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Sample records for identifies cellular cofactors

  1. Long-term inhibition of HIV-1 replication with RNA interference against cellular co-factors.

    PubMed

    Eekels, Julia J M; Geerts, Dirk; Jeeninga, Rienk E; Berkhout, Ben

    2011-01-01

    In this study we tested whether HIV-1 replication could be inhibited by stable RNAi-mediated knockdown of cellular co-factors. Cell lines capable of expressing shRNAs against 30 candidate co-factors implicated at different steps of the viral replication cycle were generated and analyzed for effects on cell viability and inhibition of HIV-1 replication. For half of these candidate co-factors we obtained knockdown cell lines that are less susceptible to virus replication. For three co-factors (ALIX, ATG16 and TRBP) the cell lines were resistant to HIV-1 replication for up to 2 months. With these cells we could test the hypothesis that HIV-1 is not able to escape from RNAi-mediated suppression of cellular co-factors, which was indeed not detected.

  2. Remaining challenges in cellular flavin cofactor homeostasis and flavoprotein biogenesis

    NASA Astrophysics Data System (ADS)

    Giancaspero, Teresa Anna; Colella, Matilde; Brizio, Carmen; Difonzo, Graziana; Fiorino, Giuseppina Maria; Leone, Piero; Brandsch, Roderich; Bonomi, Francesco; Iametti, Stefania; Barile, Maria

    2015-04-01

    The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in energetic metabolism, epigenetics, protein folding, as well as in a number of diverse regulatory processes. The problem of localisation of flavin cofactor synthesis events and in particular of the FAD synthase (EC 2.7.7.2) in HepG2 cells is addressed here by confocal microscopy in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalysed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesising activity, hFADS is able to operate as a FAD "chaperone". The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear or a mitochondrial enzyme that is lysine specific demethylase 1 (LSD1, EC 1.-.-.-) and dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4), respectively which carry out similar reactions of oxidative demethylation, assisted by tetrahydrofolate used to form 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells.

  3. Remaining challenges in cellular flavin cofactor homeostasis and flavoprotein biogenesis

    PubMed Central

    Giancaspero, Teresa A.; Colella, Matilde; Brizio, Carmen; Difonzo, Graziana; Fiorino, Giuseppina M.; Leone, Piero; Brandsch, Roderich; Bonomi, Francesco; Iametti, Stefania; Barile, Maria

    2015-01-01

    The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in a broad spectrum of biological activities, among which energetic metabolism and chromatin remodeling. Subcellular localisation of FAD synthase (EC 2.7.7.2, FADS), the second enzyme in the FAD forming pathway, is addressed here in HepG2 cells by confocal microscopy, in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalyzed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesizing activity, hFADS is able to operate as a FAD “chaperone.” The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear lysine-specific demethylase 1 (LSD1) or a mitochondrial dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4). Both enzymes carry out similar reactions of oxidative demethylation, in which tetrahydrofolate is converted into 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells. PMID:25954742

  4. Remaining challenges in cellular flavin cofactor homeostasis and flavoprotein biogenesis.

    PubMed

    Giancaspero, Teresa A; Colella, Matilde; Brizio, Carmen; Difonzo, Graziana; Fiorino, Giuseppina M; Leone, Piero; Brandsch, Roderich; Bonomi, Francesco; Iametti, Stefania; Barile, Maria

    2015-01-01

    The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in a broad spectrum of biological activities, among which energetic metabolism and chromatin remodeling. Subcellular localisation of FAD synthase (EC 2.7.7.2, FADS), the second enzyme in the FAD forming pathway, is addressed here in HepG2 cells by confocal microscopy, in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalyzed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesizing activity, hFADS is able to operate as a FAD "chaperone." The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear lysine-specific demethylase 1 (LSD1) or a mitochondrial dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4). Both enzymes carry out similar reactions of oxidative demethylation, in which tetrahydrofolate is converted into 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells.

  5. Protein-protein interactions and human cellular cofactors as new targets for HIV therapy.

    PubMed

    Tintori, Cristina; Brai, Annalaura; Fallacara, Anna Lucia; Fazi, Roberta; Schenone, Silvia; Botta, Maurizio

    2014-10-01

    Two novel approaches for the development of new drugs against AIDS are summarized each leading to the achievement of important discoveries in anti-HIV therapy. