detailed study of NOS distribution in peripheral and central neurons suggested that NOS is both intracellular and presynaptically located. Present findings confirm that NO may have a key role in the central neuronal circuits of gastropods and in sensory perception. The physiological relevance of NOS enzymes in the same organs was suggested by thermal stress experiments demonstrating that the constitutive expression of ShNOS is modulated in a time- and organ-dependent manner in response to environmental stressors. PMID:26528988

  13. β-Ketoacyl-acyl Carrier Protein Synthase I (KASI) Plays Crucial Roles in the Plant Growth and Fatty Acids Synthesis in Tobacco.

    PubMed

    Yang, Tianquan; Xu, Ronghua; Chen, Jianghua; Liu, Aizhong

    2016-01-01

    Fatty acids serve many functions in plants, but the effects of some key genes involved in fatty acids biosynthesis on plants growth and development are not well understood yet. To understand the functions of 3-ketoacyl-acyl-carrier protein synthase I (KASI) in tobacco, we isolated two KASI homologs, which we have designated NtKASI-1 and NtKASI-2. Expression analysis showed that these two KASI genes were transcribed constitutively in all tissues examined. Over-expression of NtKASI-1 in tobacco changed the fatty acid content in leaves, whereas over-expressed lines of NtKASI-2 exhibited distinct phenotypic features such as slightly variegated leaves and reduction of the fatty acid content in leaves, similar to the silencing plants of NtKASI-1 gene. Interestingly, the silencing of NtKASI-2 gene had no discernibly altered phenotypes compared to wild type. The double silencing plants of these two genes enhanced the phenotypic changes during vegetative and reproductive growth compared to wild type. These results uncovered that these two KASI genes had the partially functional redundancy, and that the KASI genes played a key role in regulating fatty acids synthesis and in mediating plant growth and development in tobacco. PMID:27509494

  14. The Structure of Rauvolfia serpentina Strictosidine Synthase Is a Novel Six-Bladed β-Propeller Fold in Plant Proteins[W

    PubMed Central

    Ma, Xueyan; Panjikar, Santosh; Koepke, Juergen; Loris, Elke; Stöckigt, Joachim

    2006-01-01

    The enzyme strictosidine synthase (STR1) from the Indian medicinal plant Rauvolfia serpentina is of primary importance for the biosynthetic pathway of the indole alkaloid ajmaline. Moreover, STR1 initiates all biosynthetic pathways leading to the entire monoterpenoid indole alkaloid family representing an enormous structural variety of ∼2000 compounds in higher plants. The crystal structures of STR1 in complex with its natural substrates tryptamine and secologanin provide structural understanding of the observed substrate preference and identify residues lining the active site surface that contact the substrates. STR1 catalyzes a Pictet-Spengler–type reaction and represents a novel six-bladed β-propeller fold in plant proteins. Structure-based sequence alignment revealed a common repetitive sequence motif (three hydrophobic residues are followed by a small residue and a hydrophilic residue), indicating a possible evolutionary relationship between STR1 and several sequence-unrelated six-bladed β-propeller structures. Structural analysis and site-directed mutagenesis experiments demonstrate the essential role of Glu-309 in catalysis. The data will aid in deciphering the details of the reaction mechanism of STR1 as well as other members of this enzyme family. PMID:16531499

  15. Glycogen synthase kinase-3-mediated phosphorylation of serine 73 targets sterol response element binding protein-1c (SREBP-1c) for proteasomal degradation.

    PubMed

    Dong, Qingming; Giorgianni, Francesco; Beranova-Giorgianni, Sarka; Deng, Xiong; O'Meally, Robert N; Bridges, Dave; Park, Edwards A; Cole, Robert N; Elam, Marshall B; Raghow, Rajendra

    2016-01-01

    Sterol regulatory element binding protein-1c (SREBP-1c) is a key transcription factor that regulates genes involved in the de novo lipid synthesis and glycolysis pathways. The structure, turnover and transactivation potential of SREBP-1c are regulated by macronutrients and hormones via a cascade of signalling kinases. Using MS, we have identified serine 73 as a novel glycogen synthase kinase-3 (GSK-3) phosphorylation site in the rat SREBP-1c purified from McA-RH7777 hepatoma cells. Our site-specific mutagenesis strategy revealed that the turnover of SREBP-1c, containing wild type, phospho-null (serine to alanine) or phospho-mimetic (serine to aspartic acid) substitutions, was differentially regulated. We show that the S73D mutant of pSREBP-1c, that mimicked a state of constitutive phosphorylation, dissociated from the SREBP-1c-SCAP complex more readily and underwent GSK-3-dependent proteasomal degradation via SCF(Fbw7) ubiquitin ligase pathway. Pharmacologic inhibition of GSK-3 or knockdown of GSK-3 by siRNA prevented accelerated degradation of SREBP-1c. As demonstrated by MS, SREBP-1c was phosphorylated in vitro by GSK-3β at serine 73. Phosphorylation of serine 73 also occurs in the intact liver. We propose that GSK-3-mediated phosphorylation of serine 73 in the rat SREBP-1c and its concomitant destabilization represents a novel mechanism involved in the inhibition of de novo lipid synthesis in the liver. PMID:26589965

  16. Glycogen synthase kinase-3-mediated phosphorylation of serine 73 targets sterol response element binding protein-1c (SREBP-1c) for proteasomal degradation

    PubMed Central

    Dong, Qingming; Giorgianni, Francesco; Beranova-Giorgianni, Sarka; Deng, Xiong; O'Meally, Robert N.; Bridges, Dave; Park, Edwards A.; Cole, Robert N.; Elam, Marshall B.; Raghow, Rajendra

    2015-01-01

    Sterol regulatory element binding protein-1c (SREBP-1c) is a key transcription factor that regulates genes involved in the de novo lipid synthesis and glycolysis pathways. The structure, turnover and transactivation potential of SREBP-1c are regulated by macronutrients and hormones via a cascade of signalling kinases. Using MS, we have identified serine 73 as a novel glycogen synthase kinase-3 (GSK-3) phosphorylation site in the rat SREBP-1c purified from McA-RH7777 hepatoma cells. Our site-specific mutagenesis strategy revealed that the turnover of SREBP-1c, containing wild type, phospho-null (serine to alanine) or phospho-mimetic (serine to aspartic acid) substitutions, was differentially regulated. We show that the S73D mutant of pSREBP-1c, that mimicked a state of constitutive phosphorylation, dissociated from the SREBP-1c–SCAP complex more readily and underwent GSK-3-dependent proteasomal degradation via SCFFbw7 ubiquitin ligase pathway. Pharmacologic inhibition of GSK-3 or knockdown of GSK-3 by siRNA prevented accelerated degradation of SREBP-1c. As demonstrated by MS, SREBP-1c was phosphorylated in vitro by GSK-3β at serine 73. Phosphorylation of serine 73 also occurs in the intact liver. We propose that GSK-3-mediated phosphorylation of serine 73 in the rat SREBP-1c and its concomitant destabilization represents a novel mechanism involved in the inhibition of de novo lipid synthesis in the liver. PMID:26589965

  17. Rho GTPase/Rho Kinase Negatively Regulates Endothelial Nitric Oxide Synthase Phosphorylation through the Inhibition of Protein Kinase B/Akt in Human Endothelial Cells

    PubMed Central

    Ming, Xiu-Fen; Viswambharan, Hema; Barandier, Christine; Ruffieux, Jean; Kaibuchi, Kozo; Rusconi, Sandro; Yang, Zhihong

    2002-01-01

    Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human umbilical vein endothelial cells (HUVECs), we show here that both active RhoA and ROCK not only downregulate eNOS gene expression as reported previously but also inhibit eNOS phosphorylation at Ser-1177 and cellular NO production with concomitant suppression of PKB activation. Moreover, coexpression of a constitutive active form of PKB restores the phosphorylation but not gene expression of eNOS in the presence of active RhoA. Furthermore, we show that thrombin inhibits eNOS phosphorylation, as well as expression via Rho/ROCK pathway. Expression of the active PKB reverses eNOS phosphorylation but has no effect on downregulation of eNOS expression induced by thrombin. Taken together, these data demonstrate that Rho/ROCK pathway negatively regulates eNOS phosphorylation through inhibition of PKB, whereas it downregulates eNOS expression independent of PKB. PMID:12446767

  18. A Nucleus-Encoded Chloroplast Protein YL1 Is Involved in Chloroplast Development and Efficient Biogenesis of Chloroplast ATP Synthase in Rice.

    PubMed

    Chen, Fei; Dong, Guojun; Wu, Limin; Wang, Fang; Yang, Xingzheng; Ma, Xiaohui; Wang, Haili; Wu, Jiahuan; Zhang, Yanli; Wang, Huizhong; Qian, Qian; Yu, Yanchun

    2016-01-01

    Chloroplast ATP synthase (cpATPase) is an importance thylakoid membrane-associated photosynthetic complex involved in the light-dependent reactions of photosynthesis. In this study, we isolated and characterized a rice (Oryza sativa) mutant yellow leaf 1 (yl1), which exhibits chlorotic leaves throughout developmental stages. The YL1 mutation showed reduced chlorophyll contents, abnormal chloroplast morphology, and decreased photochemical efficiency. Moreover, YL1 deficiency disrupts the expression of genes associated with chloroplast development and photosynthesis. Molecular and genetic analyses revealed that YL1 is a nucleus-encoded protein with a predicted transmembrane domain in its carboxyl-terminus that is conserved in the higher plant kingdom. YL1 localizes to chloroplasts and is preferentially expressed in green tissues containing chloroplasts. Immunoblot analyses showed that inactivation of YL1 leads to drastically reduced accumulation of AtpA (α) and AtpB (β), two core subunits of CF1αβ subcomplex of cpATPase, meanwhile, a severe decrease (ca. 41.7%) in cpATPase activity was observed in the yl1-1 mutant compared with the wild type. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation assays revealed a specific interaction between YL1 and AtpB subunit of cpATPase. Taken together, our results suggest that YL1 is a plant lineage-specific auxiliary factor involved in the biogenesis of the cpATPase complex, possibly via interacting with the β-subunit. PMID:27585744

  19. Dephosphorylation of the Core Clock Protein KaiC in the Cyanobacterial KaiABC Circadian Oscillator Proceeds via an ATP Synthase Mechanism

    SciTech Connect

    Egli, Martin; Mori, Tetsuya; Pattanayek, Rekha; Xu, Yao; Qin, Ximing; Johnson, Carl H.

    2014-10-02

    The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro from three proteins, KaiA, KaiB, and KaiC in the presence of ATP, to tick in a temperature-compensated manner. KaiC, the central cog of this oscillator, forms a homohexamer with 12 ATP molecules bound between its N- and C-terminal domains and exhibits unusual properties. Both the N-terminal (CI) and C-terminal (CII) domains harbor ATPase activity, and the subunit interfaces between CII domains are the sites of autokinase and autophosphatase activities. Hydrolysis of ATP correlates with phosphorylation at threonine and serine sites across subunits in an orchestrated manner, such that first T432 and then S431 are phosphorylated, followed by dephosphorylation of these residues in the same order. Although structural work has provided insight into the mechanisms of ATPase and kinase, the location and mechanism of the phosphatase have remained enigmatic. From the available experimental data based on a range of approaches, including KaiC crystal structures and small-angle X-ray scattering models, metal ion dependence, site-directed mutagenesis (i.e., E318, the general base), and measurements of the associated clock periods, phosphorylation patterns, and dephosphorylation courses as well as a lack of sequence motifs in KaiC that are typically associated with known phosphatases, we hypothesized that KaiCII makes use of the same active site for phosphorylation and dephosphorlyation. We observed that wild-type KaiC (wt-KaiC) exhibits an ATP synthase activity that is significantly reduced in the T432A/S431A mutant. We interpret the first observation as evidence that KaiCII is a phosphotransferase instead of a phosphatase and the second that the enzyme is capable of generating ATP, both from ADP and P{sub i} (in a reversal of the ATPase reaction) and from ADP and P-T432/P-S431 (dephosphorylation). This new concept regarding the mechanism of dephosphorylation is also supported by the

  20. Dephosphorylation of the core clock protein KaiC in the cyanobacterial KaiABC circadian oscillator proceeds via an ATP synthase mechanism.

    PubMed

    Egli, Martin; Mori, Tetsuya; Pattanayek, Rekha; Xu, Yao; Qin, Ximing; Johnson, Carl H

    2012-02-28

    The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro from three proteins, KaiA, KaiB, and KaiC in the presence of ATP, to tick in a temperature-compensated manner. KaiC, the central cog of this oscillator, forms a homohexamer with 12 ATP molecules bound between its N- and C-terminal domains and exhibits unusual properties. Both the N-terminal (CI) and C-terminal (CII) domains harbor ATPase activity, and the subunit interfaces between CII domains are the sites of autokinase and autophosphatase activities. Hydrolysis of ATP correlates with phosphorylation at threonine and serine sites across subunits in an orchestrated manner, such that first T432 and then S431 are phosphorylated, followed by dephosphorylation of these residues in the same order. Although structural work has provided insight into the mechanisms of ATPase and kinase, the location and mechanism of the phosphatase have remained enigmatic. From the available experimental data based on a range of approaches, including KaiC crystal structures and small-angle X-ray scattering models, metal ion dependence, site-directed mutagenesis (i.e., E318, the general base), and measurements of the associated clock periods, phosphorylation patterns, and dephosphorylation courses as well as a lack of sequence motifs in KaiC that are typically associated with known phosphatases, we hypothesized that KaiCII makes use of the same active site for phosphorylation and dephosphorlyation. We observed that wild-type KaiC (wt-KaiC) exhibits an ATP synthase activity that is significantly reduced in the T432A/S431A mutant. We interpret the first observation as evidence that KaiCII is a phosphotransferase instead of a phosphatase and the second that the enzyme is capable of generating ATP, both from ADP and P(i) (in a reversal of the ATPase reaction) and from ADP and P-T432/P-S431 (dephosphorylation). This new concept regarding the mechanism of dephosphorylation is also supported by the

  1. Dephosphorylation of the Core Clock Protein KaiC in the Cyanobacterial KaiABC Circadian Oscillator Proceeds via an ATP Synthase Mechanism

    PubMed Central

    Egli, Martin; Mori, Tetsuya; Pattanayek, Rekha; Xu, Yao; Qin, Ximing; Johnson, Carl H.

    2012-01-01

    The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro from three proteins, KaiA, KaiB and KaiC in the presence of ATP, to tick in a temperature-compensated manner. KaiC, the central cog of this oscillator, forms a homo-hexamer with twelve ATP molecules bound between its N- and C-terminal domains and exhibits unusual properties. Both the N-terminal (CI) and C-terminal (CII) domains harbor ATPase activity and the subunit interfaces between CII domains are the sites of auto-kinase and auto-phosphatase activities. Hydrolysis of ATP correlates with phosphorylation at threonine and serine sites across subunits in an orchestrated manner, such that first T432 and then S431 is phosphorylated, followed by dephosphorylation of these residues in the same order. Although structural work has provided insight into the mechanisms of ATPase and kinase, the location and mechanism of the phosphatase have remained enigmatic. From the available experimental data based on a range of approaches, including KaiC crystal structures and small angle X-ray scattering (SAXS) models, metal ion dependence, site-directed mutagenesis (i.e. E318, the general base) and measurements of the associated clock periods, phosphorylation patterns and dephosphorylation courses as well as a lack of sequence motifs in KaiC that are typically associated with known phosphatases, we hypothesized that KaiCII makes use of the same active site for phosphorylation and dephosphorlyation. We observed that wt-KaiC exhibits an ATP synthase activity that is significantly reduced in the T432A/S431A mutant. We interpret the first observation as evidence that KaiCII is a phospho-transferase instead of a phosphatase and the second that the enzyme is capable of generating ATP, both from ADP + Pi (in a reversal of the ATPase reaction), and ADP + P-T432/P-S431 (dephosphorylation). This new concept regarding the mechanism of dephosphorylation is also supported by strikingly similar make

  2. Thyroid hormone responsive protein Spot14 enhances catalysis of fatty acid synthase in lactating mammary epithelium[S

    PubMed Central

    Rudolph, Michael C.; Wellberg, Elizabeth A.; Lewis, Andrew S.; Terrell, Kristina L.; Merz, Andrea L.; Maluf, N. Karl; Serkova, Natalie J.; Anderson, Steven M.

    2014-01-01

    Thyroid hormone responsive protein Spot 14 has been consistently associated with de novo fatty acid synthesis activity in multiple tissues, including the lactating mammary gland, which synthesizes large quantities of medium chain fatty acids (MCFAs) exclusively via FASN. However, the molecular function of Spot14 remains undefined during lactation. Spot14-null mice produce milk deficient in total triglyceride and de novo MCFA that does not sustain optimal neonatal growth. The lactation defect was rescued by provision of a high fat diet to the lactating dam. Transgenic mice overexpressing Spot14 in mammary epithelium produced total milk fat equivalent to controls, but with significantly greater MCFA. Spot14-null dams have no diminution of metabolic gene expression, enzyme protein levels, or intermediate metabolites that accounts for impaired de novo MCFA. When [13C] fatty acid products were quantified in vitro using crude cytosolic lysates, native FASN activity was 1.6-fold greater in control relative to Spot14-null lysates, and add back of Spot14 partially restored activity. Recombinant FASN catalysis increased 1.4-fold and C = 14:0 yield was enhanced 4-fold in vitro following addition of Spot14. These findings implicate Spot14 as a direct protein enhancer of FASN catalysis in the mammary gland during lactation when maximal MCFA production is needed. PMID:24771867

  3. Longitudinal study of differential protein expression in an Alzheimer’s mouse model lacking inducible nitric oxide synthase

    PubMed Central

    Hoos, Michael D.; Richardson, Brenna M.; Foster, Matthew W.; Everhart, Angela; Thompson, J. Will; Moseley, M. Arthur; Colton, Carol A.

    2013-01-01

    Alzheimer’s disease (AD) is a complex neurodegenerative process that involves altered brain immune, neuronal and metabolic functions. Understanding the underlying mechanisms has relied on mouse models that mimic components of AD pathology. We used gel-free, label-free LC/MS/MS to quantify protein and phosphopeptide levels in brains of APPSwDI/NOS2−/− (CVN-AD) mice. CVN-AD mice show a full spectrum of AD-like pathology, including amyloid deposition, hyperphosphorylated and aggregated tau and neuronal loss that worsens with age. Tryptic digests, with or without phosphopeptide enrichment on an automated titanium dioxide LC system, were separated by on-line two-dimensional LC and analyzed on a Waters Synapt G2 HDMS, yielding relative expression for >950 proteins and >1100 phosphopeptides. Among differentially expressed proteins were known markers found in humans with AD, including GFAP and C1Q. Phosphorylation of connexin 43, not previously described in AD, was increased at 42 weeks, consistent with dysregulation of gap junctions and activation of astrocytes. Additional alterations in phosphoproteins suggests dysregulation of mitochondria, synaptic transmission, vesicle trafficking and innate immune pathways. These data validate the CVN-AD mouse model of AD, identify novel disease and age-related changes in the brain during disease progression and demonstrate the utility of integrating unbiased and phosphoproteomics for understanding disease processes in AD. PMID:24006891

  4. Yeast Tolerance to Various Stresses Relies on the Trehalose-6P Synthase (Tps1) Protein, Not on Trehalose*

    PubMed Central

    Petitjean, Marjorie; Teste, Marie-Ange; François, Jean M.; Parrou, Jean-Luc

    2015-01-01

    Trehalose is a stable disaccharide commonly found in nature, from bacteria to fungi and plants. For the model yeast Saccharomyces cerevisiae, claims that trehalose is a stress protectant were based indirectly either on correlation between accumulation of trehalose and high resistance to various stresses or on stress hypersensitivity of mutants deleted for TPS1, which encodes the first enzyme in trehalose biosynthetic pathway. Our goal was to investigate more directly which one, between trehalose and/or the Tps1 protein, may serve yeast cells to withstand exposure to stress. By employing an original strategy that combined the use of mutant strains expressing catalytically inactive variants of Tps1, with MAL+ yeast strains able to accumulate trehalose from an exogenous supply, we bring for the first time unbiased proof that trehalose does not protect yeast cells from dying and that the stress-protecting role of trehalose in this eukaryotic model was largely overestimated. Conversely, we identified the Tps1 protein as a key player for yeast survival in response to temperature, oxidative, and desiccation stress. We also showed by robust RT-quantitative PCR and genetic interaction analysis that the role of Tps1 in thermotolerance is not dependent upon Hsf1-dependent transcription activity. Finally, our results revealed that the Tps1 protein is essential to maintain ATP levels during heat shock. Altogether, these findings supported the idea that Tps1 is endowed with a regulatory function in energy homeostasis, which is essential to withstand adverse conditions and maintain cellular integrity. PMID:25934390

  5. Nuclear Magnetic Resonance Characterization of the Type III Secretion System Tip Chaperone Protein PcrG of Pseudomonas aeruginosa.

    PubMed

    Chaudhury, Sukanya; Nordhues, Bryce A; Kaur, Kawaljit; Zhang, Na; De Guzman, Roberto N

    2015-11-01

    Lung infection with Pseudomonas aeruginosa is the leading cause of death among cystic fibrosis patients. To initiate infection, P. aeruginosa assembles a protein nanomachine, the type III secretion system (T3SS), to inject bacterial proteins directly into target host cells. An important regulator of the P. aeruginosa T3SS is the chaperone protein PcrG, which forms a complex with the tip protein, PcrV. In addition to its role as a chaperone to the tip protein, PcrG also regulates protein secretion. PcrG homologues are also important in the T3SS of other pathogens such as Yersinia pestis, the causative agent of bubonic plague. The atomic structure of PcrG or any member of the family of tip protein chaperones is currently unknown. Here, we show by circular dichroism and nuclear magnetic resonance (NMR) spectroscopy that PcrG lacks a tertiary structure. However, it is not completely disordered but contains secondary structures dominated by two long α-helices from residue 16 to 41 and from residue 55 to 76. The helices of PcrG are partially formed, have similar backbone dynamics, and are flexible. NMR titrations show that the entire length of PcrG residues from position 9 to 76 is involved in binding to PcrV. PcrG adds to the growing list of partially folded or unstructured proteins with important roles in type III secretion. PMID:26451841

  6. Natural Antisense Transcript for Hyaluronan Synthase 2 (HAS2-AS1) Induces Transcription of HAS2 via Protein O-GlcNAcylation*

    PubMed Central

    Vigetti, Davide; Deleonibus, Sara; Moretto, Paola; Bowen, Timothy; Fischer, Jens W.; Grandoch, Maria; Oberhuber, Alexander; Love, Dona C.; Hanover, John A.; Cinquetti, Raffaella; Karousou, Eugenia; Viola, Manuela; D'Angelo, Maria Luisa; Hascall, Vincent C.; De Luca, Giancarlo; Passi, Alberto

    2014-01-01

    Changes in the microenvironment organization within vascular walls are critical events in the pathogenesis of vascular pathologies, including atherosclerosis and restenosis. Hyaluronan (HA) accumulation into artery walls supports vessel thickening and is involved in many cardiocirculatory diseases. Excessive cytosolic glucose can enter the hexosamine biosynthetic pathway, increase UDP-N-acetylglucosamine (UDP-GlcNAc) availability, and lead to modification of cytosolic proteins via O-linked attachment of the monosaccharide β-N-GlcNAc (O-GlcNAcylation) from UDP-GlcNAc by the enzyme O-GlcNAc transferase. As many cytoplasmic and nuclear proteins can be glycosylated by O-GlcNAc, we studied whether the expression of the HA synthases that synthesize HA could be controlled by O-GlcNAcylation in human aortic smooth muscle cells. Among the three HAS isoenzymes, only HAS2 mRNA increased after O-GlcNAcylation induced by glucosamine treatments or by inhibiting O-GlcNAc transferase with PUGNAC (O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate). We found that the natural antisense transcript of HAS2 (HAS2-AS1) was absolutely necessary to induce the transcription of the HAS2 gene. Moreover, we found that O-GlcNAcylation modulated HAS2-AS1 promoter activation by recruiting the NF-κB subunit p65, but not the HAS2 promoter, whereas HAS2-AS1 natural antisense transcript, working in cis, regulated HAS2 transcription by altering the chromatin structure around the HAS2 proximal promoter via O-GlcNAcylation and acetylation. These results indicate that HAS2 transcription can be finely regulated not only by recruiting transcription factors to the promoter as previously described but also by modulating chromatin accessibility by epigenetic modifications. PMID:25183006

  7. DNase I hypersensitivity sites and nuclear protein binding on the fatty acid synthase gene: identification of an element with properties similar to known glucose-responsive elements.

    PubMed Central

    Foufelle, F; Lepetit, N; Bosc, D; Delzenne, N; Morin, J; Raymondjean, M; Ferré, P

    1995-01-01

    We have shown previously that fatty acid synthase (FAS) gene expression is positively regulated by glucose in rat adipose tissue and liver. In the present study, we have identified in the first intron of the gene a sequence closely related to known glucose-responsive elements such as in the L-pyruvate kinase and S14 genes, including a putative upstream stimulatory factor/major late transcription factor (USF/MLTF) binding site (E-box) (+ 292 nt to + 297 nt). Location of this sequence corresponds to a site of hypersensitivity to DNase I which is present in the liver but not in the spleen. Moreover, using this information from a preliminary report of the present work, others have shown that a + 283 nt to + 303 nt sequence of the FAS gene can confer glucose responsiveness to a heterologous promoter. The protein binding to this region has been investigated in vitro by a combination of DNase I footprinting and gel-retardation experiments with synthetic oligonucleotides and known nuclear proteins. DNase I footprinting experiments using a + 161 nt to + 405 nt fragment of the FAS gene demonstrate that a region from + 290 nt to + 316 nt is protected by nuclear extracts from liver and spleen. This region binds two ubiquitous nuclear factors, USF/MLTF and the CAAT-binding transcription factor/nuclear factor 1 (CTF/NF1). Binding of these factors is similar in nuclear extracts from liver which does or does not express the FAS gene as observed for glucose-responsive elements in the L-pyruvate kinase and S14 genes. This suggests a posttranslational modification of a factor of the complex after glucose stimulation. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7772036

  8. Melatonin attenuated adipogenesis through reduction of the CCAAT/enhancer binding protein beta by regulating the glycogen synthase 3 beta in human mesenchymal stem cells.

    PubMed

    Rhee, Yun-Hee; Ahn, Jin-Chul

    2016-06-01

    Adipogenic differentiation is characterized by an increase in two major transcription factors: peroxisome proliferator-activated receptor gamma (PPARγ) and the CCAAT/enhancer binding protein alpha (C/EBPα). These two signals are influenced by C/EBPβ and C/EBPδ and cross-regulate each other's expression during the initial stages of adipogenesis. Melatonin has been known to act as not only a direct scavenger of free radicals but also an inhibitor of glycogen synthase kinase 3β (GSK-3β). Here, we report that melatonin inhibits the adipogenic differentiation of human mesenchymal stem cells (hMSCs) which is due to the regulations of C/EBPβ in the early stage of adipogenic differentiation. Melatonin reduced the lipid accumulation, adiponectin, and lipoprotein lipase (LPL) during the adipogenic differentiation of hMSCs. Since C/EBPβ has been associated with the activation of PPARγ and the consensus site of ERK/GSK-3β, PPARγ and β-catenin were detected by immunofluorescence staining after pretreatment of melatonin. Melatonin blocked the activation of PPARγ which induced the degradation of β-catenin. Melatonin also decreased the levels of cyclic adenosine-3,5-monophosphate (cAMP) and reactive oxygen species (ROS). The cAMP triggered the activity of C/EBPβ which is a critical inducer of PPARγ and C/EBPα activation in the early stage of adipogenic differentiation, and this is further affected by ROS production. The adipogenic marker proteins such as PPARγ, C/EBPα, C/EBPβ, and pERK were also decreased by melatonin. In summary, melatonin inhibited the cAMP synthesis through ROS reduction and the phosphorylation of the ERK/GSK-3β site which is known to be responsible for C/EBPβ activation for adipogenic differentiation in hMSCs. PMID:26797706

  9. Octacalcium phosphate crystals directly stimulate expression of inducible nitric oxide synthase through p38 and JNK mitogen-activated protein kinases in articular chondrocytes

    PubMed Central

    Ea, Hang-Korng; Uzan, Benjamin; Rey, Christian; Lioté, Frédéric

    2005-01-01

    Basic calcium phosphate (BCP) crystals, including hydroxyapatite, octacalcium phosphate (OCP) and carbonate-apatite, have been associated with severe osteoarthritis and several degenerative arthropathies. Most studies have considered the chondrocyte to be a bystander in the pathogenesis of calcium crystal deposition disease, assuming that synovial cell cytokines were the only triggers of chondrocyte activation. In the present study we identified direct activation of articular chondrocytes by OCP crystals, which are the BCP crystals with the greatest potential for inducing inflammation. OCP crystals induced nitric oxide (NO) production and inducible nitric oxide synthase (NOS) mRNA expression by isolated articular chondrocytes and cartilage fragments, in a dose-dependent manner and with variations over time. OCP crystals also induced IL-1β mRNA expression. Using pharmacological and cytokine inhibitors, we observed that OCP crystals induced NO production and inducible NOS mRNA activation were regulated at both the transcriptional and the translational levels; were independent from IL-1β gene activation; and involved p38 and c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways, as further confirmed by OCP crystal-induced p38 and JNK MAPK phosphorylation. Taken together, our data suggest that the transcriptional inducible NOS response to OCP crystals involved both the p38 and the JNK MAPK pathways, probably under the control of activator protein-1. NO, a major mediator of cartilage degradation, can be directly produced by BCP crystals in chondrocytes. Together with synovial activation, this direct mechanism may be important in the pathogenesis of destructive arthropathies triggered by microcrystals. PMID:16207333

  10. Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins

    PubMed Central

    Jin, Lin; Ham, Jong Hyun; Hage, Rosemary; Zhao, Wanying; Soto-Hernández, Jaricelis; Lee, Sang Yeol; Paek, Seung-Mann; Kim, Min Gab; Boone, Charles; Coplin, David L.; Mackey, David

    2016-01-01

    Bacterial AvrE-family Type-III effector proteins (T3Es) contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, we employed a yeast-two-hybrid screen with non-lethal fragments of WtsE and a synthetic genetic array with full-length WtsE. Together these screens indicate that WtsE targets maize protein phosphatase 2A (PP2A) heterotrimeric enzyme complexes via direct interaction with B’ regulatory subunits. AvrE1, another AvrE-family T3E from Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), associates with specific PP2A B’ subunit proteins from its susceptible host Arabidopsis that are homologous to the maize B’ subunits shown to interact with WtsE. Additionally, AvrE1 was observed to associate with the WtsE-interacting maize proteins, indicating that PP2A B’ subunits are likely conserved targets of AvrE-family T3Es. Notably, the ability of AvrE1 to promote bacterial growth and/or suppress callose deposition was compromised in Arabidopsis plants with mutations of PP2A genes. Also, chemical inhibition of PP2A activity blocked the virulence activity of both WtsE and AvrE1 in planta. The function of HopM1, a Pto DC3000 T3E that is functionally redundant to AvrE1, was also impaired in specific PP2A mutant lines, although no direct interaction with B’ subunits was observed. These results indicate that sub-component specific PP2A complexes are targeted by bacterial T3Es, including direct targeting by members of the widely conserved AvrE-family. PMID:27191168

  11. Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins.

    PubMed

    Jin, Lin; Ham, Jong Hyun; Hage, Rosemary; Zhao, Wanying; Soto-Hernández, Jaricelis; Lee, Sang Yeol; Paek, Seung-Mann; Kim, Min Gab; Boone, Charles; Coplin, David L; Mackey, David

    2016-05-01

    Bacterial AvrE-family Type-III effector proteins (T3Es) contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, we employed a yeast-two-hybrid screen with non-lethal fragments of WtsE and a synthetic genetic array with full-length WtsE. Together these screens indicate that WtsE targets maize protein phosphatase 2A (PP2A) heterotrimeric enzyme complexes via direct interaction with B' regulatory subunits. AvrE1, another AvrE-family T3E from Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), associates with specific PP2A B' subunit proteins from its susceptible host Arabidopsis that are homologous to the maize B' subunits shown to interact with WtsE. Additionally, AvrE1 was observed to associate with the WtsE-interacting maize proteins, indicating that PP2A B' subunits are likely conserved targets of AvrE-family T3Es. Notably, the ability of AvrE1 to promote bacterial growth and/or suppress callose deposition was compromised in Arabidopsis plants with mutations of PP2A genes. Also, chemical inhibition of PP2A activity blocked the virulence activity of both WtsE and AvrE1 in planta. The function of HopM1, a Pto DC3000 T3E that is functionally redundant to AvrE1, was also impaired in specific PP2A mutant lines, although no direct interaction with B' subunits was observed. These results indicate that sub-component specific PP2A complexes are targeted by bacterial T3Es, including direct targeting by members of the widely conserved AvrE-family. PMID:27191168

  12. Effect of the oral contraceptive pill on protein S and antithrombin-III levels in Malaysian women.

    PubMed

    Wong, K K; Ng, S C; Koong, P L

    Studies focusing on the relationship between oral contraceptive (OC) usage and occurrence of thromboembolism have been conducted for over 3 decades. Those studies centered on the effects OC use has on blood proteins and on measurable physiological changes that occurred in women with venous thrombosis. This article reports the findings of a study that investigated the effects of OC use on the levels of the anticoagulants antithrombin-III (AT-III), protein C (PC), and protein S (PS) in a group of Asian women. Previous studies had mostly been based on Caucasian women. Of the 21 women studied, 16 were Malaysian, 3 were Chinese, and 2 were Indian. Low-dose OCs containing 30 mcg of ethinyl estradiol and 150 mcg of either desogestrel or levonorgestrel were used. Blood was tested before OC use and 3 and 6 months after starting OC use. Levels of AT-III and PS were measured using the Laurell rocket immunoelectrophoresis technique. Statistical analysis was performed using the paired Student's t-test and an analysis of variance test. No statistically significant differences were found for the mean levels of AT-III and total PS when comparing the pre-OC with the 3- and 6-month post-OC values. Earlier studies based mostly on Caucasian women have reported lower levels of both total PS and free PS in OC users. PMID:12288974

  13. Abscission Is Regulated by the ESCRT-III Protein Shrub in Drosophila Germline Stem Cells

    PubMed Central

    Matias, Neuza Reis; Mathieu, Juliette; Huynh, Jean-René

    2015-01-01

    Abscission is the final event of cytokinesis that leads to the physical separation of the two daughter cells. Recent technical advances have allowed a better understanding of the cellular and molecular events leading to abscission in isolated yeast or mammalian cells. However, how abscission is regulated in different cell types or in a developing organism remains poorly understood. Here, we characterized the function of the ESCRT-III protein Shrub during cytokinesis in germ cells undergoing a series of complete and incomplete divisions. We found that Shrub is required for complete abscission, and that levels of Shrub are critical for proper timing of abscission. Loss or gain of Shrub delays abscission in germline stem cells (GSCs), and leads to the formation of stem-cysts, where daughter cells share the same cytoplasm as the mother stem cell and cannot differentiate. In addition, our results indicate a negative regulation of Shrub by the Aurora B kinase during GSC abscission. Finally, we found that Lethal giant discs (lgd), known to be required for Shrub function in the endosomal pathway, also regulates the duration of abscission in GSCs. PMID:25647097

  14. Phosphorylation promotes Al(iii) binding to proteins: GEGEGSGG as a case study.

    PubMed

    Grande-Aztatzi, Rafael; Formoso, Elena; Mujika, Jon I; Ugalde, Jesus M; Lopez, Xabier

    2016-03-14

    Aluminum, the third most abundant element in the Earth's crust and one of the key industrial components of our everyday life, has been associated with several neurodegenerative diseases due to its ability to promote neurofilament tangles and β-amyloid peptide aggregation. However, the experimental characterization of aluminum speciation in vivo is a difficult task. In the present study, we develop a theoretical protocol that combines molecular dynamics simulations, clustering of structures, and density functional theory for the characterization of the binding of aluminum to the synthetic neurofilament analogue octapeptide GEGEGSGG and its phosphorylated variant. Our protocol is tested with respect to previous NMR experimental data, which allows for a full interpretation of the experimental information available and its relationship with key thermodynamic quantities. Our results demonstrate the importance of phosphorylation in the ability of a peptide to bind to aluminum. Thus, phosphorylation: (i) changes the binding pattern of aluminum to GEGEGSGG, shifting the preferential binding site from the C-terminal to S6(P); (ii) increases the binding affinity by a factor of around 15 kcal mol(-1) in free energy; and (iii) may cause significant changes in the secondary structure and stiffness of the polypeptide chain, specially in the case of bidentate binding modes. Our results shed light on the possibility of aluminum to induce aggregation of β-amyloid proteins and neurofilament tangles. PMID:26891057

  15. Endothelial nitric oxide synthase negatively regulates hydrogen peroxide-stimulated AMP-activated protein kinase in endothelial cells.

    PubMed

    Jin, Benjamin Y; Sartoretto, Juliano L; Gladyshev, Vadim N; Michel, Thomas

    2009-10-13

    Hydrogen peroxide and other reactive oxygen species are intimately involved in endothelial cell signaling. In many cell types, the AMP-activated protein kinase (AMPK) has been implicated in the control of metabolic responses, but the role of endothelial cell redox signaling in the modulation of AMPK remains to be completely defined. We used RNA interference and pharmacological methods to establish that H(2)O(2) is a critical activator of AMPK in cultured bovine aortic endothelial cells (BAECs). H(2)O(2) treatment of BAECs rapidly and significantly increases the phosphorylation of AMPK. The EC(50) for H(2)O(2)-promoted phosphorylation of AMPK is 65 + or - 15 microM, within the physiological range of cellular H(2)O(2) concentrations. The Ca(2+)/calmodulin-dependent protein kinase kinase-beta (CaMKKbeta) inhibitor STO-609 abolishes H(2)O(2)-dependent AMPK activation, whereas eNOS inhibitors enhance AMPK activation. Similarly, siRNA-mediated knockdown of CaMKKbeta abrogates AMPK activation, whereas siRNA-mediated knockdown of eNOS leads to a striking increase in AMPK phosphorylation. Cellular imaging studies using the H(2)O(2) biosensor HyPer show that siRNA-mediated eNOS knockdown leads to a marked increase in intracellular H(2)O(2) generation, which is blocked by PEG-catalase. eNOS(-/-) mice show a marked increase in AMPK phosphorylation in liver and lung compared to wild-type mice. Lung endothelial cells from eNOS(-/-) mice also show a significant increase in AMPK phosphorylation. Taken together, these results establish that CaMKKbeta is critically involved in mediating the phosphorylation of AMPK promoted by H(2)O(2) in endothelial cells, and document that eNOS is an important negative regulator of AMPK phosphorylation and intracellular H(2)O(2) generation in endothelial cells. PMID:19805165

  16. Endothelial nitric oxide synthase negatively regulates hydrogen peroxide-stimulated AMP-activated protein kinase in endothelial cells

    PubMed Central

    Jin, Benjamin Y.; Sartoretto, Juliano L.; Gladyshev, Vadim N.; Michel, Thomas

    2009-01-01

    Hydrogen peroxide and other reactive oxygen species are intimately involved in endothelial cell signaling. In many cell types, the AMP-activated protein kinase (AMPK) has been implicated in the control of metabolic responses, but the role of endothelial cell redox signaling in the modulation of AMPK remains to be completely defined. We used RNA interference and pharmacological methods to establish that H2O2 is a critical activator of AMPK in cultured bovine aortic endothelial cells (BAECs). H2O2 treatment of BAECs rapidly and significantly increases the phosphorylation of AMPK. The EC50 for H2O2-promoted phosphorylation of AMPK is 65 ± 15 μM, within the physiological range of cellular H2O2 concentrations. The Ca2+/calmodulin-dependent protein kinase kinase-β (CaMKKβ) inhibitor STO-609 abolishes H2O2-dependent AMPK activation, whereas eNOS inhibitors enhance AMPK activation. Similarly, siRNA-mediated knockdown of CaMKKβ abrogates AMPK activation, whereas siRNA-mediated knockdown of eNOS leads to a striking increase in AMPK phosphorylation. Cellular imaging studies using the H2O2 biosensor HyPer show that siRNA-mediated eNOS knockdown leads to a marked increase in intracellular H2O2 generation, which is blocked by PEG-catalase. eNOS−/− mice show a marked increase in AMPK phosphorylation in liver and lung compared to wild-type mice. Lung endothelial cells from eNOS−/− mice also show a significant increase in AMPK phosphorylation. Taken together, these results establish that CaMKKβ is critically involved in mediating the phosphorylation of AMPK promoted by H2O2 in endothelial cells, and document that eNOS is an important negative regulator of AMPK phosphorylation and intracellular H2O2 generation in endothelial cells. PMID:19805165

  17. The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes.

    PubMed

    Banerjee, Sudip; Melnyk, Stepan B; Krager, Kimberly J; Aykin-Burns, Nukhet; Letzig, Lynda G; James, Laura P; Hinson, Jack A

    2015-12-01

    3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10 µM)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5-3.0 h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0 h, respectively, and increased 3NT and GSNO at 1.5-3.0 h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5-3.0 h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT. PMID:26454079

  18. Nitric Oxide Synthase (NOS) Inhibition during Porcine In Vitro Maturation Modifies Oocyte Protein S-Nitrosylation and In Vitro Fertilization

    PubMed Central

    Romero-Aguirregomezcorta, Jon; Santa, Ángela Patricia; García-Vázquez, Francisco Alberto; Coy, Pilar; Matás, Carmen

    2014-01-01

    Nitric oxide (NO) is a molecule involved in many reproductive processes. Its importance during oocyte in vitro maturation (IVM) has been demonstrated in various species although sometimes with contradictory results. The objective of this study was to determine the effect of NO during IVM of cumulus oocyte complexes and its subsequent impact on gamete interaction in porcine species. For this purpose, IVM media were supplemented with three NOS inhibitors: NG-nitro-L-arginine methyl ester (L-NAME), NG-monomethyl-L-arginine (L-NMMA) and aminoguanidine (AG). A NO donor, S-nitrosoglutathione (GSNO), was also used. The effects on the cumulus cell expansion, meiotic resumption, zona pellucida digestion time (ZPdt) and, finally, on in vitro fertilization (IVF) parameters were evaluated. The oocyte S-nitrosoproteins were also studied by in situ nitrosylation. The results showed that after 42 h of IVM, AG, L-NAME and L-NMMA had an inhibitory effect on cumulus cell expansion. Meiotic resumption was suppressed only when AG was added, with 78.7% of the oocytes arrested at the germinal vesicle state (P<0.05). Supplementation of the IVM medium with NOS inhibitors or NO donor did not enhance the efficiency of IVF, but revealed the importance of NO in maturation and subsequent fertilization. Furthermore, protein S-nitrosylation is reported for the first time as a pathway through which NO exerts its effect on porcine IVM; therefore, it would be important to determine which proteins are nitrosylated in the oocyte and their functions, in order to throw light on the mechanism of action of NO in oocyte maturation and subsequent fertilization. PMID:25542028

  19. Characterization of the Shigella and Salmonella Type III Secretion System Tip-Translocon Protein-Protein Interaction by Paramagnetic Relaxation Enhancement.

    PubMed

    Kaur, Kawaljit; Chatterjee, Srirupa; De Guzman, Roberto N

    2016-04-15

    Many Gram-negative pathogens, such as Shigella and Salmonella, assemble the type III secretion system (T3SS) to inject virulence proteins directly into eukaryotic cells to initiate infectious diseases. The needle apparatus of the T3SS consists of a base, an extracellular needle, a tip protein complex, and a translocon. The atomic structure of the assembled tip complex and the translocon is unknown. Here, we show by NMR paramagnetic relaxation enhancement (PRE) that the mixed α-β domain at the distal region of the Shigella and Salmonella tip proteins interacts with the N-terminal ectodomain of their major translocon proteins. Our results reveal the binding surfaces involved in the tip-translocon protein-protein interaction and provide insights about the assembly of the needle apparatus of the T3SS. PMID:26749041

  20. Mapping the Protein Interaction Network for TFIIB-Related Factor Brf1 in the RNA Polymerase III Preinitiation Complex

    PubMed Central

    Khoo, Seok-Kooi; Wu, Chih-Chien; Lin, Yu-Chun; Lee, Jin-Cheng

    2014-01-01

    TFIIB-related factor Brf1 is essential for RNA polymerase (Pol) III recruitment and open-promoter formation in transcription initiation. We site specifically incorporated a nonnatural amino acid cross-linker into Brf1 to map its protein interaction targets in the preinitiation complex (PIC). Our cross-linking analysis in the N-terminal domain of Brf1 indicated a pattern of multiple protein interactions reminiscent of TFIIB in the Pol active-site cleft. In addition to the TFIIB-like protein interactions, the Brf1 cyclin repeat subdomain is in contact with the Pol III-specific C34 subunit. With site-directed hydroxyl radical probing, we further revealed the binding between Brf1 cyclin repeats and the highly conserved region connecting C34 winged-helix domains 2 and 3. In contrast to the N-terminal domain of Brf1, the C-terminal domain contains extensive binding sites for TBP and Bdp1 to hold together the TFIIIB complex on the promoter. Overall, the domain architecture of the PIC derived from our cross-linking data explains how individual structural subdomains of Brf1 integrate the protein network from the Pol III active center to the promoter for transcription initiation. PMID:24277937

  1. Mapping the protein interaction network for TFIIB-related factor Brf1 in the RNA polymerase III preinitiation complex.

    PubMed

    Khoo, Seok-Kooi; Wu, Chih-Chien; Lin, Yu-Chun; Lee, Jin-Cheng; Chen, Hung-Ta

    2014-02-01

    TFIIB-related factor Brf1 is essential for RNA polymerase (Pol) III recruitment and open-promoter formation in transcription initiation. We site specifically incorporated a nonnatural amino acid cross-linker into Brf1 to map its protein interaction targets in the preinitiation complex (PIC). Our cross-linking analysis in the N-terminal domain of Brf1 indicated a pattern of multiple protein interactions reminiscent of TFIIB in the Pol active-site cleft. In addition to the TFIIB-like protein interactions, the Brf1 cyclin repeat subdomain is in contact with the Pol III-specific C34 subunit. With site-directed hydroxyl radical probing, we further revealed the binding between Brf1 cyclin repeats and the highly conserved region connecting C34 winged-helix domains 2 and 3. In contrast to the N-terminal domain of Brf1, the C-terminal domain contains extensive binding sites for TBP and Bdp1 to hold together the TFIIIB complex on the promoter. Overall, the domain architecture of the PIC derived from our cross-linking data explains how individual structural subdomains of Brf1 integrate the protein network from the Pol III active center to the promoter for transcription initiation. PMID:24277937

  2. Function of the mammalian La protein: evidence for its action in transcription termination by RNA polymerase III.

    PubMed Central

    Gottlieb, E; Steitz, J A

    1989-01-01

    We have tested the hypothesis that the mammalian La protein, which appears to be required for accurate and efficient RNA polymerase III transcription, is a transcription termination factor. Our data suggest that 3' foreshortened transcripts generated in La's absence are components of a novel transcription intermediate containing a paused polymerase. These transcripts are produced by fractionated transcription complexes, are synthesized with kinetics different from full-length transcripts, and are chasable to completion from the stalled transcription complexes. Together, these findings argue that termination by RNA polymerase III requires auxilliary factor(s) and implicate La as such a factor. Since La appears to facilitate transcript completion and release and also binds the resulting RNA product, it may be a regulator of RNA polymerase III transcription. Images PMID:2470590

  3. Cell-penetration by Co(III)cyclen-based peptide-cleaving catalysts selective for pathogenic proteins of amyloidoses.

    PubMed

    Chei, Woo Suk; Lee, Joo-Won; Kim, Jae Bum; Suh, Junghun

    2010-07-15

    Derivatives of the Co(III) complex of 1,4,7,10-tetraazacyclododecane (cyclen) with various organic pendants have been reported as target-selective peptide-cleaving catalysts, which can be exploited as catalytic drugs. In order to provide a firm basis for the catalytic drugs based on Co(III)cyclen, the ability of the Co(III)cyclen-containing peptide-cleaving catalysts to penetrate animal cells such as mouse fibroblast NIH-3T 3 or human embryonic kidney (HEK) 293 cells is demonstrated in the present study. Since the catalysts destroy pathogenic proteins for amyloidoses, results of the present study are expected to initiate extensive efforts to obtain therapeutically safe catalytic drugs for amyloidoses such as Alzheimer's disease, type 2 diabetes mellitus, Parkinson's disease, Huntington's disease, mad cow disease, and so on. PMID:20542701

  4. Immunogenicity and protective efficacy of the E. coli-expressed domain III of Japanese encephalitis virus envelope protein in mice.

    PubMed

    Alka; Bharati, Kaushik; Malik, Y P S; Vrati, Sudhanshu

    2007-12-01

    Domain III of Japanese encephalitis virus (JEV) envelope protein (E-DIII) was synthesized in E. coli as a fusion protein containing maltose-binding protein (MBP-E-DIII) or six contiguous histidine residues (His-E-DIII) at its N-terminus. MBP-E-DIII was found both in the soluble as well as the insoluble fraction of the bacterial lysate, while His-E-DIII was found exclusively in the inclusion bodies. These purified proteins were examined in mice for their immunogenicity in presence of an aluminium hydroxide based-adjuvant Alhydrogel and Freund's adjuvant. While both proteins generated anti-JEV antibodies that neutralized JEV activity in vitro, His-E-DIII generated higher antibody titers than MBP-E-DIII. Mice immunized with His-E-DIII in presence of Alhydrogel generated antibody titers similar to those induced by the commercial vaccine and protected mice against lethal JEV challenge. PMID:17377815

  5. Endothelin-1 Impairs Nitric Oxide Signaling in Endothelial Cells Through a Protein Kinase Cδ-Dependent Activation of STAT3 and Decreased Endothelial Nitric Oxide Synthase Expression

    PubMed Central

    Sud, Neetu

    2009-01-01

    In an ovine model of persistent pulmonary hypertension of the newborn (PPHN), endothelin-1 (ET-1) expression is increased, while endothelial nitric oxide synthase (eNOS) expression is decreased. However, the molecular mechanisms by which ET-1 attenuates eNOS expression in endothelial cells are not completely understood. Thus, the goal of this study was to determine if the overexpression of ET-1 decreases eNOS expression in pulmonary arterial endothelial cells isolated from fetal lambs. To increase the ET-1 expression, cells were transfected with a plasmid coding for Prepro-ET-1, a precursor of ET-1. After overexpression of Prepro-ET-1, ET-1 levels in the culture medium were significantly increased (control = 805.3 ± 69.8; Prepro-ET-1 overexpression = 1351 ± 127.9). eNOS promoter activity, protein levels, and NO generation were all significantly decreased by the overexpression of Prepro-ET-1. The decrease in transcription correlated with increased activity of protein kinase Cδ (PKCδ) and STAT3. Further, DNA binding activity of STAT3 was also increased by Prepro-ET-1 overexpression. The increase in STAT3 activity and decrease in eNOS promoter activity were inhibited by the overexpression of dominant negative mutants of PKCδ or STAT3. Further, a 2 bp mutation in the STAT3 binding site in the eNOS promoter inhibited STAT3 binding and led to enhanced promoter activity in the presence of Prepro-ET-1 overexpression. In conclusion, ET-1 secretion is increased by Prepro-ET-1 overexpression. This results in activation of PKCδ, which phosphorylates STAT3, increasing its binding to the eNOS promoter. This in turn decreases eNOS promoter activity, protein levels, and NO production. Thus, ET-1 can reduce eNOS expression and NO generation in fetal pulmonary artery endothelial cells through PKCδ-mediated activation of STAT3. PMID:19754268

  6. A Novel Periplasmic Protein, VrpA, Contributes to Efficient Protein Secretion by the Type III Secretion System in Xanthomonas spp.

    PubMed

    Zhou, Xiaofeng; Hu, Xiufang; Li, Jinyun; Wang, Nian

    2015-02-01

    Efficient secretion of type III effector proteins from the bacterial cytoplasm to host cell cytosol via a type III secretion system (T3SS) is crucial for virulence of plant-pathogenic bacterium. Our previous study revealed a conserved hypothetical protein, virulence-related periplasm protein A (VrpA), which was identified as a critical virulence factor for Xanthomonas citri subsp. citri. In this study, we demonstrate that mutation of vrpA compromises X. citri subsp. citri virulence and hypersensitive response induction. This deficiency is also observed in the X. campestris pv. campestris strain, suggesting a functional conservation of VrpA in Xanthomonas spp. Our study indicates that VrpA is required for efficient protein secretion via T3SS, which is supported by multiple lines of evidence. A CyaA reporter assay shows that VrpA is involved in type III effector secretion; quantitative reverse-transcription polymerase chain reaction analysis suggests that the vrpA mutant fails to activate citrus-canker-susceptible gene CsLOB1, which is transcriptionally activated by transcription activator-like effector PthA4; in vitro secretion study reveals that VrpA plays an important role in secretion of T3SS pilus, translocon, and effector proteins. Our data also indicate that VrpA in X. citri subsp. citri localizes to bacterial periplasmic space and the periplasmic localization is required for full function of VrpA and X. citri subsp. citri virulence. Protein-protein interaction studies show that VrpA physically interacts with periplasmic T3SS components HrcJ and HrcC. However, the mutation of VrpA does not affect T3SS gene expression. Additionally, VrpA is involved in X. citri subsp. citri tolerance of oxidative stress. Our data contribute to the mechanical understanding of an important periplasmic protein VrpA in Xanthomonas spp. PMID:25338144

  7. Acyl Carrier Protein Synthases from Gram-Negative, Gram-Positive, and Atypical Bacterial Species: Biochemical and Structural Properties and Physiological Implications

    PubMed Central

    McAllister, Kelly A.; Peery, Robert B.; Zhao, Genshi

    2006-01-01

    Acyl carrier protein (ACP) synthase (AcpS) catalyzes the transfer of the 4′-phosphopantetheine moiety from coenzyme A (CoA) onto a serine residue of apo-ACP, resulting in the conversion of apo-ACP to the functional holo-ACP. The holo form of bacterial ACP plays an essential role in mediating the transfer of acyl fatty acid intermediates during the biosynthesis of fatty acids and phospholipids. AcpS is therefore an attractive target for therapeutic intervention. In this study, we have purified and characterized the AcpS enzymes from Escherichia coli, Streptococcus pneumoniae, and Mycoplasma pneumoniae, which exemplify gram-negative, gram-positive, and atypical bacteria, respectively. Our gel filtration column chromatography and cross-linking studies demonstrate that the AcpS enzyme from M. pneumoniae, like E. coli enzyme, exhibits a homodimeric structure, but the enzyme from S. pneumoniae exhibits a trimeric structure. Our biochemical studies show that the AcpS enzymes from M. pneumoniae and S. pneumoniae can utilize both short- and long-chain acyl CoA derivatives but prefer long-chain CoA derivatives as substrates. On the other hand, the AcpS enzyme from E. coli can utilize short-chain CoA derivatives but not the long-chain CoA derivatives tested. Finally, our biochemical studies show that M. pneumoniae AcpS is kinetically a very sluggish enzyme compared with those from E. coli and S. pneumoniae. Together, the results of these studies show that the AcpS enzymes from different bacterial species exhibit different native structures and substrate specificities with regard to the utilization of CoA and its derivatives. These findings suggest that AcpS from different microorganisms plays a different role in cellular physiology. PMID:16788183

  8. Effect of the ATPase inhibitor protein IF{sub 1} on H{sup +} translocation in the mitochondrial ATP synthase complex

    SciTech Connect

    Zanotti, Franco; Gnoni, Antonio; Mangiullo, Roberto; Papa, Sergio

    2009-06-19

    The H{sup +} F{sub o}F{sub 1}-ATP synthase complex of coupling membranes converts the proton-motive force into rotatory mechanical energy to drive ATP synthesis. The F{sub 1} moiety of the complex protrudes at the inner side of the membrane, the F{sub o} sector spans the membrane reaching the outer side. The IF{sub 1} component of the mitochondrial complex is a basic 10 kDa protein, which inhibits the F{sub o}F{sub 1}-ATP hydrolase activity. The mitochondrial matrix pH is the critical factor for the inhibitory binding of the central segment of IF{sub 1} (residue 42-58) to the F{sub 1}-{alpha}/{beta} subunits. We have analyzed the effect of native purified IF{sub 1} the IF{sub 1}-(42-58) synthetic peptide and its mutants on proton conduction, driven by ATP hydrolysis or by [K{sup +}] gradients, in bovine heart inside-out submitochondrial particles and in liposome-reconstituted F{sub o}F{sub 1} complex. The results show that IF{sub 1}, and in particular its central 42-58 segment, displays different inhibitory affinity for proton conduction from the F{sub 1} to the F{sub o} side and in the opposite direction. Cross-linking of IF{sub 1} to F{sub 1}-{alpha}/{beta} subunits inhibits the ATP-driven H{sup +} translocation but enhances H{sup +} conduction in the reverse direction. These observation are discussed in terms of the rotary mechanism of the F{sub o}F{sub 1} complex.

  9. Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts.

    PubMed

    Nagaoka, Aya; Yoshida, Hiroyuki; Nakamura, Sachiko; Morikawa, Tomohiko; Kawabata, Keigo; Kobayashi, Masaki; Sakai, Shingo; Takahashi, Yoshito; Okada, Yasunori; Inoue, Shintaro

    2015-12-25

    Regulation of hyaluronan (HA) synthesis and degradation is essential to maintenance of extracellular matrix homeostasis. We recently reported that HYBID (HYaluronan-Binding protein Involved in hyaluronan Depolymerization), also called KIAA1199, plays a key role in HA depolymerization in skin and arthritic synovial fibroblasts. However, regulation of HA metabolism mediated by HYBID and HA synthases (HASs) under stimulation with growth factors remains obscure. Here we report that TGF-β1, basic FGF, EGF, and PDGF-BB commonly enhance total amount of HA in skin fibroblasts through up-regulation of HAS expression, but molecular size of newly produced HA is dependent on HYBID expression levels. Stimulation of HAS1/2 expression and suppression of HYBID expression by TGF-β1 were abrogated by blockade of the MAPK and/or Smad signaling and the PI3K-Akt signaling, respectively. In normal human skin, expression of the TGF-β1 receptors correlated positively with HAS2 expression and inversely with HYBID expression. On the other hand, TGF-β1 up-regulated HAS1/2 expression but exerted only a slight suppressive effect on HYBID expression in synovial fibroblasts from the patients with osteoarthritis or rheumatoid arthritis, resulting in the production of lower molecular weight HA compared with normal skin and synovial fibroblasts. These data demonstrate that although TGF-β1, basic FGF, EGF, and PDGF-BB enhance HA production in skin fibroblasts, TGF-β1 most efficiently contributes to production of high molecular weight HA by HAS up-regulation and HYBID down-regulation and suggests that inefficient down-regulation of HYBID by TGF-β1 in arthritic synovial fibroblasts may be linked to accumulation of depolymerized HA in synovial fluids in arthritis patients. PMID:26518873

  10. Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase

    SciTech Connect

    Hwang, Yong Pil; Kim, Hyung Gyun; Hien, Tran Thi; Jeong, Myung Ho; Jeong, Tae Cheon; Jeong, Hye Gwang

    2011-11-15

    The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-{alpha}-stimulated monocytes to endothelial cells and suppressed the TNF-{alpha} induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-{alpha}-induced nuclear factor-{kappa}B activation, which was attenuated by pretreatment with N{sup G}-nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: Black-Right-Pointing-Pointer Puerarin induced the phosphorylation of eNOS and the production of NO. Black-Right-Pointing-Pointer Puerarin activated eNOS through ER-dependent PI3-kinase and Ca{sup 2+}-dependent AMPK. Black-Right-Pointing-Pointer Puerarin-induced NO was involved in the inhibition of NF-kB activation. Black-Right-Pointing-Pointer Puerarin may help for prevention of vascular dysfunction and diabetes.

  11. Correlation of nitric oxide produced by an inducible nitric oxide synthase-like protein with enhanced expression of the phenylpropanoid pathway in Inonotus obliquus cocultured with Phellinus morii.

    PubMed

    Zhao, Yanxia; Xi, Qi; Xu, Qian; He, Meihong; Ding, Jianing; Dai, Yucheng; Keller, Nancy P; Zheng, Weifa

    2015-05-01

    Fungal interspecific interactions enhance biosynthesis of phenylpropanoid metabolites (PM), and production of nitric oxide (NO) is known to be involved in this process. However, it remains unknown which signaling pathway(s) or regulator(s) mediate fungal PM biosynthesis. In this study, we cocultured two white-rot fungi, Inonotus obliquus and Phellinus morii, to examine NO production, expression of the genes involved in phenylpropanoid metabolism and accumulation of phenylpropanoid-derived polyphenols by I. obliquus. Coculture of the two fungi caused an enhanced NO biosynthesis followed by increased transcription of the genes encoding phenylalanine ammonia lyase (PAL) and 4-coumarate CoA ligase (4CL), as well as an upregulated biosynthesis of styrylpyrone polyphenols in I. obliquus. Addition of the NO synthase (NOS) selective inhibitor aminoguanidine (AG) inhibited NO production by more than 90% followed by cease in transcription of PAL and 4Cl. Treatment of guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one did not affect NO production but suppressed transcription of PAL and 4CL and reduced accumulation of total phenolic constituents. Genome-wide analysis of I. obliquus revealed two genes encoding a constitutive and an inducible NOS-like protein, respectively (cNOSL and iNOSL). Coculture of the two fungi did not increase the expression of the cNOSL gene but triggered expression of the iNOSL gene. Cloned iNOSL from Escherichia coli shows higher activity in transferring L-arginine to NO, and this activity is lost upon AG addition. Thus, iNOSL is more responsible for NO production in I. obliquus and may act as an important regulator governing PM production during fungal interspecific interactions. PMID:25582560

  12. Follistatin-like 1, a secreted muscle protein, promotes endothelial cell function and revascularization in ischemic tissue through a nitric-oxide synthase-dependent mechanism.

    PubMed

    Ouchi, Noriyuki; Oshima, Yuichi; Ohashi, Koji; Higuchi, Akiko; Ikegami, Chiaki; Izumiya, Yasuhiro; Walsh, Kenneth

    2008-11-21

    Myogenic Akt signaling coordinates blood vessel recruitment with normal tissue growth. Here, we investigated the role of Follistatin-like 1 (Fstl1) in the regulation of endothelial cell function and blood vessel growth in muscle. Transgenic Akt1 overexpression in skeletal muscle led to myofiber growth that was coupled to an increase in muscle capillary density. Myogenic Akt signaling or ischemic hind limb surgery led to the induction of Fstl1 in muscle and increased circulating levels of Fstl1. Intramuscular administration of an adenoviral vector expressing Fstl1 (Ad-Fstl1) accelerated flow recovery and increased capillary density in the ischemic hind limbs of wild-type mice, and this was associated with an increase in endothelial nitric oxide synthase (eNOS) phosphorylation at residue Ser-1179. In cultured endothelial cells, Ad-Fstl1 stimulated migration and differentiation into network structures and inhibited apoptosis under conditions of serum deprivation. These cell responses were associated with the activating phosphorylation of Akt and eNOS. Conversely, transduction with dominant-negative Akt or LY294002 blocked Fstl1-stimulated eNOS phosphorylation and inhibited Fstl1-stimulated cellular responses. Treatment with the eNOS inhibitor N(G)-nitro-L-arginine methyl ester also reduced endothelial cell migration and differentiation induced by Ad-Fstl1. The stimulatory effect of Ad-Fstl1 on ischemic limb reperfusion was abolished in mice lacking eNOS. These data indicate that Fstl1 is a secreted muscle protein or myokine that can function to promote endothelial cell function and stimulates revascularization in response to ischemic insult through its ability to activate Akt-eNOS signaling. PMID:18718903

  13. The role of inducible nitric oxide synthase for interstitial remodeling of alveolar septa in surfactant protein D-deficient mice.

    PubMed

    Knudsen, Lars; Atochina-Vasserman, Elena N; Massa, Christopher B; Birkelbach, Bastian; Guo, Chang-Jiang; Scott, Pamela; Haenni, Beat; Beers, Michael F; Ochs, Matthias; Gow, Andrew J

    2015-11-01

    Surfactant protein D (SP-D) modulates the lung's immune system. Its absence leads to NOS2-independent alveolar lipoproteinosis and NOS2-dependent chronic inflammation, which is critical for early emphysematous remodeling. With aging, SP-D knockout mice develop an additional interstitial fibrotic component. We hypothesize that this age-related interstitial septal wall remodeling is mediated by NOS2. Using invasive pulmonary function testing such as the forced oscillation technique and quasistatic pressure-volume perturbation and design-based stereology, we compared 29-wk-old SP-D knockout (Sftpd(-/-)) mice, SP-D/NOS2 double-knockout (DiNOS) mice, and wild-type mice (WT). Structural changes, including alveolar epithelial surface area, distribution of septal wall thickness, and volumes of septal wall components (alveolar epithelium, interstitial tissue, and endothelium) were quantified. Twenty-nine-week-old Sftpd(-/-) mice had preserved lung mechanics at the organ level, whereas elastance was increased in DiNOS. Airspace enlargement and loss of surface area of alveolar epithelium coexist with increased septal wall thickness in Sftpd(-/-) mice. These changes were reduced in DiNOS, and compared with Sftpd(-/-) mice a decrease in volumes of interstitial tissue and alveolar epithelium was found. To understand the effects of lung pathology on measured lung mechanics, structural data were used to inform a computational model, simulating lung mechanics as a function of airspace derecruitment, septal wall destruction (loss of surface area), and septal wall thickening. In conclusion, NOS2 mediates remodeling of septal walls, resulting in deposition of interstitial tissue in Sftpd(-/-). Forward modeling linking structure and lung mechanics describes the complex mechanical properties by parenchymatous destruction (emphysema), interstitial remodeling (septal wall thickening), and altered recruitability of acinar airspaces. PMID:26320150

  14. Dengue-4 envelope domain III fused twice within the meningococcal P64k protein carrier induces partial protection in mice.

    PubMed

    Lazo, Laura; Zulueta, Aída; Hermida, Lisset; Blanco, Aracelys; Sánchez, Jorge; Valdés, Iris; Gil, Lázaro; López, Carlos; Romero, Yaremis; Guzmán, María G; Guillén, Gerardo

    2009-04-01

    A vaccine against dengue virus must be able to induce an effective and equivalent immune response to the four viral serotypes; however, some studies have revealed that DEN4 (dengue-virus serotype 4) induces a weaker immune response than the others in quadrivalent (tetravalent') formulations. We have previously reported the protective capacity, in a viral encephalitis murine model, of fusion protein P64k-envelope domain III of DEN1, DEN2 and DEN3. We also reported that the P64k protein can be used as a carrier in two different positions: the insertion following the first 45 amino acids and the fusion at the C-terminus. Considering the low immunogenicity described for DEN4, in the present study we obtained a novel chimaeric protein by inserting two dengue-4 envelope domains III in both sites of P64k (PD24), and hence increasing the presence of the virus in the final construct. After expression in Escherichia coli and semipurification, the protein exhibited a pattern of high molecular mass and was well recognized by human and murine polyclonal antibodies. The protein was finally evaluated in mice, Al(OH)(3) being employed as the adjuvant. Even though the animals exhibited low levels of antiviral antibodies, the recombinant protein induced significant protection against lethal challenge with dengue-4 virus. PMID:18636968

  15. Abundant class III acidic chitinase homologue in tamarind (Tamarindus indica) seed serves as the major storage protein.

    PubMed

    Rao, Devavratha H; Gowda, Lalitha R

    2008-03-26

    The phyla Leguminosae contains protease inhibitors, lectins, chitinases, and glycohydrolases as major defense proteins in their seeds. Electrophoretic analysis of the seed proteins of tamarind ( Tamarindus indica L.), an agri-waste material, indicated the unusual presence of two major proteins comparable to overexpression of recombinant proteins. These proteins were identified by amino-terminal analysis to be (1) Kunitz-type trypsin inhibitor and (2) class III endochitinase (34000 Da). These two proteins were purified to apparent homogeneity by a single-step chitin bead affinity chromatography and characterized. The Kunitz inhibitor was specific toward inhibiting trypsin with a stoichiometry of 1:1. The 33000 +/- 1000 Da protein, accounting for >50% of the total seed protein, is an acidic glycoprotein exhibiting a very low endotype hydrolytic activity toward chitin derivatives. SDS-PAGE followed by densitometry of tamarind seed germination indicates the disappearance of the chitinase with the concomitant appearance of a cysteine endopeptidase. On the basis of its abundance, accumulation without any pathogenesis-related stimulus, temporal regulation, amino acid composition, and very low enzyme activity, this 34000 Da protein designated "tamarinin" physiologically serves as the major storage protein. PMID:18298067

  16. NMR identification of the binding surfaces involved in the Salmonella and Shigella Type III secretion tip-translocon protein-protein interactions.

    PubMed

    McShan, Andrew C; Kaur, Kawaljit; Chatterjee, Srirupa; Knight, Kevin M; De Guzman, Roberto N

    2016-08-01

    The type III secretion system (T3SS) is essential for the pathogenesis of many bacteria including Salmonella and Shigella, which together are responsible for millions of deaths worldwide each year. The structural component of the T3SS consists of the needle apparatus, which is assembled in part by the protein-protein interaction between the tip and the translocon. The atomic detail of the interaction between the tip and the translocon proteins is currently unknown. Here, we used NMR methods to identify that the N-terminal domain of the Salmonella SipB translocon protein interacts with the SipD tip protein at a surface at the distal region of the tip formed by the mixed α/β domain and a portion of its coiled-coil domain. Likewise, the Shigella IpaB translocon protein and the IpaD tip protein interact with each other using similar surfaces identified for the Salmonella homologs. Furthermore, removal of the extreme N-terminal residues of the translocon protein, previously thought to be important for the interaction, had little change on the binding surface. Finally, mutations at the binding surface of SipD reduced invasion of Salmonella into human intestinal epithelial cells. Together, these results reveal the binding surfaces involved in the tip-translocon protein-protein interaction and advance our understanding of the assembly of the T3SS needle apparatus. Proteins 2016; 84:1097-1107. © 2016 Wiley Periodicals, Inc. PMID:27093649

  17. Baseline serum C-reactive protein and death from colorectal cancer in the NHANES III cohort.

    PubMed

    Swede, Helen; Hajduk, Alexandra M; Sharma, Jyoti; Rawal, Shristi; Rasool, Homaira; Vella, Anthony T; Tobet, Rebecca E; Stevens, Richard G

    2014-04-15

    Several prospective studies suggest that C-reactive protein (CRP), a nonspecific serologic marker of inflammation, might be linked to risk of colorectal cancer (CRC), whereas others have reported null or protective effects. We analyzed data from 7,072 participants (50-85 years) in the U.S. National Health and Nutrition Examination Survey III (1988-1994), a nationally representative cohort (n = 33,994; 2 months-85 years) with vital status follow-up to 2000. Hazard ratios (HRs) for mortality associated with baseline clinically raised (≥1.00 mg/dL) and intermediate (≥0.22-0.99 mg/dL) CRP levels were estimated using Cox proportional hazards regression controlling for CRC risk factors. There were 59 deaths from CRC, 106 from other obesity-related cancers (other-ORCs) and 1,130 from cardiovascular disease (CVD). Participants with clinically raised CRP at baseline were found to have a statistically significant greater risk of CRC death (HRs = 2.36-2.47) in comparison to persons with undetected levels. HRs were lower for death from other-ORC and CVD (1.82, 95% CI 1.05-3.15; 1.53, 95% CI 1.29-1.81, respectively). Intermediate CRP level was associated with a nonsignificant 10-21% increased risk for CRC death. HR for CRC death was higher among persons with a normal BMI (2.16, 95% 0.96-4.87, p = 0.06) compared to those who were overweight (1.22, 95% CI 0.53-2.78) or obese (1.23, 95% CI, 0.37-4.08). A similar pattern was observed for waist circumference. This effect modification suggests that the impact of chronic inflammation may be independent of excess body fat. Future research is recommended to confirm emerging data that elevated serologic CRP might reflect underlying colonic inflammation. PMID:24122448

  18. Identification and Characterization of Putative Translocated Effector Proteins of the Edwardsiella ictaluri Type III Secretion System.

    PubMed

    Dubytska, Lidiya P; Rogge, Matthew L; Thune, Ronald L

    2016-01-01

    Edwardsiella ictaluri, a major pathogen in channel catfish aquaculture, encodes a type III secretion system (T3SS) that is essential for intracellular replication and virulence. Previous work identified three putative T3SS effectors in E. ictaluri, and in silico analysis of the E. ictaluri genome identified six additional putative effectors, all located on the chromosome outside the T3SS pathogenicity island. To establish active translocation by the T3SS, we constructed translational fusions of each effector to the amino-terminal adenylate cyclase (AC) domain of the Bordetella pertussis adenylate cyclase toxin CyaA. When translocated through the membrane of the Edwardsiella-containing vacuole (ECV), the cyclic AMP produced by the AC domain in the presence of calmodulin in the host cell cytoplasm can be measured. Results showed that all nine effectors were translocated from E. ictaluri in the ECV to the cytoplasm of the host cells in the wild-type strain but not in a T3SS mutant, indicating that translocation is dependent on the T3SS machinery. This confirms that the E. ictaluri T3SS is similar to the Salmonella pathogenicity island 2 T3SS in that it translocates effectors through the membrane of the bacterial vacuole directly into the host cell cytoplasm. Additional work demonstrated that both initial acidification and subsequent neutralization of the ECV were necessary for effector translocation, except for two of them that did not require neutralization. Single-gene mutants constructed for seven of the individual effectors were all attenuated for replication in CCO cells, but only three were replication deficient in head kidney-derived macrophages (HKDM). IMPORTANCE The bacterial pathogen Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), an economically significant disease of farm-raised channel catfish. Commercial catfish production accounts for the majority of the total fin fish aquaculture in the United States, with almost 300,000

  19. Identification and Characterization of Putative Translocated Effector Proteins of the Edwardsiella ictaluri Type III Secretion System

    PubMed Central

    Dubytska, Lidiya P.; Rogge, Matthew L.

    2016-01-01

    ABSTRACT Edwardsiella ictaluri, a major pathogen in channel catfish aquaculture, encodes a type III secretion system (T3SS) that is essential for intracellular replication and virulence. Previous work identified three putative T3SS effectors in E. ictaluri, and in silico analysis of the E. ictaluri genome identified six additional putative effectors, all located on the chromosome outside the T3SS pathogenicity island. To establish active translocation by the T3SS, we constructed translational fusions of each effector to the amino-terminal adenylate cyclase (AC) domain of the Bordetella pertussis adenylate cyclase toxin CyaA. When translocated through the membrane of the Edwardsiella-containing vacuole (ECV), the cyclic AMP produced by the AC domain in the presence of calmodulin in the host cell cytoplasm can be measured. Results showed that all nine effectors were translocated from E. ictaluri in the ECV to the cytoplasm of the host cells in the wild-type strain but not in a T3SS mutant, indicating that translocation is dependent on the T3SS machinery. This confirms that the E. ictaluri T3SS is similar to the Salmonella pathogenicity island 2 T3SS in that it translocates effectors through the membrane of the bacterial vacuole directly into the host cell cytoplasm. Additional work demonstrated that both initial acidification and subsequent neutralization of the ECV were necessary for effector translocation, except for two of them that did not require neutralization. Single-gene mutants constructed for seven of the individual effectors were all attenuated for replication in CCO cells, but only three were replication deficient in head kidney-derived macrophages (HKDM). IMPORTANCE The bacterial pathogen Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), an economically significant disease of farm-raised channel catfish. Commercial catfish production accounts for the majority of the total fin fish aquaculture in the United States, with almost 300,000

  20. Structural basis for the endoribonuclease activity of the type III-A CRISPR-associated protein Csm6

    PubMed Central

    Niewoehner, Ole; Jinek, Martin

    2016-01-01

    Prokaryotic CRISPR–Cas systems provide an RNA-guided mechanism for genome defense against mobile genetic elements such as viruses and plasmids. In type III-A CRISPR–Cas systems, the RNA-guided multisubunit Csm effector complex targets both single-stranded RNAs and double-stranded DNAs. In addition to the Csm complex, efficient anti-plasmid immunity mediated by type III-A systems also requires the CRISPR-associated protein Csm6. Here we report the crystal structure of Csm6 from Thermus thermophilus and show that the protein is a ssRNA-specific endoribonuclease. The structure reveals a dimeric architecture generated by interactions involving the N-terminal CARF and C-terminal HEPN domains. HEPN domain dimerization leads to the formation of a composite ribonuclease active site. Consistently, mutations of invariant active site residues impair catalytic activity in vitro. We further show that the ribonuclease activity of Csm6 is conserved across orthologs, suggesting that it plays an important functional role in CRISPR–Cas systems. The dimer interface of the CARF domains features a conserved electropositive pocket that may function as a ligand-binding site for allosteric control of ribonuclease activity. Altogether, our work suggests that Csm6 proteins provide an auxiliary RNA-targeting interference mechanism in type III-A CRISPR–Cas systems that operates in conjunction with the RNA- and DNA-targeting endonuclease activities of the Csm effector complex. PMID:26763118

  1. Structure of the Type III Secretion Effector Protein ExoU in Complex with Its Chaperone SpcU

    PubMed Central

    Halavaty, Andrei S.; Borek, Dominika; Tyson, Gregory H.; Veesenmeyer, Jeff L.; Shuvalova, Ludmilla; Minasov, George; Otwinowski, Zbyszek

    2012-01-01

    Disease causing bacteria often manipulate host cells in a way that facilitates the infectious process. Many pathogenic gram-negative bacteria accomplish this by using type III secretion systems. In these complex secretion pathways, bacterial chaperones direct effector proteins to a needle-like secretion apparatus, which then delivers the effector protein into the host cell cytosol. The effector protein ExoU and its chaperone SpcU are components of the Pseudomonas aeruginosa type III secretion system. Secretion of ExoU has been associated with more severe infections in both humans and animal models. Here we describe the 1.92 Å X-ray structure of the ExoU–SpcU complex, a full-length type III effector in complex with its full-length cognate chaperone. Our crystallographic data allow a better understanding of the mechanism by which ExoU kills host cells and provides a foundation for future studies aimed at designing inhibitors of this potent toxin. PMID:23166655

  2. Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A

    NASA Technical Reports Server (NTRS)

    Boo, Yong Chool; Sorescu, George; Boyd, Nolan; Shiojima, Ichiro; Walsh, Kenneth; Du, Jie; Jo, Hanjoong

    2002-01-01

    Recently, we have shown that shear stress stimulates NO(*) production by the protein kinase B/Akt (Akt)-dependent mechanisms in bovine aortic endothelial cells (BAEC) (Go, Y. M., Boo, Y. C., Park, H., Maland, M. C., Patel, R., Pritchard, K. A., Jr., Fujio, Y., Walsh, K., Darley-Usmar, V., and Jo, H. (2001) J. Appl. Physiol. 91, 1574-1581). Akt has been believed to regulate shear-dependent production of NO(*) by directly phosphorylating endothelial nitric-oxide synthase (eNOS) at the Ser(1179) residue (eNOS-S(1179)), but a critical evaluation using specific inhibitors or dominant negative mutants (Akt(AA) or Akt(AAA)) has not been reported. In addition, other kinases, including protein kinase A (PKA) and AMP kinase have also shown to phosphorylate eNOS-S(1179). Here, we show that shear-dependent phosphorylation of eNOS-S(1179) is mediated by an Akt-independent, but a PKA-dependent, mechanism. Expression of Akt(AA) or Akt(AAA) in BAEC by using recombinant adenoviral constructs inhibited phosphorylation of eNOS-S(1179) if cells were stimulated by vascular endothelial growth factor (VEGF), but not by shear stress. As shown before, expression of Akt(AA) inhibited shear-dependent NO(*) production, suggesting that Akt is still an important regulator in NO production. Further studies showed that a selective inhibitor of PKA, H89, inhibited shear-dependent phosphorylation of eNOS-S(1179) and NO(*) production. In contrast, H89 did not inhibit phosphorylation of eNOS-S(1179) induced by expressing a constitutively active Akt mutant (Akt(Myr)) in BAEC, showing that the inhibitor did not affect the Akt pathway. 8-Bromo-cAMP alone phosphorylated eNOS-S(1179) within 5 min without activating Akt, in an H89-sensitive manner. Collectively, these results demonstrate that shear stimulates phosphorylation of eNOS-S(1179) in a PKA-dependent, but Aktindependent manner, whereas the NO(*) production is regulated by the mechanisms dependent on both PKA and Akt. A coordinated interaction

  3. Age-dependent changes in nitric oxide synthase activity and protein expression in striata of mice transgenic for the Huntington's disease mutation.

    PubMed

    Pérez-Severiano, Francisca; Escalante, Bruno; Vergara, Paula; Ríos, Camilo; Segovia, José

    2002-09-27

    Huntington's disease (HD) is an autosomal hereditary neurodegenerative disorder caused by an abnormal expansion of the CAG repeats that code for a polyglutamine tract in a novel protein called huntingtin (htt). Both patients and experimental animals exhibit oxidative damage in specific areas of the brain, particularly the striatum. Nitric oxide (NO) is involved in many different physiological processes, and under pathological conditions it may promote oxidative damage through the formation of the highly reactive metabolite peroxynitrite; however, it may also play a role protecting cells from oxidative damage. We previously showed a correlation between the progression of the neurological phenotype and striatal oxidative damage in a line of transgenic mice, R6/1, which expresses a human mutated htt exon 1 with 116 CAG repeats. The purpose of the present work was to explore the participation of NO in the progressive oxidative damage that occurs in the striata of R6/1 mice. We analyzed the role of NO by measuring the activity of nitric oxide synthase (NOS) in the striata of transgenic and control mice at different ages. There was no difference in NOS activity between transgenic and wild-type mice at 11 weeks of age. In contrast, 19-week-old transgenic mice showed a significant increase in NOS activity, compared with same age controls. By 35 weeks of age, there was a decrease in NOS activity in transgenic mice when compared with wild-type controls. NOS protein expression was also determined in 11-, 19- and 35-week-old transgenic mice and wild-type littermates. Our results show increased neuronal NOS expression in 19-week-old transgenic mice, followed by a decreased level in 35-week-old mice, compared with controls, a phenomenon that parallels the changes in NOS enzyme activity. The present results suggest that NO is involved in the process leading to striatal oxidative damage and that it is associated with the onset of the progressive neurological phenotype in mice

  4. Refolding, crystallization and preliminary X-ray structural studies of the West Nile virus envelope (E) protein domain III

    SciTech Connect

    Yuan, Fang; Lou, Zhiyong; Li, Xiaofeng; Chen, Yu Wai; Bell, John I.; Rao, Zihe; Gao, George F.

    2005-04-01

    Domain III of the West Nile virus envelope protein, the putative receptor-binding domain, was reported to be intrinsically unstable and has defied previous crystallization attempts. It has now been purified from inclusion bodies by protein refolding and was crystallized using the hanging-drop vapour-diffusion method at 291 K. Domain III of the West Nile virus envelope protein, the putative receptor-binding domain, is a major virion-surface determinant for virulence. This protein was reported to be intrinsically unstable and has defied previous crystallization attempts. It has now been purified from inclusion bodies by protein refolding and was crystallized using the hanging-drop vapour-diffusion method at 291 K. The crystals belong to space group P222{sub 1}, with unit-cell parameters a = 52.6, b = 59.7, c = 95.0 Å. A complete data set was collected to 2.8 Å at 100 K with Cu Kα X-rays from a rotating-anode generator.

  5. Interactions of Xanthomonas type-III effector proteins with the plant ubiquitin and ubiquitin-like pathways

    PubMed Central

    Üstün, Suayib; Börnke, Frederik

    2014-01-01

    In eukaryotes, regulated protein turnover is required during many cellular processes, including defense against pathogens. Ubiquitination and degradation of ubiquitinated proteins via the ubiquitin–proteasome system (UPS) is the main pathway for the turnover of intracellular proteins in eukaryotes. The extensive utilization of the UPS in host cells makes it an ideal pivot for the manipulation of cellular processes by pathogens. Like many other Gram-negative bacteria, Xanthomonas species secrete a suite of type-III effector proteins (T3Es) into their host cells to promote virulence. Some of these T3Es exploit the plant UPS to interfere with immunity. This review summarizes T3E examples from the genus Xanthomonas with a proven or suggested interaction with the host UPS or UPS-like systems and also discusses the apparent paradox that arises from the presence of T3Es that inhibit the UPS in general while others rely on its activity for their function. PMID:25566304

  6. Mutational Analysis of Glycogen Synthase Kinase 3β Protein Kinase Together with Kinome-Wide Binding and Stability Studies Suggests Context-Dependent Recognition of Kinases by the Chaperone Heat Shock Protein 90

    PubMed Central

    Pasculescu, Adrian; Dai, Anna Yue; Williton, Kelly; Taylor, Lorne; Savitski, Mikhail M.; Bantscheff, Marcus; Woodgett, James R.; Pawson, Tony; Colwill, Karen

    2016-01-01

    The heat shock protein 90 (HSP90) and cell division cycle 37 (CDC37) chaperones are key regulators of protein kinase folding and maturation. Recent evidence suggests that thermodynamic properties of kinases, rather than primary sequences, are recognized by the chaperones. In concordance, we observed a striking difference in HSP90 binding between wild-type (WT) and kinase-dead (KD) glycogen synthase kinase 3β (GSK3β) forms. Using model cell lines stably expressing these two GSK3β forms, we observed no interaction between WT GSK3β and HSP90, in stark contrast to KD GSK3β forming a stable complex with HSP90 at a 1:1 ratio. In a survey of 91 ectopically expressed kinases in DLD-1 cells, we compared two parameters to measure HSP90 dependency: static binding and kinase stability following HSP90 inhibition. We observed no correlation between HSP90 binding and reduced stability of a kinase after pharmacological inhibition of HSP90. We expanded our stability study to >50 endogenous kinases across four cell lines and demonstrated that HSP90 dependency is context dependent. These observations suggest that HSP90 binds to its kinase client in a particular conformation that we hypothesize to be associated with the nucleotide-processing cycle. Lastly, we performed proteomics profiling of kinases and phosphopeptides in DLD-1 cells to globally define the impact of HSP90 inhibition on the kinome. PMID:26755559

  7. SepD/SepL-Dependent Secretion Signals of the Type III Secretion System Translocator Proteins in Enteropathogenic Escherichia coli

    PubMed Central

    Deng, Wanyin; Yu, Hong B.; Li, Yuling

    2015-01-01

    ABSTRACT The type III protein secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE) is essential for the pathogenesis of attaching/effacing bacterial pathogens, including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and Citrobacter rodentium. These pathogens use the T3SS to sequentially secrete three categories of proteins: the T3SS needle and inner rod protein components; the EspA, EspB, and EspD translocators; and many LEE- and non-LEE-encoded effectors. SepD and SepL are essential for translocator secretion, and mutations in either lead to hypersecretion of effectors. However, how SepD and SepL control translocator secretion and secretion hierarchy between translocators and effectors is poorly understood. In this report, we show that the secreted T3SS components, the translocators, and both LEE- and non-LEE-encoded effectors all carry N-terminal type III secretion and translocation signals. These signals all behave like those of the effectors and are sufficient for mediating type III secretion and translocation by wild-type EPEC and hypersecretion by the sepD and sepL mutants. Our results extended previous observations and suggest that the secretion hierarchy of the different substrates is determined by a signal other than the N-terminal secretion signal. We identified a domain located immediately downstream of the N-terminal secretion signal in the translocator EspB that is required for SepD/SepL-dependent secretion. We further demonstrated that this EspB domain confers SepD/SepL- and CesAB-dependent secretion on the secretion signal of effector EspZ. Our results thus suggest that SepD and SepL control and regulate secretion hierarchy between translocators and effectors by recognizing translocator-specific export signals. IMPORTANCE Many bacterial pathogens use a syringe-like protein secretion apparatus, termed the type III protein secretion system (T3SS), to secrete and inject numerous proteins directly into

  8. Functional Prostacyclin Synthase Promoter Polymorphisms. Impact in Pulmonary Arterial Hypertension

    PubMed Central

    Cornelius, Amber R.; Lu, Xiao; Conklin, David S.; Del Rosario, Mark J.; Lowe, Anita M.; Elos, Mihret T.; Fettig, Lynsey M.; Wong, Randall E.; Hara, Naoko; Cogan, Joy D.; Phillips, John A.; Taylor, Matthew R.; Graham, Brian B.; Tuder, Rubin M.; Loyd, James E.; Geraci, Mark W.

    2014-01-01

    Rationale: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by elevated pulmonary artery pressure, vascular remodeling, and ultimately right ventricular heart failure. PAH can have a genetic component (heritable PAH), most often through mutations of bone morphogenetic protein receptor 2, and idiopathic and associated forms. Heritable PAH is not completely penetrant within families, with approximately 20% concurrence of inactivating bone morphogenetic protein receptor 2 mutations and delayed onset of PAH disease. Because one of the treatment options is using prostacyclin analogs, we hypothesized that prostacyclin synthase promoter sequence variants associated with increased mRNA expression may play a protective role in the bone morphogenetic protein receptor 2 unaffected carriers. Objectives: To characterize the range of prostacyclin synthase promoter variants and assess their transcriptional activities in PAH-relevant cell types. To determine the distribution of prostacyclin synthase promoter variants in PAH, unaffected carriers in heritable PAH families, and control populations. Methods: Polymerase chain reaction approaches were used to genotype prostacyclin synthase promoter variants in more than 300 individuals. Prostacyclin synthase promoter haplotypes’ transcriptional activities were determined with luciferase reporter assays. Measurements and Main Results: We identified a comprehensive set of prostacyclin synthase promoter variants and tested their transcriptional activities in PAH-relevant cell types. We demonstrated differences of prostacyclin synthase promoter activities dependent on their haplotype. Conclusions: Prostacyclin synthase promoter sequence variants exhibit a range of transcriptional activities. We discovered a significant bias for more active prostacyclin synthase promoter variants in unaffected carriers as compared with affected patients with PAH. PMID:24605778

  9. Novel homodimeric and heterodimeric rat gamma-hydroxybutyrate synthases that associate with the Golgi apparatus define a distinct subclass of aldo-keto reductase 7 family proteins.

    PubMed Central

    Kelly, Vincent P; Sherratt, Philip J; Crouch, Dorothy H; Hayes, John D

    2002-01-01

    -length human AKR7A2 is composed of 359 amino acids and also possesses an additional N-terminal domain. On the basis of these observations, we conclude that AKR7 proteins can be divided into two subfamilies, one of which is a Golgi-associated GHB synthase with a unique, previously unrecognized, N-terminal domain that is absent from other AKR proteins. PMID:12071861

  10. Generation and characterization of potential dengue vaccine candidates based on domain III of the envelope protein and the capsid protein of the four serotypes of dengue virus.

    PubMed

    Suzarte, Edith; Marcos, Ernesto; Gil, Lázaro; Valdés, Iris; Lazo, Laura; Ramos, Yassel; Pérez, Yusleidi; Falcón, Viviana; Romero, Yaremis; Guzmán, María G; González, Sirenia; Kourí, Juan; Guillén, Gerardo; Hermida, Lisset

    2014-07-01

    Dengue is currently one of the most important arthropod-borne diseases, causing up to 25,000 deaths annually. There is currently no vaccine to prevent dengue virus infection, which needs a tetravalent vaccine approach. In this work, we describe the cloning and expression in Escherichia coli of envelope domain III-capsid chimeric proteins (DIIIC) of the four dengue serotypes as a tetravalent dengue vaccine candidate that is potentially able to generate humoral and cellular immunity. The recombinant proteins were purified to more than 85 % purity and were recognized by anti-dengue mouse and human sera. Mass spectrometry analysis verified the identity of the proteins and the correct formation of the intracatenary disulfide bond in the domain III region. The chimeric DIIIC proteins were also serotype-specific, and in the presence of oligonucleotides, they formed aggregates that were visible by electron microscopy. These results support the future use of DIIIC recombinant chimeric proteins in preclinical studies in mice for assessing their immunogenicity and efficacy. PMID:24420159

  11. Cloning, purification, and properties of Candida albicans thymidylate synthase.

    PubMed Central

    Singer, S C; Richards, C A; Ferone, R; Benedict, D; Ray, P

    1989-01-01

    The thymidylate synthase (TS) gene was isolated from a genomic Candida albicans library by functional complementation of a Saccharomyces cerevisiae strain deficient in TS. The gene was localized on a 4-kilobase HindIII DNA fragment and was shown to be expressed in a Thy- strain of Escherichia coli. The nucleotide sequence of the TS gene predicted a protein of 315 amino acids with a molecular weight of 36,027. The gene was cloned into a T7 expression vector in E. coli, allowing purification of large amounts of C. albicans TS. It was also purified from a wild-type C. albicans strain. Comparison of several enzyme properties including analysis of amino-terminal amino acid sequences showed the native and cloned C. albicans TS to be the same. PMID:2646281

  12. The Deinococcus radiodurans DR1245 Protein, a DdrB Partner Homologous to YbjN Proteins and Reminiscent of Type III Secretion System Chaperones

    SciTech Connect

    Norais, Cédric; Servant, Pascale; Bouthier-de-la-Tour, Claire; Coureux, Pierre-Damien; Ithurbide, Solenne; Vannier, Françoise; Guerin, Philippe P.; Dulberger, Charles L.; Satyshur, Kenneth A.; Keck, James L.; Armengaud, Jean; Cox, Michael M.; Sommer, Suzanne

    2013-02-18

    The bacterium Deinococcus radiodurans exhibits an extreme resistance to ionizing radiation. A small subset of Deinococcus genus-specific genes were shown to be up-regulated upon exposure to ionizing radiation and to play a role in genome reconstitution. These genes include an SSB-like protein called DdrB. Here, we identified a novel protein encoded by the dr1245gene as an interacting partner of DdrB. A strain devoid of the DR1245 protein is impaired in growth, exhibiting a generation time approximately threefold that of the wild type strain while radioresistance is not affected. We determined the three-dimensional structure of DR1245, revealing a relationship with type III secretion system chaperones and YbjN family proteins. Thus, DR1245 may display some chaperone activity towards DdrB and possibly other substrates.

  13. A protein fragment of streptococcal cell surface antigen I/II which prevents adhesion of Streptococcus mutans.

    PubMed Central

    Munro, G H; Evans, P; Todryk, S; Buckett, P; Kelly, C G; Lehner, T

    1993-01-01

    Attachment of Streptococcus mutans to the tooth surface involves a cell surface protein with an M(r) of 185,000, termed streptococcal antigen (SA) I/II. Four overlapping fragments of the gene encoding SA I/II were amplified by polymerase chain reaction, cloned, and expressed in Escherichia coli. The recombinant polypeptides were assayed for adhesion-binding activity to salivary receptors and for recognition by a panel of monoclonal antibodies (MAbs) raised against SA I/II. Two of the MAbs which are known to prevent colonization of S. mutans in vivo bound the recombinant polypeptide comprising residues 816 to 1161. In vitro adhesion of S. mutans to saliva-coated hydroxyapatite beads was also inhibited specifically by a polypeptide (residues 816 to 1213) encompassing the same region. The evidence from the MAbs preventing colonization of S. mutans and the adherence inhibition assay suggests that an adhesion-binding activity resides within the portion of SA I/II comprising residues 816 to 1213, which is highly conserved among oral streptococcal species. Images PMID:7691754

  14. Guanylate binding protein-1 mediates EGFRvIII and promotes glioblastoma growth in vivo but not in vitro

    PubMed Central

    Cheng, Yanwei; Mukasa, Akitaki; Ma, Jiawei; Hong, Lei; Yu, Shuye; Sun, Lili; Huang, Qiang; Purow, Benjamin; Li, Ming

    2016-01-01

    Glioblastoma multiforme (GBM) is the most common and deadly primary brain tumor in adults. Epidermal growth factor receptor (EGFR) is frequently amplified and mutated in GBM. We previously reported that Guanylate binding protein-1 (GBP1) is a novel transcriptional target gene of EGFR and plays a role in GBM invasion. Here we demonstrate that GBP1 can also be induced by EGFRvIII at the transcriptional level through the p38 MAPK/Yin Yang 1 (YY1) signaling pathway. Silencing of GBP1 by RNA interference significantly inhibits EGFRvIII-mediated GBM cell proliferation in vitro and in a mouse model. Overexpression of GBP1 has no obvious effect on glioblastoma cell proliferation in vitro. In contrast, in an orthotopic glioma mouse model GBP1 overexpression significantly promotes glioma growth and reduces survival rate of glioma-bearing mice by increasing cell proliferation and decreasing cell apoptosis in tumor. Clinically, GBP1 expression is elevated in human GBM tumors and positively correlates with EGFRvIII status in GBM specimens, and its expression is inversely correlated with the survival rate of GBM patients. Taken together, these results reveal that GBP1 may serve as a potential therapeutic target for GBMs with EGFRvIII mutation. PMID:26848767

  15. Bioinformatics-enabled identification of the HrpL regulon and type III secretion system effector proteins of Pseudomonas syringae pv. phaseolicola 1448A

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ability of Pseudomonas syringae pv. phaseolicola to cause halo blight of bean is dependent on its ability to translocate effector proteins into host cells via the Hrp type III secretion system (T3SS). To identity genes encoding type III effectors and other potential virulence factors that are r...

  16. Terpene synthases are widely distributed in bacteria

    PubMed Central

    Yamada, Yuuki; Kuzuyama, Tomohisa; Komatsu, Mamoru; Shin-ya, Kazuo; Omura, Satoshi; Cane, David E.; Ikeda, Haruo

    2015-01-01

    Odoriferous terpene metabolites of bacterial origin have been known for many years. In genome-sequenced Streptomycetaceae microorganisms, the vast majority produces the degraded sesquiterpene alcohol geosmin. Two minor groups of bacteria do not produce geosmin, with one of these groups instead producing other sesquiterpene alcohols, whereas members of the remaining group do not produce any detectable terpenoid metabolites. Because bacterial terpene synthases typically show no significant overall sequence similarity to any other known fungal or plant terpene synthases and usually exhibit relatively low levels of mutual sequence similarity with other bacterial synthases, simple correlation of protein sequence data with the structure of the cyclized terpene product has been precluded. We have previously described a powerful search method based on the use of hidden Markov models (HMMs) and protein families database (Pfam) search that has allowed the discovery of monoterpene synthases of bacterial origin. Using an enhanced set of HMM parameters generated using a training set of 140 previously identified bacterial terpene synthase sequences, a Pfam search of 8,759,463 predicted bacterial proteins from public databases and in-house draft genome data has now revealed 262 presumptive terpene synthases. The biochemical function of a considerable number of these presumptive terpene synthase genes could be determined by expression in a specially engineered heterologous Streptomyces host and spectroscopic identification of the resulting terpene products. In addition to a wide variety of terpenes that had been previously reported from fungal or plant sources, we have isolated and determined the complete structures of 13 previously unidentified cyclic sesquiterpenes and diterpenes. PMID:25535391

  17. Long-term memory of visually cued fear conditioning: roles of the neuronal nitric oxide synthase gene and cyclic AMP response element-binding protein.

    PubMed

    Kelley, J B; Anderson, K L; Altmann, S L; Itzhak, Y

    2011-02-01

    Nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) has a role in late-phase long-term potentiation (LTP) and long-term memory (LTM) formation. Our recent studies implicated NO signaling in contextual and auditory cued fear conditioning. The present study investigated the role of NO signaling in visually cued fear conditioning. First, visually cued fear conditioning was investigated in wild-type (WT) and nNOS knockout (KO) mice. Second, the effects of pharmacological modulators of NO signaling on the acquisition of visually cued fear conditioning were investigated. Third, plasma levels of corticosterone were measured to determine a relationship between physiological and behavioral responses to fear conditioning. Fourth, levels of extracellular signal-related kinase (ERK1/2) and cyclic AMP response element binding protein (CREB) phosphorylation, downstream of NO signaling, were determined in the amygdala as potential correlates of fear learning. Mice underwent single or multiple (4) spaced trainings that consisted of a visual cue (blinking light) paired with footshock. WT mice acquired cued and contextual LTM following single and multiple trainings. nNOS KO mice acquired neither cued nor contextual LTM following a single training; however, multiple trainings improved contextual but not cued LTM. The selective nNOS inhibitor S-methyl-thiocitrulline (SMTC) impaired cued and contextual LTM in WT mice. The NO donor molsidomine recovered contextual LTM but had no effect on cued LTM in nNOS KO mice. Re-exposure to the visual cue 24 h posttraining elicited freezing response and a marked increase in plasma corticosterone levels in WT but not nNOS KO mice. The expression of CREB phosphorylation (Ser-133) was significantly higher in naive nNOS KO mice than in WT counterparts, and pharmacological modulators of NO had significant effects on levels of CREB phosphorylation and expression. These findings suggest that visual cue-dependent LTM is impaired in nNOS KO

  18. Recombinant synthesis and solution characterization of the protein, apolipophorin-III: An amphipathec biopolymer for the removal of foulants from aqueous systems

    SciTech Connect

    Kahalley, J.; McCormick, C.L.; Cannon, G.

    1996-10-01

    The amphipathic biopolymer, apolipophorin-III, functions in the tobacco hornworm to shuttle hydrophobic and amphipathic materials through the aqueous hemolymph of the insect. The function of this protein in vivo suggests that it has the potential to act as a remediative agent for contaminated waters. The protein has been recombinantly produced and purified. Apo-III has been shown to partition to an oil water interface in a concentration dependent manner. In addition, this protein has been shown to sparingly phase transfer hydrocarbons into aqueous solutions. The association of Apo-III with a hydrophobic, fluorescent probe exhibits dramatic changes in response to pH suggesting a trigger mechanism for the capture and release of foulants in water. Site specific modification of the protein is currently underway in an effort to improve the sequestration and emulsification behavior of apo-III.

  19. ULK3 regulates cytokinetic abscission by phosphorylating ESCRT-III proteins.

    PubMed

    Caballe, Anna; Wenzel, Dawn M; Agromayor, Monica; Alam, Steven L; Skalicky, Jack J; Kloc, Magdalena; Carlton, Jeremy G; Labrador, Leticia; Sundquist, Wesley I; Martin-Serrano, Juan

    2015-01-01

    The endosomal sorting complexes required for transport (ESCRT) machinery mediates the physical separation between daughter cells during cytokinetic abscission. This process is regulated by the abscission checkpoint, a genome protection mechanism that relies on Aurora B and the ESCRT-III subunit CHMP4C to delay abscission in response to chromosome missegregation. In this study, we show that Unc-51-like kinase 3 (ULK3) phosphorylates and binds ESCRT-III subunits via tandem MIT domains, and thereby, delays abscission in response to lagging chromosomes, nuclear pore defects, and tension forces at the midbody. Our structural and biochemical studies reveal an unusually tight interaction between ULK3 and IST1, an ESCRT-III subunit required for abscission. We also demonstrate that IST1 phosphorylation by ULK3 is an essential signal required to sustain the abscission checkpoint and that ULK3 and CHMP4C are functionally linked components of the timer that controls abscission in multiple physiological situations. PMID:26011858

  20. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    SciTech Connect

    Gou, Ke-Mian; Chang, Chia-Chun; Shen, Qing-Ji; Sung, Li-Ying; Liu, Ji-Long

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  1. Dengue virus envelope domain III protein based on a tetravalent antigen secreted from insect cells: Potential use for serological diagnosis.

    PubMed

    Niu, Guoyu; Pang, Zheng; Guan, Chun; Qi, Jun; Li, Dexin

    2015-04-01

    In the present study, we developed a tetravalent protein by connecting the receptor-binding envelope domain III (EDIII) of the four dengue virus serotypes in the order of D1-D3-D4-D2. Using a baculovirus expression system, the protein was secreted into the supernatant of infected sf9 cells in a stable form with preserved native conformation. Using immobilized affinity chromatography, the recombinant EDIII (rEDIII) protein was purified with a yield of 300μg per 10(6) cells. The purity and reactivity of the protein were determined via SDS-PAGE and Western blot respectively. A MAC-ELISA method based on the secreted rEDIII protein was subsequently established and evaluated using a panel of pre-characterized dengue IgM-positive and -negative human sera. We obtained a specificity of 100% and sensitivity of 93% using this method. Our data collectively suggest that the secreted tetravalent rEDIII protein has potential utility in the diagnosis of dengue virus infections. PMID:25697685

  2. ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis

    PubMed Central

    Cardona-López, Ximena; Cuyas, Laura; Marín, Elena; Irigoyen, María Luisa; Gil, Erica; Puga, María Isabel; Bligny, Richard; Nussaume, Laurent; Geldner, Niko; Paz-Ares, Javier

    2015-01-01

    Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life. PMID:26342016

  3. Biophysical Characterization of the Type III Secretion Tip Proteins and the Tip Proteins Attached to Bacterium-Like Particles

    PubMed Central

    Choudhari, Shyamal P.; Chen, Xiaotong; Kim, Jae Hyun; van Roosmalen, Maarten L.; Greenwood, Jamie C.; Joshi, Sangeeta B.; Picking, William D.; Leenhouts, Kees; Middaugh, C. Russell; Picking, Wendy L.

    2014-01-01

    Bacterium-like particles (BLPs), derived from Lactococcus lactis, offer a self-adjuvanting delivery vehicle for subunit protein vaccines. Proteins can be specifically loaded onto the BLPs via a peptidoglycan anchoring domain (PA). In this study, the tip proteins IpaD, SipD and LcrV belonging to type three secretion systems of Shigella flexneri, Salmonella enterica and Yersinia enterocolitica, respectively, were fused to the PA and loaded onto the BLPs. Herein, we biophysically characterized these nine samples and condensed the spectroscopic results into three-index empirical phase diagrams (EPDs). The EPDs show distinctions between the IpaD/SipD and LcrV subfamilies of tip proteins, based on their physical stability, even upon addition of the PA. Upon attachment to the BLPs, the BLPs become defining moiety in the spectroscopic measurements, leaving the tip proteins to have a subtle yet modulating effect on the structural integrity of the tip proteins-BLPs binding. In summary, this work provides a comprehensive view of physical stability of the tip proteins and tip protein-BLPs and serves as a baseline for screening of excipients to increase the stability of the tip protein-BLPs for future vaccine formulation. PMID:24916512

  4. An insulin-binding protein from the venom of a solitary wasp Eumenes pomiformis binds to apolipophorin III in lepidopteran hemolymph.

    PubMed

    Baek, Ji Hyeong; Lee, Si Hyeock; Kim, Woe-Yeon; Kim, Min Gab

    2016-03-01

    EpIBP, an insulin-like peptide-binding protein, is a major protein component of the venom of a solitary hunting wasp, Eumenes pomiformis. To evaluate the bioactivity, bacteria-expressed EpIBP was injected into Spodoptera exigua larvae, resulting in a higher survival rate and reduced loss of body weight under starvation conditions than control larvae. EpIBP was found to interact with apolipophorin III (apoLp III), implying that EpIBP might function by altering the apoLp III-mediated metabolism of prey. PMID:26748153

  5. Human dipeptidyl peptidase III regulates G-protein coupled receptor-dependent Ca2+ concentration in human embryonic kidney 293T cells.

    PubMed

    Prajapati, Subhash C; Singh, Ratnakar; Chauhan, Shyam S

    2016-06-01

    The precise biological function of human dipeptidyl peptidase III (hDPP III) is poorly understood. Using luciferase reporter constructs responsive to change in Ca2+ and/or cAMP and Fura 2-AM fluorometric assay, we show a significant decrease in intracellular Ca2+ following hDPP III overexpression and angiotensin II stimulation in angiotensin II type 1 receptor (G-protein coupled receptor, GPCR) expressing HEK293T cells. Silencing the expression of hDPP III by siRNA reversed the effect of hDPP III overexpression with a concomitant increase in Ca2+. These results, for the first time, show involvement of hDPP III in GPCR dependent Ca2+ regulation in HEK293T cells. PMID:26887037

  6. SIRT3 Deacetylates Ceramide Synthases: IMPLICATIONS FOR MITOCHONDRIAL DYSFUNCTION AND BRAIN INJURY.

    PubMed

    Novgorodov, Sergei A; Riley, Christopher L; Keffler, Jarryd A; Yu, Jin; Kindy, Mark S; Macklin, Wendy B; Lombard, David B; Gudz, Tatyana I

    2016-01-22

    Experimental evidence supports the role of mitochondrial ceramide accumulation as a cause of mitochondrial dysfunction and brain injury after stroke. Herein, we report that SIRT3 regulates mitochondrial ceramide biosynthesis via deacetylation of ceramide synthase (CerS) 1, 2, and 6. Reciprocal immunoprecipitation experiments revealed that CerS1, CerS2, and CerS6, but not CerS4, are associated with SIRT3 in cerebral mitochondria. Furthermore, CerS1, -2, and -6 are hyperacetylated in the mitochondria of SIRT3-null mice, and SIRT3 directly deacetylates the ceramide synthases in a NAD(+)-dependent manner that increases enzyme activity. Investigation of the SIRT3 role in mitochondrial response to brain ischemia/reperfusion (IR) showed that SIRT3-mediated deacetylation of ceramide synthases increased enzyme activity and ceramide accumulation after IR. Functional studies demonstrated that absence of SIRT3 rescued the IR-induced blockade of the electron transport chain at the level of complex III, attenuated mitochondrial outer membrane permeabilization, and decreased reactive oxygen species generation and protein carbonyls in mitochondria. Importantly, Sirt3 gene ablation reduced the brain injury after IR. These data support the hypothesis that IR triggers SIRT3-dependent deacetylation of ceramide synthases and the elevation of ceramide, which could inhibit complex III, leading to increased reactive oxygen species generation and brain injury. The results of these studies highlight a novel mechanism of SIRT3 involvement in modulating mitochondrial ceramide biosynthesis and suggest an important role of SIRT3 in mitochondrial dysfunction and brain injury after experimental stroke. PMID:26620563

  7. Cobalt(III) Protoporphyrin Activates the DGCR8 Protein and Can Compensate microRNA Processing Deficiency.

    PubMed

    Barr, Ian; Weitz, Sara H; Atkin, Talia; Hsu, PeiKen; Karayiorgou, Maria; Gogos, Joseph A; Weiss, Shimon; Guo, Feng

    2015-06-18

    Processing of microRNA primary transcripts (pri-miRNAs) is highly regulated and defects in the processing machinery play a key role in many human diseases. In 22q11.2 deletion syndrome (22q11.2DS), heterozygous deletion of DiGeorge critical region gene 8 (DGCR8) causes a processing deficiency, which contributes to abnormal brain development. The DGCR8 protein is the RNA-binding partner of Drosha RNase, both essential for processing canonical pri-miRNAs. To identify an agent that can compensate reduced DGCR8 expression, we screened for metalloporphyrins that can mimic the natural DGCR8 heme cofactor. We found that Co(III) protoporphyrin IX (PPIX) stably binds DGCR8 and activates it for pri-miRNA processing in vitro and in HeLa cells. Importantly, treating cultured Dgcr8(+/-) mouse neurons with Co(III)PPIX can compensate the pri-miRNA processing defects. Co(III)PPIX is effective at concentrations as low as 0.2 μM and is not degraded by heme degradation enzymes, making it useful as a research tool and a potential therapeutic. PMID:26091172

  8. SINC, a type III secreted protein of Chlamydia psittaci, targets the inner nuclear membrane of infected cells and uninfected neighbors

    PubMed Central

    Mojica, Sergio A.; Hovis, Kelley M.; Frieman, Matthew B.; Tran, Bao; Hsia, Ru-ching; Ravel, Jacques; Jenkins-Houk, Clifton; Wilson, Katherine L.; Bavoil, Patrik M.

    2015-01-01

    SINC, a new type III secreted protein of the avian and human pathogen Chlamydia psittaci, uniquely targets the nuclear envelope of C. psittaci–infected cells and uninfected neighboring cells. Digitonin-permeabilization studies of SINC-GFP–transfected HeLa cells indicate that SINC targets the inner nuclear membrane. SINC localization at the nuclear envelope was blocked by importazole, confirming SINC import into the nucleus. Candidate partners were identified by proximity to biotin ligase-fused SINC in HEK293 cells and mass spectrometry (BioID). This strategy identified 22 candidates with high confidence, including the nucleoporin ELYS, lamin B1, and four proteins (emerin, MAN1, LAP1, and LBR) of the inner nuclear membrane, suggesting that SINC interacts with host proteins that control nuclear structure, signaling, chromatin organization, and gene silencing. GFP-SINC association with the native LEM-domain protein emerin, a conserved component of nuclear “lamina” structure, or with a complex containing emerin was confirmed by GFP pull down. Our findings identify SINC as a novel bacterial protein that targets the nuclear envelope with the capability of globally altering nuclear envelope functions in the infected host cell and neighboring uninfected cells. These properties may contribute to the aggressive virulence of C. psittaci. PMID:25788290

  9. ULK3 regulates cytokinetic abscission by phosphorylating ESCRT-III proteins

    PubMed Central

    Caballe, Anna; Wenzel, Dawn M; Agromayor, Monica; Alam, Steven L; Skalicky, Jack J; Kloc, Magdalena; Carlton, Jeremy G; Labrador, Leticia; Sundquist, Wesley I; Martin-Serrano, Juan

    2015-01-01

    The endosomal sorting complexes required for transport (ESCRT) machinery mediates the physical separation between daughter cells during cytokinetic abscission. This process is regulated by the abscission checkpoint, a genome protection mechanism that relies on Aurora B and the ESCRT-III subunit CHMP4C to delay abscission in response to chromosome missegregation. In this study, we show that Unc-51-like kinase 3 (ULK3) phosphorylates and binds ESCRT-III subunits via tandem MIT domains, and thereby, delays abscission in response to lagging chromosomes, nuclear pore defects, and tension forces at the midbody. Our structural and biochemical studies reveal an unusually tight interaction between ULK3 and IST1, an ESCRT-III subunit required for abscission. We also demonstrate that IST1 phosphorylation by ULK3 is an essential signal required to sustain the abscission checkpoint and that ULK3 and CHMP4C are functionally linked components of the timer that controls abscission in multiple physiological situations. DOI: http://dx.doi.org/10.7554/eLife.06547.001 PMID:26011858

  10. EXTRACELLULAR PROTEINS INVOLVED IN SOYBEAN CULTIVAR-SPECIFIC NODULATION ARE ASSOCIATED WITH PILUS-LIKE SURFACE APPENDANGES AND EXPORTED BY A TYPE III PROTEIN SECRETION SYSTEM IN SINORHIZOBIUM FREDII USDA257

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several Gram-negative plant and animal pathogenic bacteria have evolved a type III secretion system (TTSS) to deliver effector proteins directly into the host cell cytosol. Sinorhizobium fredii USDA257, a symbiont of soybean and many other legumes, secretes signal-responsive proteins (SR proteins) ...

  11. Plasticity of a critical antigenic determinant in the West Nile virus NY99 envelope protein domain III.

    PubMed

    Plante, Jessica A; Torres, Maricela; Huang, Claire Y-H; Beasley, David W C

    2016-09-01

    West Nile virus (WNV) is a mosquito-borne flavivirus that causes febrile illness, encephalitis, and occasionally death in humans. The envelope protein is the main component of the WNV virion surface, and domain III of the envelope protein (EIII) is both a putative receptor binding domain and a target of highly specific, potently neutralizing antibodies. Envelope E-332 (E-332) is known to have naturally occurring variation and to be a key determinant of neutralization for anti-EIII antibodies. A panel of viruses containing all possible amino acid substitutions at E-332 was constructed. E-332 was found to be highly tolerant of mutation, and almost all of these changes had large impacts on antigenicity of EIII but only limited effects on growth or virulence phenotypes. PMID:27284640

  12. A Novel Mechanism of Regulating the ATPase VPS4 by Its Cofactor LIP5 and the Endosomal Sorting Complex Required for Transport (ESCRT)-III Protein CHMP5*

    PubMed Central

    Vild, Cody J.; Li, Yan; Guo, Emily Z.; Liu, Yuan; Xu, Zhaohui

    2015-01-01

    Disassembly of the endosomal sorting complex required for transport (ESCRT) machinery from biological membranes is a critical final step in cellular processes that require the ESCRT function. This reaction is catalyzed by VPS4, an AAA-ATPase whose activity is tightly regulated by a host of proteins, including LIP5 and the ESCRT-III proteins. Here, we present structural and functional analyses of molecular interactions between human VPS4, LIP5, and the ESCRT-III proteins. The N-terminal domain of LIP5 (LIP5NTD) is required for LIP5-mediated stimulation of VPS4, and the ESCRT-III protein CHMP5 strongly inhibits the stimulation. Both of these observations are distinct from what was previously described for homologous yeast proteins. The crystal structure of LIP5NTD in complex with the MIT (microtubule-interacting and transport)-interacting motifs of CHMP5 and a second ESCRT-III protein, CHMP1B, was determined at 1 Å resolution. It reveals an ESCRT-III binding induced moderate conformational change in LIP5NTD, which results from insertion of a conserved CHMP5 tyrosine residue (Tyr182) at the core of LIP5NTD structure. Mutation of Tyr182 partially relieves the inhibition displayed by CHMP5. Together, these results suggest a novel mechanism of VPS4 regulation in metazoans, where CHMP5 functions as a negative allosteric switch to control LIP5-mediated stimulation of VPS4. PMID:25637630

  13. Pore-forming activity of type III system-secreted proteins leads to oncosis of Pseudomonas aeruginosa-infected macrophages.

    PubMed

    Dacheux, D; Goure, J; Chabert, J; Usson, Y; Attree, I

    2001-04-01

    The Pseudomonas aeruginosa cystic fibrosis isolate CHA induces type III secretion system-dependent but ExoU-independent oncosis of neutrophils and macrophages. Time-lapse microscopy of the infection process revealed the rapid accumulation of motile bacteria around infected cells undergoing the process of oncosis, a phenomenon we termed pack swarming. Characterization of the non-chemotactic CHAcheZ mutant showed that pack swarming is a bacterial chemotactic response to infected macrophages. A non-cytotoxic mutant, lacking the type III-secreted proteins PcrV, PopB and PopD, was able to pack swarm only in the presence of the parental strain CHA or when macrophages were pretreated with the pore-forming toxin streptolysin O. Interaction of P. aeruginosa with red blood cells (RBCs) showed that the contact-dependent haemolysis provoked by CHA requires secretion via the type III system and the PcrV, PopB/PopD proteins. The pore inserted into RBC membrane was estimated from osmoprotection experiments to be between 2.8 and 3.5 nm. CHA-infected macrophages could be protected from cell lysis with PEG3350, indicating that the pore introduced into RBC and macrophage membranes is of similar size. The time course uptake of the vital fluorescent dye, Yo-Pro-1, into infected macrophages confirmed that the formation of transmembrane pores by CHA precedes cellular oncosis. Therefore, CHA-induced macrophage death results from a pore-forming activity that is dependent on the intact pcrGVHpopBD operon. PMID:11298277

  14. High expression of glycolytic and pigment proteins is associated with worse clinical outcome in stage III melanoma.

    PubMed

    Falkenius, Johan; Lundeberg, Joakim; Johansson, Hemming; Tuominen, Rainer; Frostvik-Stolt, Marianne; Hansson, Johan; Egyhazi Brage, Suzanne

    2013-12-01

    There are insufficient numbers of prognostic factors available for prediction of clinical outcome in patients with stage III malignant cutaneous melanoma, even when known adverse pathological risk factors, such as macrometastasis, number of lymph node metastases, and ulceration are taken into consideration. The aim of this study was therefore to identify additional prognostic factors to better predict patients with a high risk of relapse, thus enabling us to better determine the need for adjuvant treatment in stage III disease. An RNA oligonucleotide microarray study was performed on first regional lymph node metastases in 42 patients with stage III melanoma: 23 patients with short-term survival (≤ 13 months) and 19 with long-term survival (≥ 60 months), to identify genes associated with clinical outcome. Candidate genes were validated by real-time PCR and immunohistochemical analysis. Several gene ontology (GO) categories were highly significantly differentially expressed including glycolysis (GO: 0006096; P<0.001) and the pigment biosynthetic process (GO: 0046148; P<0.001), in which overexpression was associated with short-disease-specific survival. Three overexpressed glycolytic genes, GAPDHS, GAPDH, and PKM2, and two pigment-related genes, TYRP1 and OCA2, were selected for validation. A significant difference in GAPDHS protein expression between short- and long-term survivors (P=0.021) and a trend for PKM2 (P=0.093) was observed in univariate analysis. Positive expression of at least two of four proteins (GAPDHS, GAPDH, PKM2, TYRP1) in immunohistochemical analysis was found to be an independent adverse prognostic factor for disease-specific survival (P=0.011). Our results indicate that this prognostic panel in combination with established risk factors may contribute to an improved prediction of patients with a high risk of relapse. PMID:24128789

  15. Dynamic behavior of Arabidopsis eIF4A-III, putative core protein of exon junction complex: fast relocation to nucleolus and splicing speckles under hypoxia.

    PubMed

    Koroleva, O A; Calder, G; Pendle, A F; Kim, S H; Lewandowska, D; Simpson, C G; Jones, I M; Brown, J W S; Shaw, P J

    2009-05-01

    Here, we identify the Arabidopsis thaliana ortholog of the mammalian DEAD box helicase, eIF4A-III, the putative anchor protein of exon junction complex (EJC) on mRNA. Arabidopsis eIF4A-III interacts with an ortholog of the core EJC component, ALY/Ref, and colocalizes with other EJC components, such as Mago, Y14, and RNPS1, suggesting a similar function in EJC assembly to animal eIF4A-III. A green fluorescent protein (GFP)-eIF4A-III fusion protein showed localization to several subnuclear domains: to the nucleoplasm during normal growth and to the nucleolus and splicing speckles in response to hypoxia. Treatment with the respiratory inhibitor sodium azide produced an identical response to the hypoxia stress. Treatment with the proteasome inhibitor MG132 led to accumulation of GFP-eIF4A-III mainly in the nucleolus, suggesting that transition of eIF4A-III between subnuclear domains and/or accumulation in nuclear speckles is controlled by proteolysis-labile factors. As revealed by fluorescence recovery after photobleaching analysis, the nucleoplasmic fraction was highly mobile, while the speckles were the least mobile fractions, and the nucleolar fraction had an intermediate mobility. Sequestration of eIF4A-III into nuclear pools with different mobility is likely to reflect the transcriptional and mRNA processing state of the cell. PMID:19435936

  16. Genetic characterization of conserved charged residues in the bacterial flagellar type III export protein FlhA.

    PubMed

    Hara, Noritaka; Namba, Keiichi; Minamino, Tohru

    2011-01-01

    For assembly of the bacterial flagellum, most of flagellar proteins are transported to the distal end of the flagellum by the flagellar type III protein export apparatus powered by proton motive force (PMF) across the cytoplasmic membrane. FlhA is an integral membrane protein of the export apparatus and is involved in an early stage of the export process along with three soluble proteins, FliH, FliI, and FliJ, but the energy coupling mechanism remains unknown. Here, we carried out site-directed mutagenesis of eight, highly conserved charged residues in putative juxta- and trans-membrane helices of FlhA. Only Asp-208 was an essential acidic residue. Most of the FlhA substitutions were tolerated, but resulted in loss-of-function in the ΔfliH-fliI mutant background, even with the second-site flhB(P28T) mutation that increases the probability of flagellar protein export in the absence of FliH and FliI. The addition of FliH and FliI allowed the D45A, R85A, R94K and R270A mutant proteins to work even in the presence of the flhB(P28T) mutation. Suppressor analysis of a flhA(K203W) mutation showed an interaction between FlhA and FliR. Taken all together, we suggest that Asp-208 is directly involved in PMF-driven protein export and that the cooperative interactions of FlhA with FlhB, FliH, FliI, and FliR drive the translocation of export substrate. PMID:21811603

  17. RpoS and quorum sensing control expression and polar localization of Vibrio cholerae chemotaxis cluster III proteins in vitro and in vivo

    PubMed Central

    Ringgaard, Simon; Hubbard, Troy; Mandlik, Anjali; Davis, Brigid M.; Waldor, Matthew K.

    2015-01-01

    Summary The diarrheal pathogen Vibrio cholerae contains 3 gene clusters that encode chemotaxis-related proteins, but only cluster II appears to be required for chemotaxis. Here, we present the first characterization of V. cholerae's “cluster III” chemotaxis system. We found that cluster III proteins assemble into foci at bacterial poles, like those formed by cluster II proteins, but the two systems assemble independently and do not colocalize. Cluster III proteins are expressed in vitro during stationary phase and in conjunction with growth arrest linked to carbon starvation. This expression, as well as expression in vivo in suckling rabbits, is dependent upon RpoS. V. cholerae's CAI-1 quorum sensing (QS) system is also required for cluster III expression in stationary phase and modulates its expression in vivo, but is not required for cluster III expression in response to carbon starvation. Surprisingly, even though the CAI-1 and AI-2 QS systems are thought to feed into the same signaling pathway, the AI-2 system inhibited cluster III gene expression, revealing that the outputs of the two QS systems are not always the same. The distinctions between genetic determinants of cluster III expression in vitro and in vivo highlight the distinctive nature of the in vivo environment. PMID:25989366

  18. An energy transduction mechanism used in bacterial flagellar type III protein export

    PubMed Central

    Minamino, Tohru; Morimoto, Yusuke V.; Hara, Noritaka; Namba, Keiichi

    2011-01-01

    Flagellar proteins of bacteria are exported by a specific export apparatus. FliI ATPase forms a complex with FliH and FliJ and escorts export substrates from the cytoplasm to the export gate complex, which is made up of six membrane proteins. The export gate complex utilizes proton motive force across the cytoplasmic membrane for protein translocation, but the mechanism remains unknown. Here we show that the export gate complex by itself is a proton–protein antiporter that uses the two components of proton motive force, Δψ and ΔpH, for different steps of the protein export process. However, in the presence of FliH, FliI and FliJ, a specific binding of FliJ with an export gate membrane protein, FlhA, is brought about by the FliH–FliI complex, which turns the export gate into a highly efficient, Δψ-driven protein export apparatus. PMID:21934659

  19. The tomato terpene synthase gene family.

    PubMed

    Falara, Vasiliki; Akhtar, Tariq A; Nguyen, Thuong T H; Spyropoulou, Eleni A; Bleeker, Petra M; Schauvinhold, Ines; Matsuba, Yuki; Bonini, Megan E; Schilmiller, Anthony L; Last, Robert L; Schuurink, Robert C; Pichersky, Eran

    2011-10-01

    Compounds of the terpenoid class play numerous roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of cultivated tomato (Solanum lycopersicum) contains 44 terpene synthase (TPS) genes, including 29 that are functional or potentially functional. Of these 29 TPS genes, 26 were expressed in at least some organs or tissues of the plant. The enzymatic functions of eight of the TPS proteins were previously reported, and here we report the specific in vitro catalytic activity of 10 additional tomato terpene synthases. Many of the tomato TPS genes are found in clusters, notably on chromosomes 1, 2, 6, 8, and 10. All TPS family clades previously identified in angiosperms are also present in tomato. The largest clade of functional TPS genes found in tomato, with 12 members, is the TPS-a clade, and it appears to encode only sesquiterpene synthases, one of which is localized to the mitochondria, while the rest are likely cytosolic. A few additional sesquiterpene synthases are encoded by TPS-b clade genes. Some of the tomato sesquiterpene synthases use z,z-farnesyl diphosphate in vitro as well, or more efficiently than, the e,e-farnesyl diphosphate substrate. Genes encoding monoterpene synthases are also prevalent, and they fall into three clades: TPS-b, TPS-g, and TPS-e/f. With the exception of two enzymes involved in the synthesis of ent-kaurene, the precursor of gibberellins, no other tomato TPS genes could be demonstrated to encode diterpene synthases so far. PMID:21813655

  20. Molecular characterization of the thi3 gene involved in thiamine biosynthesis in Zea mays: cDNA sequence and enzymatic and structural properties of the recombinant bifunctional protein with 4-amino-5-hydroxymethyl-2-methylpyrimidine (phosphate) kinase and thiamine monophosphate synthase activities.

    PubMed

    Rapala-Kozik, Maria; Olczak, Mariusz; Ostrowska, Katarzyna; Starosta, Agata; Kozik, Andrzej

    2007-12-01

    A thiamine biosynthesis gene, thi3, from maize Zea mays has been identified through cloning and sequencing of cDNA and heterologous overexpression of the encoded protein, THI3, in Escherichia coli. The recombinant THI3 protein was purified to homogeneity and shown to possess two essentially different enzymatic activities of HMP(-P) [4-amino-5-hydroxymethyl-2-methylpyrimidine (phosphate)] kinase and TMP (thiamine monophosphate) synthase. Both activities were characterized in terms of basic kinetic constants, with interesting findings that TMP synthase is uncompetitively inhibited by excess of one of the substrates [HMP-PP (HMP diphosphate)] and ATP. A bioinformatic analysis of the THI3 sequence suggested that these activities were located in two distinct, N-terminal kinase and C-terminal synthase, domains. Models of the overall folds of THI3 domains and the arrangements of active centre residues were obtained with the SWISS-MODEL protein modelling server, on the basis of the known three-dimensional structures of Salmonella enterica serotype Typhimurium HMP(-P) kinase and Bacillus subtilis TMP synthase. The essential roles of Gln98 and Met134 residues for HMP kinase activity and of Ser444 for TMP synthase activity were experimentally confirmed by site-directed mutagenesis. PMID:17696876

  1. A Tick Gut Protein with Fibronectin III Domains Aids Borrelia burgdorferi Congregation to the Gut during Transmission

    PubMed Central

    Schuijt, Tim J.; Boder, Eric; Hovius, Joppe W.; Fikrig, Erol

    2014-01-01

    Borrelia burgdorferi transmission to the vertebrate host commences with growth of the spirochete in the tick gut and migration from the gut to the salivary glands. This complex process, involving intimate interactions of the spirochete with the gut epithelium, is pivotal to transmission. We utilized a yeast surface display library of tick gut proteins to perform a global screen for tick gut proteins that might interact with Borrelia membrane proteins. A putative fibronectin type III domain-containing tick gut protein (Ixofin3D) was most frequently identified from this screen and prioritized for further analysis. Immunization against Ixofin3D and RNA interference-mediated reduction in expression of Ixofin3D resulted in decreased spirochete burden in tick salivary glands and in the murine host. Microscopic examination showed decreased aggregation of spirochetes on the gut epithelium concomitant with reduced expression of Ixofin3D. Our observations suggest that the interaction between Borrelia and Ixofin3D facilitates spirochete congregation to the gut during transmission, and provides a “molecular exit” direction for spirochete egress from the gut. PMID:25102051

  2. Mathematical aspects of molecular replacement. III. Properties of space groups preferred by proteins in the Protein Data Bank.

    PubMed

    Chirikjian, G; Sajjadi, S; Toptygin, D; Yan, Y

    2015-03-01

    The main goal of molecular replacement in macromolecular crystallography is to find the appropriate rigid-body transformations that situate identical copies of model proteins in the crystallographic unit cell. The search for such transformations can be thought of as taking place in the coset space Γ\\G where Γ is the Sohncke group of the macromolecular crystal and G is the continuous group of rigid-body motions in Euclidean space. This paper, the third in a series, is concerned with viewing nonsymmorphic Γ in a new way. These space groups, rather than symmorphic ones, are the most common ones for protein crystals. Moreover, their properties impact the structure of the space Γ\\G. In particular, nonsymmorphic space groups contain both Bieberbach subgroups and symmorphic subgroups. A number of new theorems focusing on these subgroups are proven, and it is shown that these concepts are related to the preferences that proteins have for crystallizing in different space groups, as observed in the Protein Data Bank. PMID:25727867

  3. A Substrate-Fusion Protein Is Trapped inside the Type III Secretion System Channel in Shigella flexneri

    PubMed Central

    Dohlich, Kim; Zumsteg, Anna Brotcke; Goosmann, Christian; Kolbe, Michael

    2014-01-01

    The Type III Secretion System (T3SS) is a macromolecular complex used by Gram-negative bacteria to secrete effector proteins from the cytoplasm across the bacterial envelope in a single step. For many pathogens, the T3SS is an essential virulence factor that enables the bacteria to interact with and manipulate their respective host. A characteristic structural feature of the T3SS is the needle complex (NC). The NC resembles a syringe with a basal body spanning both bacterial membranes and a long needle-like structure that protrudes from the bacterium. Based on the paradigm of a syringe-like mechanism, it is generally assumed that effectors and translocators are unfolded and secreted from the bacterial cytoplasm through the basal body and needle channel. Despite extensive research on T3SS, this hypothesis lacks experimental evidence and the mechanism of secretion is not fully understood. In order to elucidate details of the T3SS secretion mechanism, we generated fusion proteins consisting of a T3SS substrate and a bulky protein containing a knotted motif. Because the knot cannot be unfolded, these fusions are accepted as T3SS substrates but remain inside the NC channel and obstruct the T3SS. To our knowledge, this is the first time substrate fusions have been visualized together with isolated NCs and we demonstrate that substrate proteins are secreted directly through the channel with their N-terminus first. The channel physically encloses the fusion protein and shields it from a protease and chemical modifications. Our results corroborate an elementary understanding of how the T3SS works and provide a powerful tool for in situ-structural investigations in the future. This approach might also be applicable to other protein secretion systems that require unfolding of their substrates prior to secretion. PMID:24453973

  4. (R)-citramalate synthase in methanogenic archaea.

    PubMed

    Howell, D M; Xu, H; White, R H

    1999-01-01

    The Methanococcus jannaschii gene MJ1392 was cloned, and its protein product was hyperexpressed in Escherichia coli. The resulting protein was purified and shown to catalyze the condensation of pyruvate and acetyl coenzyme A, with the formation of (R)-citramalate. Thus, this gene (cimA) encodes an (R)-citramalate synthase (CimA). This is the first identification of this enzyme, which is likely involved in the biosynthesis of isoleucine. PMID:9864346

  5. Antithrombin III and its interaction with heparin. Comparison of the human, bovine, and porcine proteins by /sup 1/H NMR spectroscopy

    SciTech Connect

    Gettins, P.

    1987-03-10

    /sup 1/H NMR has been used to characterize and compare the structures of antithrombin III from human, bovine, and porcine plasma as well as to investigate the interactions of each of these proteins with heparin fragments of defined length. The amino acid compositions of the three proteins are very similar, which is reflected in the gross features of their /sup 1/H NMR spectra. Human antithrombin III has five histidine residues, bovine has six, and porcine has five. The C(2) proton from each of these residues gives a narrow resonance and titrates with pH; the pK/sub a/'s are in the range 5.15-7.25. It is concluded that all histidines in each protein are surface residues with considerable independent mobility. The carbohydrate chains in each protein also give sharp resonances consistent with a surface location and motional flexibility. The /sup 1/H spectra are sensitive to heparin binding. Although heparin resonances obscure protein resonances in the region 3.2-6.0 ppm, difference spectra between antithrombin III with and without heparin show clear perturbation of a small number of aromatic and aliphatic protein protons. For human antithrombin III, it was shown that heparin fragments 8, 10, and 16 sugar residues in length result in almost identical perturbations to the protein. In contrast, tetrasaccharide results in fewer perturbations. Significantly, intact high molecular weight heparin causes the same spectral perturbations as the 16-residue fragment. These data are discussed in terms of requirements for heparin binding.

  6. Synthesis, molecular modeling and biological evaluation of β-ketoacyl-acyl carrier protein synthase III (FabH) as novel antibacterial agents.

    PubMed

    Zhang, Hong-Jia; Zhu, Di-Di; Li, Zi-Lin; Sun, Juan; Zhu, Hai-Liang

    2011-08-01

    A series of novel cinnamic acid secnidazole ester derivatives have been designed and synthesized, and their biological activities were also evaluated as potential inhibitors of FabH. These compounds were assayed for antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus. Compounds with potent antibacterial activities were tested for their E. coli FabH inhibitory activity. Compound 3n showed the most potent antibacterial activity with MIC of 1.56-6.25 μg/mL against the tested bacterial strains and exhibited the most potent E. coli FabH inhibitory activity with IC₅₀ of 2.5 μM. Docking simulation was performed to position compound 3n into the E. coli FabH active site to determine the probable binding conformation. PMID:21741250

  7. THI1, a Thiamine Thiazole Synthase, Interacts with Ca2+-Dependent Protein Kinase CPK33 and Modulates the S-Type Anion Channels and Stomatal Closure in Arabidopsis1[OPEN

    PubMed Central

    Li, Chun-Long; Wang, Mei; Wu, Xiao-Meng; Chen, Dong-Hua; Lv, Hong-Jun; Shen, Jian-Lin; Qiao, Zhu; Zhang, Wei

    2016-01-01

    Thiamine is required for both plant growth and development. Here, the involvement of a thiamine thiazole synthase, THI1, has been demonstrated in both guard cell abscisic acid (ABA) signaling and the drought response in Arabidopsis (Arabidopsis thaliana). THI1 overexpressors proved to be more sensitive to ABA than the wild type with respect to both the activation of guard cell slow type anion channels and stomatal closure; this effectively reduced the rate of water loss from the plant and thereby enhanced its level of drought tolerance. A yeast two-hybrid strategy was used to screen a cDNA library from epidermal strips of leaves for THI1 regulatory factors, and identified CPK33, a Ca2+-dependent protein kinase, as interactor with THI1 in a plasma membrane-delimited manner. Loss-of-function cpk33 mutants were hypersensitive to ABA activation of slow type anion channels and ABA-induced stomatal closure, while the CPK33 overexpression lines showed opposite phenotypes. CPK33 kinase activity was essential for ABA-induced stomatal closure. Consistent with their contrasting regulatory role over stomatal closure, THI1 suppressed CPK33 kinase activity in vitro. Together, our data reveal a novel regulatory role of thiamine thiazole synthase to kinase activity in guard cell signaling. PMID:26662273

  8. 2.0 Angstrom Structure of Prostaglandin H2 Synthase-1 Reconstituted with a Manganese Porphyrin Cofactor

    SciTech Connect

    Gupta,K.; Selinsky, B.; Loll, P.

    2006-01-01

    Prostaglandin H{sub 2} synthase (EC 1.14.99.1) is a clinically important drug target that catalyzes two key steps in the biosynthesis of the eicosanoid hormones. The enzyme contains spatially distinct cyclooxygenase and peroxidase active sites, both of which require a heme cofactor. Substitution of ferric heme by Mn{sup III} protoporphyrin IX greatly diminishes the peroxidase activity, but has little effect on the cyclooxygenase activity. Here, the 2.0 Angstrom resolution crystal structure of the Mn{sup III} form of ovine prostaglandin H{sub 2} synthase-1 is described (R = 21.8%, R{sub free} = 23.7%). Substitution of Mn{sup III} for Fe{sup III} causes no structural perturbations in the protein. However, the out-of-plane displacement of the manganese ion with respect to the porphyrin is greater than that of the iron by approximately 0.2 Angstroms. This perturbation may help to explain the altered catalytic properties of the manganese enzyme.

  9. Maf1 protein, repressor of RNA polymerase III, indirectly affects tRNA processing.

    PubMed

    Karkusiewicz, Iwona; Turowski, Tomasz W; Graczyk, Damian; Towpik, Joanna; Dhungel, Nripesh; Hopper, Anita K; Boguta, Magdalena

    2011-11-11

    Maf1 is negative regulator of RNA polymerase III in yeast. We observed high levels of both primary transcript and end-matured, intron-containing pre-tRNAs in the maf1Δ strain. This pre-tRNA accumulation could be overcome by transcription inhibition, arguing against a direct role of Maf1 in tRNA maturation and suggesting saturation of processing machinery by the increased amounts of primary transcripts. Saturation of the tRNA exportin, Los1, is one reason why end-matured intron-containing pre-tRNAs accumulate in maf1Δ cells. However, it is likely possible that other components of the processing pathway are also limiting when tRNA transcription is increased. According to our model, Maf1-mediated transcription control and nuclear export by Los1 are two major stages of tRNA biosynthesis that are regulated by environmental conditions in a coordinated manner. PMID:21940626

  10. Maf1 Protein, Repressor of RNA Polymerase III, Indirectly Affects tRNA Processing*

    PubMed Central

    Karkusiewicz, Iwona; Turowski, Tomasz W.; Graczyk, Damian; Towpik, Joanna; Dhungel, Nripesh; Hopper, Anita K.; Boguta, Magdalena

    2011-01-01

    Maf1 is negative regulator of RNA polymerase III in yeast. We observed high levels of both primary transcript and end-matured, intron-containing pre-tRNAs in the maf1Δ strain. This pre-tRNA accumulation could be overcome by transcription inhibition, arguing against a direct role of Maf1 in tRNA maturation and suggesting saturation of processing machinery by the increased amounts of primary transcripts. Saturation of the tRNA exportin, Los1, is one reason why end-matured intron-containing pre-tRNAs accumulate in maf1Δ cells. However, it is likely possible that other components of the processing pathway are also limiting when tRNA transcription is increased. According to our model, Maf1-mediated transcription control and nuclear export by Los1 are two major stages of tRNA biosynthesis that are regulated by environmental conditions in a coordinated manner. PMID:21940626

  11. On the principle of ion selectivity in Na+/H+-coupled membrane proteins: Experimental and theoretical studies of an ATP synthase rotor

    PubMed Central

    Leone, Vanessa; Pogoryelov, Denys; Meier, Thomas; Faraldo-Gómez, José D.

    2015-01-01

    Numerous membrane transporters and enzymes couple their mechanisms to the permeation of Na+ or H+, thereby harnessing the energy stored in the form of transmembrane electrochemical potential gradients to sustain their activities. The molecular and environmental factors that control and modulate the ion specificity of most of these systems are, however, poorly understood. Here, we use isothermal titration calorimetry to determine the Na+/H+ selectivity of the ion-driven membrane rotor of an F-type ATP synthase. Consistent with earlier theoretical predictions, we find that this rotor is significantly H+ selective, although not sufficiently to be functionally coupled to H+, owing to the large excess of Na+ in physiological settings. The functional Na+ specificity of this ATP synthase thus results from two opposing factors, namely its inherent chemical selectivity and the relative availability of the coupling ion. Further theoretical studies of this membrane rotor, and of two others with a much stronger and a slightly weaker H+ selectivity, indicate that, although the inherent selectivity of their ion-binding sites is largely set by the balance of polar and hydrophobic groups flanking a conserved carboxylic side chain, subtle variations in their structure and conformational dynamics, for a similar chemical makeup, can also have a significant contribution. We propose that the principle of ion selectivity outlined here may provide a rationale for the differentiation of Na+- and H+-coupled systems in other families of membrane transporters and enzymes. PMID:25713346

  12. Essential role of the donor acyl carrier protein in stereoselective chain translocation to a fully reducing module of the nanchangmycin polyketide synthase.

    PubMed

    Guo, Xun; Liu, Tiangang; Deng, Zixin; Cane, David E

    2012-01-31

    Incubation of recombinant module 2 of the polyether nanchangmycin synthase (NANS), carrying an appended thioesterase domain, with the ACP-bound substrate (2RS)-2-methyl-3-ketobutyryl-NANS_ACP1 (2-ACP1) and methylmalonyl-CoA in the presence of NADPH gave diastereomerically pure (2S,4R)-2,4-dimethyl-5-ketohexanoic acid (4a). These results contrast with the previously reported weak discrimination by NANS module 2+TE between the enantiomers of the corresponding N-acetylcysteamine-conjugated substrate analogue (±)-2-methyl-3-ketobutyryl-SNAC (2-SNAC), which resulted in formation of a 5:3 mixture of 4a and its (2S,4S)-diastereomer 4b. Incubation of NANS module 2+TE with 2-ACP1 in the absence of NADPH gave unreduced 3,5,6-trimethyl-4-hydroxypyrone (3) with a k(cat) of 4.4 ± 0.9 min⁻¹ and a k(cat)/K(m) of 67 min⁻¹ mM⁻¹, corresponding to a ∼2300-fold increase compared to the k(cat)/K(m) for the diffusive substrate 2-SNAC. Covalent tethering of the 2-methyl-3-ketobutyryl thioester substrate to the NANS ACP1 domain derived from the natural upstream PKS module of the nanchangmycin synthase significantly enhanced both the stereospecificity and the kinetic efficiency of the sequential polyketide chain translocation and condensation reactions catalyzed by the ketosynthase domain of NANS module 2. PMID:22229794

  13. Interaction between carbon metabolism and phosphate accumulation is revealed by a mutation of a cellulose synthase-like protein, CSLF6

    PubMed Central

    Jin, Cheng; Fang, Chuanying; Yuan, Hui; Wang, Shouchuang; Wu, Yangyang; Liu, Xianqing; Zhang, Yuanyuan; Luo, Jie

    2015-01-01

    Phosphorus is an essential macronutrient for plant growth and development. However, the network that affects phosphate (Pi) accumulation in crops is not well established. It is reported here that OsCSLF6, a member of the cellulose synthase-like family (CSLF), which is found only in grasses, is involved in Pi accumulation. The oscslf6 mutants (oscslf6-1 and oscslf6-2) display Pi toxic symptoms and increased Pi accumulation in both roots and shoots under the Pi-sufficient condition, which correlate with the induced expression of Pi transporters in the knockout mutants. Consistent with the over-accumulation of Pi, a significant decrease in primary root length, adventitious root length, and adventitious root number were observed in the oscslf6 mutants when compared with the wild type (WT) under Pi-sufficient conditions. In addition, the sucrose (Suc) level was increased in the oscslf6 mutants and the expression of sucrose synthases (OsSUS4/5) and sucrose transporters (OsSUT1/2/4/OsSweet14) genes were also induced in the shoots of oscslf6 mutants, suggesting that OsCSLF6 may play a role in affecting Pi accumulation by affecting the level of carbon metabolism. PMID:25740927

  14. On the principle of ion selectivity in Na+/H+-coupled membrane proteins: experimental and theoretical studies of an ATP synthase rotor.

    PubMed

    Leone, Vanessa; Pogoryelov, Denys; Meier, Thomas; Faraldo-Gómez, José D

    2015-03-10

    Numerous membrane transporters and enzymes couple their mechanisms to the permeation of Na(+) or H(+), thereby harnessing the energy stored in the form of transmembrane electrochemical potential gradients to sustain their activities. The molecular and environmental factors that control and modulate the ion specificity of most of these systems are, however, poorly understood. Here, we use isothermal titration calorimetry to determine the Na(+)/H(+) selectivity of the ion-driven membrane rotor of an F-type ATP synthase. Consistent with earlier theoretical predictions, we find that this rotor is significantly H(+) selective, although not sufficiently to be functionally coupled to H(+), owing to the large excess of Na(+) in physiological settings. The functional Na(+) specificity of this ATP synthase thus results from two opposing factors, namely its inherent chemical selectivity and the relative availability of the coupling ion. Further theoretical studies of this membrane rotor, and of two others with a much stronger and a slightly weaker H(+) selectivity, indicate that, although the inherent selectivity of their ion-binding sites is largely set by the balance of polar and hydrophobic groups flanking a conserved carboxylic side chain, subtle variations in their structure and conformational dynamics, for a similar chemical makeup, can also have a significant contribution. We propose that the principle of ion selectivity outlined here may provide a rationale for the differentiation of Na(+)- and H(+)-coupled systems in other families of membrane transporters and enzymes. PMID:25713346

  15. Observing single FoF1-ATP synthase at work using an improved fluorescent protein mNeonGreen as FRET donor

    NASA Astrophysics Data System (ADS)

    Heitkamp, Thomas; Deckers-Hebestreit, Gabriele; Börsch, Michael

    2016-02-01

    Adenosine triphosphate (ATP) is the universal chemical energy currency for cellular activities provided mainly by the membrane enzyme FoF1-ATP synthase in bacteria, chloroplasts and mitochondria. Synthesis of ATP is accompanied by subunit rotation within the enzyme. Over the past 15 years we have developed a variety of single-molecule FRET (smFRET) experiments to monitor catalytic action of individual bacterial enzymes in vitro. By specifically labeling rotating and static subunits within a single enzyme we were able to observe three-stepped rotation in the F1 motor, ten-stepped rotation in the Fo motor and transient elastic deformation of the connected rotor subunits. However, the spatial and temporal resolution of motor activities measured by smFRET were limited by the photophysics of the FRET fluorophores. Here we evaluate the novel FRET donor mNeonGreen as a fusion to FoF1-ATP synthase and compare it to the previously used fluorophore EGFP. Topics of this manuscript are the biochemical purification procedures and the activity measurements of the fully functional mutant enzyme.

  16. Mapping the protein-binding sites for iridium(iii)-based CO-releasing molecules.

    PubMed

    Caterino, Marco; Petruk, Ariel A; Vergara, Alessandro; Ferraro, Giarita; Marasco, Daniela; Doctorovich, Fabio; Estrin, Dario A; Merlino, Antonello

    2016-07-26

    A combination of mass spectrometry, Raman microspectroscopy, circular dichroism and X-ray crystallography has been used to obtain detailed information on the reaction of an iridium-based CO-releasing molecule (Ir-CORM), Cs2IrCl5CO, with a model protein, bovine pancreatic ribonuclease. The results show that Ir-compound fragments bind to the N-terminal amine and close to histidine and methionine side chains, and the CO ligand is retained for a long time. The data provide helpful information for identifying protein targets for Ir-CORMs and for studying the mechanism that allows them to exhibit their interesting biological properties. PMID:27411388

  17. Mitochondrial citrate synthase crystals: novel finding in Sengers syndrome caused by acylglycerol kinase (AGK) mutations.

    PubMed

    Siriwardena, Komudi; Mackay, Nevena; Levandovskiy, Valeriy; Blaser, Susan; Raiman, Julian; Kantor, Paul F; Ackerley, Cameron; Robinson, Brian H; Schulze, Andreas; Cameron, Jessie M

    2013-01-01

    We report on two families with Sengers syndrome and mutations in the acylglycerol kinase gene (AGK). In the first family, two brothers presented with vascular strokes, lactic acidosis, cardiomyopathy and cataracts, abnormal muscle cell histopathology and mitochondrial function. One proband had very abnormal mitochondria with citrate synthase crystals visible in electron micrographs, associated with markedly high citrate synthase activity. Exome sequencing was used to identify mutations in the AGK gene in the index patient. Targeted sequencing confirmed the same homozygous mutation (c.3G>A, p.M1I) in the brother. The second family had four affected members, of which we examined two. They also presented with similar clinical symptoms, but no strokes. Postmortem heart and skeletal muscle tissues showed low complex I, III and IV activities in the heart, but normal in the muscle. Skin fibroblasts showed elevated lactate/pyruvate ratios and low complex I+III activity. Targeted sequencing led to identification of a homozygous c.979A>T, p.K327* mutation. AGK is located in the mitochondria and phosphorylates monoacylglycerol and diacylglycerol to lysophosphatidic acid and phosphatidic acid. Disruption of these signaling molecules affects the mitochondria's response to superoxide radicals, resulting in oxidative damage to mitochondrial DNA, lipids and proteins, and stimulation of cellular detoxification pathways. High levels of manganese superoxide dismutase protein were detected in all four affected individuals, consistent with increased free radical damage. Phosphatidic acid is also involved in the synthesis of phospholipids and its loss will result in changes to the lipid composition of the inner mitochondrial membrane. These effects manifest as cataract formation in the eye, respiratory chain dysfunction and cardiac hypertrophy in heart tissue. These two pedigrees confirm that mutation of AGK is responsible for the severe neonatal presentation of Sengers syndrome. The

  18. Differential expression of biphenyl synthase gene family members in fire-blight-infected apple 'Holsteiner Cox'.

    PubMed

    Chizzali, Cornelia; Gaid, Mariam M; Belkheir, Asma K; Hänsch, Robert; Richter, Klaus; Flachowsky, Henryk; Peil, Andreas; Hanke, Magda-Viola; Liu, Benye; Beerhues, Ludger

    2012-02-01

    Fire blight, caused by the bacterium Erwinia amylovora, is a devastating disease of apple (Malus × domestica). The phytoalexins of apple are biphenyls and dibenzofurans, whose carbon skeleton is formed by biphenyl synthase (BIS), a type III polyketide synthase. In the recently published genome sequence of apple 'Golden Delicious', nine BIS genes and four BIS gene fragments were detected. The nine genes fall into four subfamilies, referred to as MdBIS1 to MdBIS4. In a phylogenetic tree, the BIS amino acid sequences from apple and Sorbus aucuparia formed an individual cluster within the clade of the functionally diverse type III polyketide synthases. cDNAs encoding MdBIS1 to MdBIS4 were cloned from fire-blight-infected shoots of apple 'Holsteiner Cox,' heterologously expressed in Escherichia coli, and functionally analyzed. Benzoyl-coenzyme A and salicoyl-coenzyme A were the preferred starter substrates. In response to inoculation with E. amylovora, the BIS3 gene was expressed in stems of cv Holsteiner Cox, with highest transcript levels in the transition zone between necrotic and healthy tissues. The transition zone was the accumulation site of biphenyl and dibenzofuran phytoalexins. Leaves contained transcripts for BIS2 but failed to form immunodetectable amounts of BIS protein. In cell cultures of apple 'Cox Orange,' expression of the BIS1 to BIS3 genes was observed after the addition of an autoclaved E. amylovora suspension. Using immunofluorescence localization under a confocal laser-scanning microscope, the BIS3 protein in the transition zone of stems was detected in the parenchyma of the bark. Dot-shaped immunofluorescence was confined to the junctions between neighboring cortical parenchyma cells. PMID:22158676

  19. Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems.

    PubMed

    Gottschamel, Johanna; Lössl, Andreas; Ruf, Stephanie; Wang, Yanliang; Skaugen, Morten; Bock, Ralph; Clarke, Jihong Liu

    2016-07-01

    Dengue fever is a disease in many parts of the tropics and subtropics and about half the world's population is at risk of infection according to the World Health Organization. Dengue is caused by any of the four related dengue virus serotypes DEN-1, -2, -3 and -4, which are transmitted to people by Aedes aegypti mosquitoes. Currently there is only one vaccine (Dengvaxia(®)) available (limited to a few countries) on the market since 2015 after half a century's intensive efforts. Affordable and accessible vaccines against dengue are hence still urgently needed. The dengue envelop protein domain III (EDIII), which is capable of eliciting serotype-specific neutralizing antibodies, has become the focus for subunit vaccine development. To contribute to the development of an accessible and affordable dengue vaccine, in the current study we have used plant-based vaccine production systems to generate a dengue subunit vaccine candidate in tobacco. Chloroplast genome engineering was applied to express serotype-specific recombinant EDIII proteins in tobacco chloroplasts using both constitutive and ethanol-inducible expression systems. Expression of a tetravalent antigen fusion construct combining EDIII polypeptides from all four serotypes was also attempted. Transplastomic EDIII-expressing tobacco lines were obtained and homoplasmy was verified by Southern blot analysis. Northern blot analyses showed expression of EDIII antigen-encoding genes. EDIII protein accumulation levels varied for the different recombinant EDIII proteins and the different expression systems, and reached between 0.8 and 1.6 % of total cellular protein. Our study demonstrates the suitability of the chloroplast compartment as a production site for an EDIII-based vaccine candidate against dengue fever and presents a Gateway(®) plastid transformation vector for inducible transgene expression. PMID:27116001

  20. Sequence and structural analysis of surface protein antigen I/II (SpaA) of Streptococcus sobrinus.

    PubMed Central

    LaPolla, R J; Haron, J A; Kelly, C G; Taylor, W R; Bohart, C; Hendricks, M; Pyati, J P; Graff, R T; Ma, J K; Lehner, T

    1991-01-01

    Streptococcal antigen I/II or the surface protein antigen A (SpaA) of Streptococcus sobrinus is an adhesin which mediates binding of the organism to tooth surfaces. The complete sequence of the gene which encodes SpaA has been determined. The gene consists of 4,584 bp and encodes a protein of 1,528 amino acid residues. The deduced amino acid sequence shows extensive homology with those of the cell surface adhesins from Streptococcus mutans serotypes c and f and from Streptococcus sanguis. Structural analysis of the N-terminal region (residues 50 to 550), which is rich in alanine and includes four tandem repeats of an 82-residue sequence, suggests that it adopts an alpha-helical coiled-coil conformation. Cell surface hydrophobicity may be associated with this region. The C-terminal region is more conserved and includes two tandem repeats of a 39-residue proline-rich sequence. A further proline-rich sequence in this region is predicted to span the cell wall. Although a hydrophobic sequence is present in the C-terminal region, it appears to be too short to span the cell membrane. Anchoring of SpaA in the cell membrane may therefore require some form of posttranslational modification or association with another membrane protein. PMID:1855987

  1. RNA polymerase III drives alternative splicing of the potassium channel–interacting protein contributing to brain complexity and neurodegeneration

    PubMed Central

    Massone, Sara; Vassallo, Irene; Castelnuovo, Manuele; Fiorino, Gloria; Gatta, Elena; Robello, Mauro; Borghi, Roberta; Tabaton, Massimo; Russo, Claudio; Dieci, Giorgio; Cancedda, Ranieri

    2011-01-01

    Alternative splicing generates protein isoforms that are conditionally or differentially expressed in specific tissues. The discovery of factors that control alternative splicing might clarify the molecular basis of biological and pathological processes. We found that IL1-α−dependent up-regulation of 38A, a small ribonucleic acid (RNA) polymerase III–transcribed RNA, drives the synthesis of an alternatively spliced form of the potassium channel–interacting protein (KCNIP4). The alternative KCNIP4 isoform cannot interact with the γ-secretase complex, resulting in modification of γ-secretase activity, amyloid precursor protein processing, and increased secretion of β-amyloid enriched in the more toxic Aβ x-42 species. Notably, synthesis of the variant KCNIP4 isoform is also detrimental to brain physiology, as it results in the concomitant blockade of the fast kinetics of potassium channels. This alternative splicing shift is observed at high frequency in tissue samples from Alzheimer’s disease patients, suggesting that RNA polymerase III cogenes may be upstream determinants of alternative splicing that significantly contribute to homeostasis and pathogenesis in the brain. PMID:21624954

  2. Web-Based Computational Chemistry Education with CHARMMing III: Reduction Potentials of Electron Transfer Proteins

    PubMed Central

    Perrin, B. Scott; Miller, Benjamin T.; Schalk, Vinushka; Woodcock, H. Lee; Brooks, Bernard R.; Ichiye, Toshiko

    2014-01-01

    A module for fast determination of reduction potentials, E°, of redox-active proteins has been implemented in the CHARMM INterface and Graphics (CHARMMing) web portal (www.charmming.org). The free energy of reduction, which is proportional to E°, is composed of an intrinsic contribution due to the redox site and an environmental contribution due to the protein and solvent. Here, the intrinsic contribution is selected from a library of pre-calculated density functional theory values for each type of redox site and redox couple, while the environmental contribution is calculated from a crystal structure of the protein using Poisson-Boltzmann continuum electrostatics. An accompanying lesson demonstrates a calculation of E°. In this lesson, an ionizable residue in a [4Fe-4S]-protein that causes a pH-dependent E° is identified, and the E° of a mutant that would test the identification is predicted. This demonstration is valuable to both computational chemistry students and researchers interested in predicting sequence determinants of E° for mutagenesis. PMID:25058418

  3. Web-based computational chemistry education with CHARMMing III: Reduction potentials of electron transfer proteins.

    PubMed

    Perrin, B Scott; Miller, Benjamin T; Schalk, Vinushka; Woodcock, H Lee; Brooks, Bernard R; Ichiye, Toshiko

    2014-07-01

    A module for fast determination of reduction potentials, E°, of redox-active proteins has been implemented in the CHARMM INterface and Graphics (CHARMMing) web portal (www.charmming.org). The free energy of reduction, which is proportional to E°, is composed of an intrinsic contribution due to the redox site and an environmental contribution due to the protein and solvent. Here, the intrinsic contribution is selected from a library of pre-calculated density functional theory values for each type of redox site and redox couple, while the environmental contribution is calculated from a crystal structure of the protein using Poisson-Boltzmann continuum electrostatics. An accompanying lesson demonstrates a calculation of E°. In this lesson, an ionizable residue in a [4Fe-4S]-protein that causes a pH-dependent E° is identified, and the E° of a mutant that would test the identification is predicted. This demonstration is valuable to both computational chemistry students and researchers interested in predicting sequence determinants of E° for mutagenesis. PMID:25058418

  4. Structure of thiocyanate hydrolase: a new nitrile hydratase family protein with a novel five-coordinate cobalt(III) center.

    PubMed

    Arakawa, Takatoshi; Kawano, Yoshiaki; Kataoka, Shingo; Katayama, Yoko; Kamiya, Nobuo; Yohda, Masafumi; Odaka, Masafumi

    2007-03-01

    Thiocyanate hydrolase (SCNase) of Thiobacillus thioparus THI115 is a cobalt(III)-containing enzyme catalyzing the degradation of thiocyanate to carbonyl sulfide and ammonia. We determined the crystal structures of the apo- and native SCNases at a resolution of 2.0 A. SCNases in both forms had a conserved hetero-dodecameric structure, (alphabetagamma)(4). Four alphabetagamma hetero-trimers were structurally equivalent. One alphabetagamma hetero-trimer was composed of the core domain and the betaN domain, which was located at the center of the molecule and linked the hetero-trimers with novel quaternary interfaces. In both the apo- and native SCNases, the core domain was structurally conserved between those of iron and cobalt-types of nitrile hydratase (NHase). Native SCNase possessed the post-translationally modified cysteine ligands, gammaCys131-SO(2)H and gammaCys133-SOH like NHases. However, the low-spin cobalt(III) was found to be in the distorted square-pyramidal geometry, which had not been reported before in any protein. The size as well as the electrostatic properties of the substrate-binding pocket was totally different from NHases with respect to the charge distribution and the substrate accessibility, which rationally explains the differences in the substrate preference between SCNase and NHase. PMID:17222425

  5. New Light on NO Bonding in Fe(III) Heme Proteins from Resonance Raman Spectroscopy and DFT Modeling

    PubMed Central

    Soldatova, Alexandra V.; Ibrahim, Mohammed; Olson, John S.; Czernuszewicz, Roman S.; Spiro, Thomas G.

    2010-01-01

    Visible and ultraviolet resonance Raman (RR) spectra are reported for FeIII(NO) adducts of myoglobin variants with altered polarity in the distal heme pockets. The stretching frequencies of the FeIII–NO and N–O bonds, νFeN and νNO, are negatively correlated, consistent with backbonding. However, the correlation shifts to lower νNO for variants lacking a distal histidine. DFT modeling reproduces the shifted correlations, and shows the shift to be associated with the loss of a lone-pair donor interaction from the distal histidine that selectively strengthens the N–O bond. However, when the model contains strongly electron-withdrawing substituents at the heme β-positions, νFeN and νNO become positively correlated. This effect results from FeIII–N–O bending, which is induced by lone pair donation to the NNO atom. Other mechanisms for bending are discussed, which likewise lead to a positive νFeN/νNO correlation, including thiolate ligation in heme proteins and electron-donating meso-substituents in heme models. The νFeN/νNO data for the Fe(III) complexes are reporters of heme pocket polarity and the accessibility of lone pair, Lewis base donors. Implications for biologically important processes, including NO binding, reductive nitrosylation and NO reduction, are discussed. PMID:20218710

  6. β-III spectrin underpins ankyrin R function in Purkinje cell dendritic trees: protein complex critical for sodium channel activity is impaired by SCA5-associated mutations.

    PubMed

    Clarkson, Yvonne L; Perkins, Emma M; Cairncross, Callum J; Lyndon, Alastair R; Skehel, Paul A; Jackson, Mandy

    2014-07-15

    Beta III spectrin is present throughout the elaborate dendritic tree of cerebellar Purkinje cells and is required for normal neuronal morphology and cell survival. Spinocerebellar ataxia type 5 (SCA5) and spectrin associated autosomal recessive cerebellar ataxia type 1 are human neurodegenerative diseases involving progressive gait ataxia and cerebellar atrophy. Both disorders appear to result from loss of β-III spectrin function. Further elucidation of β-III spectrin function is therefore needed to understand disease mechanisms and identify potential therapeutic options. Here, we report that β-III spectrin is essential for the recruitment and maintenance of ankyrin R at the plasma membrane of Purkinje cell dendrites. Two SCA5-associated mutations of β-III spectrin both reduce ankyrin R levels at the cell membrane. Moreover, a wild-type β-III spectrin/ankyrin-R complex increases sodium channel levels and activity in cell culture, whereas mutant β-III spectrin complexes fail to enhance sodium currents. This suggests impaired ability to form stable complexes between the adaptor protein ankyrin R and its interacting partners in the Purkinje cell dendritic tree is a key mechanism by which mutant forms of β-III spectrin cause ataxia, initially by Purkinje cell dysfunction and exacerbated by subsequent cell death. PMID:24603075

  7. Chemiluminescence detection of amino acids, peptides, and proteins using tris-2,2 prime -bipyridine ruthenium(III)

    SciTech Connect

    Li He; Cox, K.A.; Danielson, N.D. )

    1990-01-01

    The feasibility of using the tris-2-2{prime}-bipyridine ruthenium(III) chemiluminescent (CL) reaction for the detection of amino acids, peptides, and proteins has been studied. Detection limits of the amino acids as determined by flow injection analysis (FIA) ranged from 20 pmol of proline to 50 nmol of asparagine. In general, amino acids containing secondary amine groups yielded the strongest responses. A reaction mechanism for Ru(bpy){sub 3}{sup 3+} chemiluminescence of aliphatic amines has been proposed. Studies of peptide molecules and poly-prolines showed that the peptide bond barely contributes to the detection signals. The separation of hydroxyproline and proline in synthetic collagen by HPLC with Ru(bpy){sub 3}{sup 3+} chemiluminescence detection has been shown to be possible.

  8. LAP6/POLYKETIDE SYNTHASE A and LAP5/POLYKETIDE SYNTHASE B encode hydroxyalkyl α-pyrone synthases required for pollen development and sporopollenin biosynthesis in Arabidopsis thaliana.

    PubMed

    Kim, Sung Soo; Grienenberger, Etienne; Lallemand, Benjamin; Colpitts, Che C; Kim, Sun Young; Souza, Clarice de Azevedo; Geoffroy, Pierrette; Heintz, Dimitri; Krahn, Daniel; Kaiser, Markus; Kombrink, Erich; Heitz, Thierry; Suh, Dae-Yeon; Legrand, Michel; Douglas, Carl J

    2010-12-01

    Plant type III polyketide synthases (PKSs) catalyze the condensation of malonyl-CoA units with various CoA ester starter molecules to generate a diverse array of natural products. The fatty acyl-CoA esters synthesized by Arabidopsis thaliana ACYL-COA SYNTHETASE5 (ACOS5) are key intermediates in the biosynthesis of sporopollenin, the major constituent of exine in the outer pollen wall. By coexpression analysis, we identified two Arabidopsis PKS genes, POLYKETIDE SYNTHASE A (PKSA) and PKSB (also known as LAP6 and LAP5, respectively) that are tightly coexpressed with ACOS5. Recombinant PKSA and PKSB proteins generated tri-and tetraketide α-pyrone compounds in vitro from a broad range of potential ACOS5-generated fatty acyl-CoA starter substrates by condensation with malonyl-CoA. Furthermore, substrate preference profile and kinetic analyses strongly suggested that in planta substrates for both enzymes are midchain- and ω-hydroxylated fatty acyl-CoAs (e.g., 12-hydroxyoctadecanoyl-CoA and 16-hydroxyhexadecanoyl-CoA), which are the products of sequential actions of anther-specific fatty acid hydroxylases and acyl-CoA synthetase. PKSA and PKSB are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the PKS genes displayed pollen exine layer defects, and a double pksa pksb mutant was completely male sterile, with no apparent exine. These results show that hydroxylated α-pyrone polyketide compounds generated by the sequential action of ACOS5 and PKSA/B are potential and previously unknown sporopollenin precursors. PMID:21193570

  9. A net carbohydrate and protein system for evaluating cattle diets: III. Cattle requirements and diet adequacy.

    PubMed

    Fox, D G; Sniffen, C J; O'Connor, J D; Russell, J B; Van Soest, P J

    1992-11-01

    The Cornell Net Carbohydrate and Protein System (CNCPS) has equations for predicting nutrient requirements, feed intake, and feed utilization over wide variations in cattle (frame size, body condition, and stage of growth), feed carbohydrate and protein fractions and their digestion and passage rates, and environmental conditions. Independent data were used to validate the ability of the CNCPS to predict responses compared to National Research Council (NRC) systems. With DMI in steers, the CNCPS had a 12% lower standard error of the Y estimate (Sy.x) and three percentage units less bias than the NRC system. For DMI in heifers, both systems had a similar Sy.x but the NRC had four percentage units less bias. With lactating dairy cows' DMI, the CNCPS had a 12% lower Sy.x. Observed NEm requirement averaged 5% under NRC and 6% under CNCPS predicted values at temperatures above 9 degrees C but were 18% over NRC and 9% under CNCPS at temperatures under 9 degrees C. Energy retained was predicted with an R2 of .80 and .95 and a bias of 8 and 4% for the NRC and CNCPS, respectively. Protein retained was predicted with an R2 of .75 and .85 with a bias of 0 and -1% for NRC and CNCPS, respectively. Biases due to frame size, implant, or NEg were small. Body condition scores predicted body fat percentage in dairy cows with an R2 of .93 and a Sy.x of 2.35% body fat. The CNCPS predicted metabolizable protein allowable ADG with a bias of 1.6% with a Sy.x of .07 kg compared to values of -30% and .10 kg, respectively for the NRC system. PMID:1334063

  10. A primer on molecular biology for imagers: III. Proteins: structure and function.

    PubMed

    Pandit, Sunil D; Li, King C P

    2004-04-01

    This article along with the first 2 in this series (4,12) completes the discussion on the key molecules and process inside the cell namely, DNA, RNA, and proteins. These 3 articles provide a very basic foundation for understanding molecular biology concepts and summarize some of the work of numerous scientists over the past century. We understand these processes far better now than we did in the past, but clearly this knowledge is by no means complete and a number of basic scientists are working hard to elucidate and understand the fundamental mechanisms that operate within a cell. Genes and gene products work with each other in complex, interconnected pathways, and in perfect harmony to make a functional cell, tissue, and an organism as a whole. There is a lot of cross-talk that happens between different proteins that interact with various other proteins, DNA, and RNA to establish pathways, networks, and molecular systems as a team working to perfection. The past 15 years have seen the rapid development of systems biology approaches. We live in an era that emphasizes multi-disciplinary, cross-functional teams to perform science rather than individual researchers working on the bench on a very specific problem. Global approaches have become more common and the amount of data generated must be managed by trained bioinformatics personnel and large computers. In our subsequent articles, we will discuss these global approaches and the areas of genomics, functional genomics, and proteomics that have revolutionized the way we perform science. PMID:15109017

  11. Geranyl diphosphate synthase from mint

    SciTech Connect

    Croteau, Rodney Bruce; Wildung, Mark Raymond; Burke, Charles Cullen; Gershenzon, Jonathan

    1999-01-01

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

  12. Geranyl diphosphate synthase from mint

    SciTech Connect

    Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

    1999-03-02

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

  13. The LcrG Tip Chaperone Protein of the Yersinia pestis Type III Secretion System Is Partially Folded.

    PubMed

    Chaudhury, Sukanya; de Azevedo Souza, Clarice; Plano, Gregory V; De Guzman, Roberto N

    2015-09-25

    The type III secretion system (T3SS) is essential in the pathogenesis of Yersinia pestis, the causative agent of plague. A small protein, LcrG, functions as a chaperone to the tip protein LcrV, and the LcrG-LcrV interaction is important in regulating protein secretion through the T3SS. The atomic structure of the LcrG family is currently unknown. However, because of its predicted helical propensity, many have suggested that the LcrG family forms a coiled-coil structure. Here, we show by NMR and CD spectroscopy that LcrG lacks a tertiary structure and it consists of three partially folded α-helices spanning residues 7-38, 41-46, and 58-73. NMR titrations of LcrG with LcrV show that the entire length of a truncated LcrG (residues 7-73) is involved in binding to LcrV. However, there is regional variation in how LcrG binds to LcrV. The C-terminal region of a truncated LcrG (residues 52-73) shows tight binding interaction with LcrV while the N-terminal region (residues 7-51) shows weaker interaction with LcrV. This suggests that there are at least two binding events when LcrG binds to LcrV. Biological assays and mutagenesis indicate that the C-terminal region of LcrG (residues 52-73) is important in blocking protein secretion through the T3SS. Our results reveal structural and mechanistic insights into the atomic conformation of LcrG and how it binds to LcrV. PMID:26259880

  14. COX7A2L Is a Mitochondrial Complex III Binding Protein that Stabilizes the III2+IV Supercomplex without Affecting Respirasome Formation.

    PubMed

    Pérez-Pérez, Rafael; Lobo-Jarne, Teresa; Milenkovic, Dusanka; Mourier, Arnaud; Bratic, Ana; García-Bartolomé, Alberto; Fernández-Vizarra, Erika; Cadenas, Susana; Delmiro, Aitor; García-Consuegra, Inés; Arenas, Joaquín; Martín, Miguel A; Larsson, Nils-Göran; Ugalde, Cristina

    2016-08-30

    Mitochondrial respiratory chain (MRC) complexes I, III, and IV associate into a variety of supramolecular structures known as supercomplexes and respirasomes. While COX7A2L was originally described as a supercomplex-specific factor responsible for the dynamic association of complex IV into these structures to adapt MRC function to metabolic variations, this role has been disputed. Here, we further examine the functional significance of COX7A2L in the structural organization of the mammalian respiratory chain. As in the mouse, human COX7A2L binds primarily to free mitochondrial complex III and, to a minor extent, to complex IV to specifically promote the stabilization of the III2+IV supercomplex without affecting respirasome formation. Furthermore, COX7A2L does not affect the biogenesis, stabilization, and function of the individual oxidative phosphorylation complexes. These data show that independent regulatory mechanisms for the biogenesis and turnover of different MRC supercomplex structures co-exist. PMID:27545886

  15. Structural Features Reminiscent of ATP-Driven Protein Translocases Are Essential for the Function of a Type III Secretion-Associated ATPase

    PubMed Central

    Kato, Junya; Lefebre, Matthew

    2015-01-01

    ABSTRACT Many bacterial pathogens and symbionts utilize type III secretion systems to interact with their hosts. These machines have evolved to deliver bacterial effector proteins into eukaryotic target cells to modulate a variety of cellular functions. One of the most conserved components of these systems is an ATPase, which plays an essential role in the recognition and unfolding of proteins destined for secretion by the type III pathway. Here we show that structural features reminiscent of other ATP-driven protein translocases are essential for the function of InvC, the ATPase associated with a Salmonella enterica serovar Typhimurium type III secretion system. Mutational and functional analyses showed that a two-helix-finger motif and a conserved loop located at the entrance of and within the predicted pore formed by the hexameric ATPase are essential for InvC function. These findings provide mechanistic insight into the function of this highly conserved component of type III secretion machines. IMPORTANCE Type III secretion machines are essential for the virulence or symbiotic relationships of many bacteria. These machines have evolved to deliver bacterial effector proteins into host cells to modulate cellular functions, thus facilitating bacterial colonization and replication. An essential component of these machines is a highly conserved ATPase, which is necessary for the recognition and secretion of proteins destined to be delivered by the type III secretion pathway. Using modeling and structure and function analyses, we have identified structural features of one of these ATPases from Salmonella enterica serovar Typhimurium that help to explain important aspects of its function. PMID:26170413

  16. Haemocyanins in spiders, III. Chemical and physical properties of the proteins in Dugesiella and Cupiennius blood.

    PubMed

    Markl, J; Schmid, R; Czichos-Tiedt, S; Linzen, B

    1976-12-01

    The haemolymph of the tarantulas, Dugesiella (Eurypelma) californica and Dugesiella (Eurypelma) helluo contains high molecular weight haemocyanin (80-82% of total blood proteins) and a second protein not related to haemocyanin (18-20%). In the Lycosid spider, Cupiennius salei, haemocyanin (75% of total blood protein) occurs in two states of association. The haemocyanins were isolated by ultracentrifugation, gel filtration, isoelectric focusing, or preparative gel electrophoresis. Their sedimentation constants are 36.7 S (both tarantulas), 23.4 S and 15.9 S (Cupiennius). After alkaline dissociation, polypeptides sedimenting at 5.8 S (D. californica) and 4.7 S (Cupiennius) were obtained. The molecular weight of the intact functional subunit is (by sedimentation equilibrium) 70 300 (D. californica) and 69 900 (Cupiennius). Copper analysis results in closely similar values. By sodium dodecylsulphate gel electrophoresis, molecular weights of 71 000 (D. californica), 72 000 (Cupiennius) and 74 000 (D. helluo) were obtained. Denaturation with various agents did not lead to smaller polypeptides. The amino acid composition of the haemocyanins was determined (Table 1). The amino end group is blocked. The haemocyanins contain 1.2-1.5% of neutral carbohydrates and 0.3-0.5% of glucosamine (possibly acetylated). The neutral carbohydrates were identified with glucose, mannose, fucose, and arabinose, glucose being the dominant species. Neuraminic acid was not detected. The haemocyanins of the three species cannot be distinguished by their carbohydrate moieties, while there is a significant difference in amino acid composition between tarantula and Cupiennius haemocyanins. The second, non-respiratory protein isolated from spider blood sediments with 16.1 S (Dugesiella) or 15.9 S (Cupiennius). Its isoelectric point is at pH 5.5 It is stable in weakly alkaline solutions but can be denatured to yield polypeptide chains with molecular weights of 95 000 and 110 000. The amino acid

  17. Biosynthesis of riboflavin: an unusual riboflavin synthase of Methanobacterium thermoautotrophicum.

    PubMed Central

    Eberhardt, S; Korn, S; Lottspeich, F; Bacher, A

    1997-01-01

    Riboflavin synthase was purified by a factor of about 1,500 from cell extract of Methanobacterium thermoautotrophicum. The enzyme had a specific activity of about 2,700 nmol mg(-1) h(-1) at 65 degrees C, which is relatively low compared to those of riboflavin synthases of eubacteria and yeast. Amino acid sequences obtained after proteolytic cleavage had no similarity with known riboflavin synthases. The gene coding for riboflavin synthase (designated ribC) was subsequently cloned by marker rescue with a ribC mutant of Escherichia coli. The ribC gene of M. thermoautotrophicum specifies a protein of 153 amino acid residues. The predicted amino acid sequence agrees with the information gleaned from Edman degradation of the isolated protein and shows 67% identity with the sequence predicted for the unannotated reading frame MJ1184 of Methanococcus jannaschii. The ribC gene is adjacent to a cluster of four genes with similarity to the genes cbiMNQO of Salmonella typhimurium, which form part of the cob operon (this operon contains most of the genes involved in the biosynthesis of vitamin B12). The amino acid sequence predicted by the ribC gene of M. thermoautotrophicum shows no similarity whatsoever to the sequences of riboflavin synthases of eubacteria and yeast. Most notably, the M. thermoautotrophicum protein does not show the internal sequence homology characteristic of eubacterial and yeast riboflavin synthases. The protein of M. thermoautotrophicum can be expressed efficiently in a recombinant E. coli strain. The specific activity of the purified, recombinant protein is 1,900 nmol mg(-1) h(-1) at 65 degrees C. In contrast to riboflavin synthases from eubacteria and fungi, the methanobacterial enzyme has an absolute requirement for magnesium ions. The 5' phosphate of 6,7-dimethyl-8-ribityllumazine does not act as a substrate. The findings suggest that riboflavin synthase has evolved independently in eubacteria and methanobacteria. PMID:9139911

  18. Novel fold of VirA, a type III secretion system effector protein from Shigella flexneri

    SciTech Connect

    Davis, Jamaine; Wang, Jiawei; Tropea, Joseph E.; Zhang, Di; Dauter, Zbigniew; Waugh, David S.; Wlodawer, Alexander

    2009-01-28

    VirA, a secreted effector protein from Shigella sp., has been shown to be necessary for its virulence. It was also reported that VirA might be related to papain-like cysteine proteases and cleave {alpha}-tubulin, thus facilitating intracellular spreading. We have now determined the crystal structure of VirA at 3.0 {angstrom} resolution. The shape of the molecule resembles the letter 'V,' with the residues in the N-terminal third of the 45-kDa molecule (some of which are disordered) forming one clearly identifiable domain, and the remainder of the molecule completing the V-like structure. The fold of VirA is unique and does not resemble that of any known protein, including papain, although its N-terminal domain is topologically similar to cysteine protease inhibitors such as stefin B. Analysis of the sequence conservation between VirA and its Escherichia coli homologs EspG and EspG2 did not result in identification of any putative protease-like active site, leaving open a possibility that the biological function of VirA in Shigella virulence may not involve direct proteolytic activity.

  19. Neuronal nitric-oxide synthase localization mediated by a ternary complex with synapsin and CAPON

    PubMed Central

    Jaffrey, Samie R.; Benfenati, Fabio; Snowman, Adele M.; Czernik, Andrew J.; Snyder, Solomon H.

    2002-01-01

    The specificity of the reactions of nitric oxide (NO) with its neuronal targets is determined in part by the precise localizations of neuronal NO synthase (nNOS) within the cell. The targeting of nNOS is mediated by adapter proteins that interact with its PDZ domain. Here, we show that the nNOS adapter protein, CAPON, interacts with synapsins I, II, and III through an N-terminal phosphotyrosine-binding domain interaction, which leads to a ternary complex comprising nNOS, CAPON, and synapsin I. The significance of this ternary complex is demonstrated by changes in subcellular localization of nNOS in mice harboring genomic deletions of both synapsin I and synapsin II. These results suggest a mechanism for specific actions of NO at presynaptic sites. PMID:11867766

  20. Computational design and selections for an engineered, thermostable terpene synthase

    PubMed Central

    Diaz, Juan E; Lin, Chun-Shi; Kunishiro, Kazuyoshi; Feld, Birte K; Avrantinis, Sara K; Bronson, Jonathan; Greaves, John; Saven, Jeffery G; Weiss, Gregory A

    2011-01-01

    Terpenoids include structurally diverse antibiotics, flavorings, and fragrances. Engineering terpene synthases for control over the synthesis of such compounds represents a long sought goal. We report computational design, selections, and assays of a thermostable mutant of tobacco 5-epi-aristolochene synthase (TEAS) for the catalysis of carbocation cyclization reactions at elevated temperatures. Selection for thermostability included proteolytic digestion followed by capture of intact proteins. Unlike the wild-type enzyme, the mutant TEAS retains enzymatic activity at 65°C. The thermostable terpene synthase variant denatures above 80°C, approximately twice the temperature of the wild-type enzyme. PMID:21739507

  1. Endothelial Nitric Oxide Synthase T-786C Mutation, Prothrombin Gene Mutation (G-20210-A) and Protein S Deficiency Could Lead to Myocardial Infarction in a Very Young Male Adult

    PubMed Central

    Klincheva, Milka; Vilarova, Elena Ambarkova; Angjusheva, Tanja; Milev, Ivan; Idoski, Enver; Mitrev, Zan

    2016-01-01

    INTRODUCTION: Myocardial infarction is a rare medical event in young people. The main reasons include congenital coronary abnormalities, coronary artery spasm, and coronary thrombosis due to hypercoagulable states (hereditary and acquired). AIM: We present a case of a young male adult with myocardial infarction caused by a combination of gene mutations and anticoagulation protein deficiency. CASE PRESENTATION: A 19 years old young man was admitted to our hospital complaining of chest pain during the last two weeks. The patient did not have any known cardiovascular risk factors, except a positive family anamnesis. Subacute inferior nonST segment myocardial infarction was diagnosed according to the patient’s history, electrocardiographic and laboratory findings. Coronary angiography revealed suboclusive thrombus in the proximal, medial and distal part of the right coronary artery (TIMI 2). Percutaneous coronary intervention was performed. Anticoagulant and antiagregant therapy (heparin, acetilsalicilic acid and clopidogrel) according to protocol was started. The hospital stay was uneventful. Homozygous endothelial nitric oxid synthase (eNOS) T-786-C mutation, heterozygote prothrombin gene mutation (G-20210-A), and protein S deficiency were verified from the thrombophilia testing. Other trombophilic tests were normal. Three months after discharge from hospital another coronary angiography was performed. It revealed normal coronary arteries. Four years after the attack, the patient is free of symptoms and another cardiovascular event. CONCLUSION: Combination of genetic mutations and anticoagulation protein deficiency could be a reasonable cause for myocardial infarction in a very young male adult without any other cardiovascular risk factors. PMID:27275349

  2. Nitric oxide (NO) pretreatment increases cytokine-induced NO production in cultured rat hepatocytes by suppressing GTP cyclohydrolase I feedback inhibitory protein level and promoting inducible NO synthase dimerization.

    PubMed

    Park, Joon-Hong; Na, Hee-Jun; Kwon, Young-Guen; Ha, Kwon-Soo; Lee, Seon-Jin; Kim, Chun-Ki; Lee, Kwang-Soon; Yoneyama, Toshie; Hatakeyama, Kazuyuki; Kim, Peter K M; Billiar, Timothy R; Kim, Young-Myeong

    2002-12-01

    Nitric oxide (NO) regulates the biological activity of many enzymes and other functional proteins as well as gene expression. In this study, we tested whether pretreatment with NO regulates NO production in response to cytokines in cultured rat hepatocytes. Hepatocytes were recovered in fresh medium for 24 h following pretreatment with the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) and stimulated to express the inducible NO synthase (iNOS) with interleukin-1beta and interferon-gamma or transfected with the human iNOS gene. NO pretreatment resulted in a significant increase in NO production without changing iNOS expression for both conditions. This effect, which did not occur in macrophages and smooth muscle cells, was inhibited when NO was scavenged using red blood cells. Pretreatment with oxidized SNAP, 8-Br-cGMP, NO(2)(-), or NO(3)(-) did not increase the cytokine-induced NO production. SNAP pretreatment increased cytosolic iNOS activity measured only in the absence of exogenous tetrahydrobiopterin (BH(4)). SNAP pretreatment suppressed the level of GTP cyclohydrolase I (GTPCHI) feedback regulatory protein (GFRP) and increased GTPCHI activity without changing GTPCHI protein level. SNAP pretreatment also increased total cellular levels of biopterin and active iNOS dimer. These results suggest that SNAP pretreatment increased NO production from iNOS by elevating cellular BH(4) levels and promoting iNOS subunit dimerization through the suppression of GFRP levels and subsequent activation of GTPCHI. PMID:12359727

  3. Effect of Cheonggukjang supplementation upon hepatic acyl-CoA synthase, carnitine palmitoyltransferase I, acyl-CoA oxidase and uncoupling protein 2 mRNA levels in C57BL/6J mice fed with high fat diet

    PubMed Central

    Soh, Ju-Ryoun; Shin, Dong-Hwa; Kwon, Dae Young

    2007-01-01

    This study investigated the effect of Cheonggukjang on mRNA levels of hepatic acyl-CoA synthase (ACS), carnitine palmitoyltransferase I (CPT-I), acyl-CoA oxidase (ACO) and uncoupling protein 2 (UCP2), and on serum lipid profiles in C57BL/6J mice. Thirty male C57BL/6J mice were divided into three groups; normal diet (ND), high fat diet (HD) and high fat diet with 40% Cheonggukjang (HDC). Energy intake was significantly higher in the HDC group than in the ND and HD groups. The HDC group normalized in weight gain, epididymal and back fat (g/100 g) accumulation which are increased by high fat diet. Serum concentrations of triglyceride and total cholesterol in the HDC were significantly lower than those in the HD group. These results were confirmed by hepatic mRNA expression of enzymes and protein (ACS, CPT-1, ACO, UCP2) which is related with lipid metabolism by RT-PCR. Hepatic CPT-I, ACO and UCP2 mRNA expression was increased by Cheonggukjang supplementation. We demonstrated that Cheonggukjang supplement leads to increased mRNA expressions of enzymes and protein involved in fatty acid oxidation in liver, reduced accumulation of body fat and improvement of serum lipids in high fat diet fed mice. PMID:18850232

  4. Lipopolysaccharide induces inducible nitric oxide synthase-dependent podocyte dysfunction via a hypoxia-inducible factor 1α and cell division control protein 42 and Ras-related C3 botulinum toxin substrate 1 pathway

    PubMed Central

    Mashmoushi, Ahmad K.; Oates, Jim C.

    2015-01-01

    Urine protein loss in immune complex-mediated diseases such as lupus nephritis is associated with podocyte foot process effacement (podocytopathy) but is not always dependent on glomerular immune complex deposition. Several murine and human studies have associated lupus nephritis with inducible nitric oxide synthase (iNOS) expression in what appear to be podocytes. This study was conducted to determine mechanisms of immune-complex-independent and iNOS-dependent podocyte dysfunction. Conditionally immortalized podocytes were cultured with lipopolysaccharide (LPS) and nitric oxide (NO), superoxide (SO), or peroxynitrite donors in the presence or absence of inhibitors of iNOS, reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase or monocyte chemotactic protein 1 (MCP-1), or with sepiapterin to increase coupling of iNOS homodimers. Podocyte NO, SO, and MCP-1 production and nitrotyrosine modifications were determined. The podocytopathy phenotype was determined by measuring cell motility and membrane permeability to albumin. This study determined that NO produced by iNOS is sufficient and necessary to induce podocytopathy. NO probably induces this phenotype via hypoxia-inducible factor 1α and cell division control protein 42 and Ras-related C3 botulinum toxin substrate 1 pathways. With LPS stimulation, neither SO nor peroxynitrite produced by uncoupled iNOS or NADPH oxidase nor MCP-1 was sufficient to induce the full phenotype. This study supports the notion that iNOS may induce autocrine podocyte dysfunction. Thus, targeting iNOS or the pathways of its induction may have therapeutic benefit. PMID:25765888

  5. A role for the TATA-box-binding protein component of the transcription factor IID complex as a general RNA polymerase III transcription factor.

    PubMed Central

    White, R J; Jackson, S P; Rigby, P W

    1992-01-01

    The major class of vertebrate genes transcribed by RNA polymerase (EC 2.7.7.6) III, which includes 5S rRNA genes, tRNA genes, and the adenovirus VA genes, is characterized by split internal promoters and no absolute dependence upon specific upstream sequences. Fractionation experiments have shown that transcription of such genes requires two general RNA polymerase III-specific factors, TFIIIB and TFIIIC. We now demonstrate that a third general factor is also employed by these genes. This is the TATA-box-binding protein originally identified as being a component of the general RNA polymerase II transcription factor TFIID. This protein is involved in the transcription by RNA polymerase III of every template tested, even though the promoters of VA and most vertebrate tRNA and 5S rRNA genes do not contain recognizable TATA elements. Images PMID:1542692

  6. Effect of Protein Incorporation on the Nanostructure of the Bicontinuous Microemulsion Phase of Winsor-III Systems: A Small-Angle Neutron Scattering Study

    SciTech Connect

    Hayes, Douglas G.; Gomez del Rio, Javier A.; Ye, Ran; Urban, Volker S.; Pingali, Sai Venkatesh; O’Neill, Hugh M.

    2015-01-20

    Small-angle neutron scattering (SANS) analysis using the Teubner₋Strey model has been employed to evaluate the effect of protein incorporation into the middle, bicontinuous microemulsion (BμE) phase of Winsor-III (WIII) systems formed by an aerosol-OT (AOT)/alkyl ethoxylate mixed surfactant system to understand better the extraction of proteins into and out of BμEs and to study the effect of proteins on a system that serves as a biomimetic analog of cell membranes. Under conditions of high salinity, the incorporation of positively charged proteins cytochrome c, lysozyme, and α-chymotrypsin, near their solubilization limit in the BμEs promoted the release of water and oil from the BμEs, a decrease in the quasi-periodic repeat distance (d), an increase in ordering (a decrease in the amphiphilicity factor, fa) for the surfactant monolayers, and a decrease in the surface area per surfactant headgroup, suggesting that the proteins affected the self-assembly of components in the BμE phase and produced Debye shielding of AOTs sulfonate headgroup. For WIII systems possessing lower salinity, cytochrome c reduced the efficiency of surfactant in the BμE phase, noted by increases in d and fa, suggesting that the enzyme and AOT underwent ion pairing. We find that the results of this study demonstrate the importance of ionic strength to modulate proteinsurfactant interactions, which in turn will control the release of proteins encapsulated in the BμEs, relevant to WIII-based protein extraction and controlled release from BμE delivery systems, and demonstrate the utility of BμEs as a model system to understand the effect of proteins on biomembranes.

  7. Construction and characterization of isogenic mutants of Streptococcus mutans deficient in major surface protein antigen P1 (I/II).

    PubMed Central

    Lee, S F; Progulske-Fox, A; Erdos, G W; Piacentini, D A; Ayakawa, G Y; Crowley, P J; Bleiweis, A S

    1989-01-01

    The gene (spaP) coding for the Streptococcus mutans major surface protein antigen P1 (or I/II) has been cloned into Escherichia coli (S. F. Lee, A. Progulske-Fox, and A. S. Bleiweis, Infect. Immun. 56:2114-2119, 1988). In the present study, this gene has been disrupted in vitro by insertional inactivation with pVA981, which carries a Tcr marker, and transformed into S. mutans NG8 (serotype c) by electroporation. Upon homologous recombination, the defective spaP was integrated into the genome as demonstrated by Southern hybridization analysis. One Tcr mutant, designated 834, selected by its nonreactivity with anti-P1 monoclonal antibodies, was found to lack the cell surface fuzzy layer which was clearly present on the parent cells. Analysis of extracellular fluids, sodium dodecyl sulfate-solubilized membranes, and cytoplasmic fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that 834 had protein profiles identical to the parent. However, a 185-kilodalton protein which reacts with anti-P1 antibodies was missing from the wall of 834, suggesting that spaP has been specifically inactivated. This mutant displayed levels of glucosyltransferase and fructosyltransferase activities similar to those of the parent. It was much less hydrophobic than the parent. S. mutans NG8 aggregated readily in the presence of clarified whole saliva or a high-molecular-weight salivary agglutinin. This strain also adhered to agglutinin-coated hydroxyapatite. The P1-negative mutants, however, did not display these two properties, suggesting that P1 may play a role in saliva-mediated aggregation and adherence. Images PMID:2807526

  8. Dengue E Protein Domain III-Based DNA Immunisation Induces Strong Antibody Responses to All Four Viral Serotypes

    PubMed Central

    Chan, Kuan Rong; Tan, Hwee Cheng; Bestagno, Marco; Ooi, Eng Eong; Burrone, Oscar R.

    2015-01-01

    Dengue virus (DENV) infection is a major emerging disease widely distributed throughout the tropical and subtropical regions of the world affecting several millions of people. Despite constants efforts, no specific treatment or effective vaccine is yet available. Here we show a novel design of a DNA immunisation strategy that resulted in the induction of strong antibody responses with high neutralisation titres in mice against all four viral serotypes. The immunogenic molecule is an engineered version of the domain III (DIII) of the virus E protein fused to the dimerising CH3 domain of the IgG immunoglobulin H chain. The DIII sequences were also codon-optimised for expression in mammalian cells. While DIII alone is very poorly secreted, the codon-optimised fusion protein is rightly expressed, folded and secreted at high levels, thus inducing strong antibody responses. Mice were immunised using gene-gun technology, an efficient way of intradermal delivery of the plasmid DNA, and the vaccine was able to induce neutralising titres against all serotypes. Additionally, all sera showed reactivity to a recombinant DIII version and the recombinant E protein produced and secreted from mammalian cells in a mono-biotinylated form when tested in a conformational ELISA. Sera were also highly reactive to infective viral particles in a virus-capture ELISA and specific for each serotype as revealed by the low cross-reactive and cross-neutralising activities. The serotype specific sera did not induce antibody dependent enhancement of infection (ADE) in non-homologous virus serotypes. A tetravalent immunisation protocol in mice showed induction of neutralising antibodies against all four dengue serotypes as well. PMID:26218926

  9. Crystal structure of riboflavin synthase

    SciTech Connect

    Liao, D.-I.; Wawrzak, Z.; Calabrese, J.C.; Viitanen, P.V.; Jordan, D.B.

    2010-03-05

    Riboflavin synthase catalyzes the dismutation of two molecules of 6,7-dimethyl-8-(1'-D-ribityl)-lumazine to yield riboflavin and 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine. The homotrimer of 23 kDa subunits has no cofactor requirements for catalysis. The enzyme is nonexistent in humans and is an attractive target for antimicrobial agents of organisms whose pathogenicity depends on their ability to biosynthesize riboflavin. The first three-dimensional structure of the enzyme was determined at 2.0 {angstrom} resolution using the multiwavelength anomalous diffraction (MAD) method on the Escherichia coli protein containing selenomethionine residues. The homotrimer consists of an asymmetric assembly of monomers, each of which comprises two similar {beta} barrels and a C-terminal {alpha} helix. The similar {beta} barrels within the monomer confirm a prediction of pseudo two-fold symmetry that is inferred from the sequence similarity between the two halves of the protein. The {beta} barrels closely resemble folds found in phthalate dioxygenase reductase and other flavoproteins. The three active sites of the trimer are proposed to lie between pairs of monomers in which residues conserved among species reside, including two Asp-His-Ser triads and dyads of Cys-Ser and His-Thr. The proposed active sites are located where FMN (an analog of riboflavin) is modeled from an overlay of the {beta} barrels of phthalate dioxygenase reductase and riboflavin synthase. In the trimer, one active site is formed, and the other two active sites are wide open and exposed to solvent. The nature of the trimer configuration suggests that only one active site can be formed and be catalytically competent at a time.

  10. Inhibition of a type III secretion system by the deletion of a short loop in one of its membrane proteins

    SciTech Connect

    Meshcheryakov, Vladimir A.; Kitao, Akio; Matsunami, Hideyuki; Samatey, Fadel A.

    2013-05-01

    Crystal structures of the cytoplasmic domain of FlhB from S. typhimurium and A. aeolicus were solved at 2.45 and 2.55 Å resolution, respectively. The deletion of a short loop in the cytoplasmic domain of Salmonella FlhB completely abolishes secretion by the type III secretion system. A molecular-dynamics simulation shows that the deletion of the loop affects the flexibility of a linker between the transmembrane and cytoplasmic domains of FlhB. The membrane protein FlhB is a highly conserved component of the flagellar secretion system. It is composed of an N-terminal transmembrane domain and a C-terminal cytoplasmic domain (FlhB{sub C}). Here, the crystal structures of FlhB{sub C} from Salmonella typhimurium and Aquifex aeolicus are described at 2.45 and 2.55 Å resolution, respectively. These flagellar FlhB{sub C} structures are similar to those of paralogues from the needle type III secretion system, with the major difference being in a linker that connects the transmembrane and cytoplasmic domains of FlhB. It was found that deletion of a short flexible loop in a globular part of Salmonella FlhB{sub C} leads to complete inhibition of secretion by the flagellar secretion system. Molecular-dynamics calculations demonstrate that the linker region is the most flexible part of FlhB{sub C} and that the deletion of the loop reduces this flexibility. These results are in good agreement with previous studies showing the importance of the linker in the function of FlhB and provide new insight into the relationship between the different parts of the FlhB{sub C} molecule.

  11. Distribution of chromium species in a Cr-polluted soil: presence of Cr(III) in glomalin related protein fraction.

    PubMed

    Gil-Cardeza, María L; Ferri, Alejandro; Cornejo, Pablo; Gomez, Elena

    2014-09-15

    The accumulation of Cr in soil could be highly toxic to human health; therefore Cr soil distribution was studied in rhizosphere soils from Ricinus communis and Conium maculatum and bare soil (BS) from an industrial and urban area in Argentina. Total Cr, Cr(VI) and Cr(III) concentrations were determined in 3 soil fractions: total, extractable and associated to total-glomalin-related protein (T-GRSP). BS had the highest total Cr and total Cr(VI) concentrations. Total Cr(VI) concentration from both rhizosphere soils did not differ from the allowed value for residential area in Argentina (8 μg Cr(VI) g(-1) soil), while total Cr(VI) in BS was 1.8 times higher. Total Cr concentration in all the soils was higher than the allowed value (250 μg Cr g(-1) soil). Extractable and associated to T-GRSP Cr(VI) concentrations were below the detection limit. Cr(III) bound to T-GRSP was the highest in the BS. These findings are in agreement with a long term effect of glomalin in sequestrating Cr. In both plant species, total Cr was higher in root than in shoot and both species presented arbuscular mycorrhizal fungi (AMF). As far as we know, this is the first study that reports the presence of Cr in T-GRSP fraction of soil organic matter. These findings suggest that Cr mycorrhizostabilization could be a predominant mechanism used by R. communis and C. maculatum to diminish Cr soil concentration. Nevertheless, further research is needed to clarify the contribution of native AMF isolated from R. communis and C. maculatum rhizosphere to the Cr phytoremediation process. PMID:25000578

  12. Correlating levels of type III secretion and secreted proteins with fecal shedding of Escherichia coli O157:H7 in cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The locus of enterocyte effacement (LEE) encodes a type III secretion system (T3SS) for secreting factors that enable Escherichia coli O157:H7 to produce attaching and effacing lesions (A/E) on epithelial cells. The importance of LEE-encoded proteins in intestinal colonization of cattle is well-stud...

  13. A luminescent cyclometalated gold(iii)-avidin conjugate with a long-lived emissive excited state that binds to proteins and DNA and possesses anti-proliferation capacity.

    PubMed

    Tsai, Johnson Lui-Lui; Chan, Anna On-Yee; Che, Chi-Ming

    2015-05-18

    Here we describe a luminescent cyclometalated Au(iii)-avidin conjugate that exhibits a 520 nm emission with a lifetime of 1.8 μs in PBS solution in open air. The conjugate stains proteins and DNA and can inhibit cancer cell proliferation. PMID:25896112

  14. Subcutaneous and intranasal immunization with type III secreted proteins can prevent colonization and shedding of Escherichia coli O157:H7 in mice.

    PubMed

    Babiuk, Shawn; Asper, David J; Rogan, Dragan; Mutwiri, George K; Potter, Andrew A

    2008-07-01

    Type III secreted proteins from Escherichia coli O157:H7 are involved in the attachment of the organism to mammalian cells and have been shown to be effective vaccine components capable of reducing colonization of cattle by the organism. In the current study, we used a streptomycin-treated mouse model to evaluate the efficacy of subcutaneous vs intranasal administration of the vaccine. Following immunization, mice were infected with E. coli O157:H7 and feces were monitored for shedding. Immune responses against EspA and Tir were also monitored. Subcutaneous immunization of mice with type III secreted proteins induced significant EspA- and Tir-specific serum IgG antibodies but did not significantly induce any antigen-specific IgA in feces, whereas intranasal immunization elicited significant EspA- and Tir-specific serum IgG antibodies with some animals developing antigen-specific IgA in feces. Only mice that were immunized intranasally with formulations containing mucosal adjuvants, either cholera toxin or CpG-containing oligonucleotides, showed decreased E. coli O157:H7 shedding following experimental infection. Mice immunized subcutaneously with type III secreted proteins did not shed E. coli in feces. These results demonstrate the potential for the use of type III secreted proteins in mucosal vaccine formulations to prevent colonization and shedding of E. coli O157:H7. PMID:18487034

  15. Anti-dengue virus nonstructural protein 1 antibodies cause NO-mediated endothelial cell apoptosis via ceramide-regulated glycogen synthase kinase-3β and NF-κB activation.

    PubMed

    Chen, Chia-Ling; Lin, Chiou-Feng; Wan, Shu-Wen; Wei, Li-Shiung; Chen, Mei-Chun; Yeh, Trai-Ming; Liu, Hsiao-Sheng; Anderson, Robert; Lin, Yee-Shin

    2013-08-15

    Immunopathogenetic mechanisms of dengue virus (DENV) infection are involved in hemorrhagic syndrome resulting from thrombocytopenia, coagulopathy, and vasculopathy. We have proposed a mechanism of molecular mimicry in which Abs against DENV nonstructural protein 1 (NS1) cross-react with human endothelial cells and cause NF-κB-regulated immune activation and NO-mediated apoptosis. However, the signaling pathway leading to NF-κB activation after the binding of anti-DENV NS1 Abs to endothelial cells is unresolved. In this study, we found that anti-DENV NS1 Abs caused the formation of lipid raftlike structures, and that disrupting lipid raft formation by methyl-β-cyclodextrin decreased NO production and apoptosis. Treatment with anti-DENV NS1 Abs elevated ceramide generation in lipid rafts. Pharmacological inhibition of acid sphingomyelinase (aSMase) decreased anti-DENV NS1 Ab-mediated ceramide and NO production, as well as apoptosis. Exogenous ceramide treatment induced biogenesis of inducible NO synthase (iNOS)/NO and apoptosis through an NF-κB-regulated manner. Furthermore, activation of glycogen synthase kinase-3β (GSK-3β) was required for ceramide-induced NF-κB activation and iNOS expression. Notably, anti-DENV NS1 Abs caused GSK-3β-mediated NF-κB activation and iNOS expression, which were regulated by aSMase. Moreover, pharmacological inhibition of GSK-3β reduced hepatic endothelial cell apoptosis in mice passively administered anti-DENV NS1 Abs. These results suggest that anti-DENV NS1 Abs bind to the endothelial cell membrane and cause NO production and apoptosis via a mechanism involving the aSMase/ceramide/GSK-3β/NF-κB/iNOS/NO signaling pathway. PMID:23851680

  16. Chrysanthemyl Diphosphate Synthase Operates in Planta as a Bifunctional Enzyme with Chrysanthemol Synthase Activity*

    PubMed Central

    Yang, Ting; Gao, Liping; Hu, Hao; Stoopen, Geert; Wang, Caiyun; Jongsma, Maarten A.

    2014-01-01

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1′-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 μm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ∼100 μm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12–0.16 μg h−1 g−1 fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate. PMID:25378387

  17. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity.

    PubMed

    Yang, Ting; Gao, Liping; Hu, Hao; Stoopen, Geert; Wang, Caiyun; Jongsma, Maarten A

    2014-12-26

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1'-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 μm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ∼100 μm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12-0.16 μg h(-1) g(-1) fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate. PMID:25378387

  18. UncI protein can mediate ring-assembly of c-subunits of F{sub o}F{sub 1}-ATP synthase in vitro

    SciTech Connect

    Ozaki, Yoko; Suzuki, Toshiharu; Kuruma, Yutetsu; Ueda, Takuya; Yoshida, Masasuke

    2008-03-14

    In F{sub o}F{sub 1}-ATP synthase, multimeric c-subunits are assembled to a ring (c-ring) in the membranes that rotates as protons flow across F{sub o}. We recently reported that assembly of c-ring of Propionigenium modestum in the membranes of Escherichia coli cells required P. modestum UncI, a product of the conserved uncI gene in the F{sub o}F{sub 1} operon. However, cooperation with endogenous factors in E. coli remained unclear. Here, P. modestum c-subunit was synthesized in vitro in the presence of liposomes. When c-subunit alone was synthesized, it did not form c-ring. However, when c-subunit and P. modestum UncI were synthesized together, c-ring was formed. Fusion of the two kinds of liposomes, one containing only unassembled c-subunit and the other only UncI, resulted in gradual formation of c-ring. Thus, UncI alone can mediate in vitro post-translational c-ring assembly.

  19. Bacillus anthracis endospores regulate ornithine decarboxylase and inducible nitric oxide synthase through ERK1/2 and p38 mitogen-activated protein kinases.

    PubMed

    Porasuphatana, Supatra; Cao, Guan-Liang; Tsai, Pei; Tavakkoli, Fatemeh; Huwar, Theresa; Baillie, Les; Cross, Alan S; Shapiro, Paul; Rosen, Gerald M

    2010-12-01

    Interactions between Bacillus anthracis (B. anthracis) and host cells are of particular interest given the implications of anthrax as a biological weapon. Inhaled B. anthracis endospores encounter alveolar macrophages as the first line of defense in the innate immune response. Yet, the consequences of this interaction remain unclear. We have demonstrated that B. anthracis uses arginase, inherent in the endospores, to reduce the ability of macrophages to produce nitric oxide ((•)NO) from inducible nitric oxide synthase (NOS2) by competing for L-arginine, producing L-ornithine at the expense of (•)NO. In the current study, we used genetically engineered B. anthracis endospores to evaluate the contribution of germination and the lethal toxin (LT) in mediating signaling pathways responsible for the induction of NOS2 and ornithine decarboxylase (ODC), which is the rate-limiting enzyme in the conversion of L-ornithine into polyamines. We found that induction of NOS2 and ODC expression in macrophages exposed to B. anthracis occurs through the activation of p38 and ERK1/2 MAP kinases, respectively. Optimal induction of NOS2 was observed following exposure to germination-competent endospores, whereas ODC induction occurred irrespective of the endospores' germination capabilities and was more prominent in macrophages exposed to endospores lacking LT. Our findings suggest that activation of kinase signaling cascades that determine macrophage defense responses against B. anthracis infection occurs through distinct mechanisms. PMID:20440620

  20. Localization of Nitric Oxide Synthase-containing Neurons in the Bat Visual Cortex and Co-localization with Calcium-binding Proteins

    PubMed Central

    Gu, Ya-Nan; Kim, Hang-Gu; Jeon, Chang-Jin

    2015-01-01

    Microchiroptera (microbats) is a suborder of bats thought to have degenerated vision. However, many recent studies have shown that they have visual ability. In this study, we labeled neuronal nitric oxide synthase (nNOS)—the synthesizing enzyme of the gaseous non-synaptic neurotransmitter nitric oxide—and co-localized it with calbindin D28K (CB), calretinin (CR), and parvalbumin (PV) in the visual cortex of the greater horseshoe bat (Rhinolophus ferrumequinum, a species of microbats). nNOS-immunoreactive (IR) neurons were found in all layers of the visual cortex. Intensely labeled neurons were most common in layer IV, and weakly labeled neurons were most common in layer VI. Majority of the nNOS-IR neurons were round- or oval-type neurons; no pyramidal-type neurons were found. None of these neurons co-localized with CB, CR, or PV. However, the synthesis of nitric oxide in the bat visual cortex by nNOS does not depend on CB, CR, or PV. PMID:26379314

  1. Transcriptional regulation of fksA, a β-1,3-glucan synthase gene, by the APSES protein StuA during Aspergillus nidulans development.

    PubMed

    Park, Bum-Chan; Park, Yun-Hee; Yi, Soohyun; Choi, Yu Kyung; Kang, Eun-Hye; Park, Hee-Moon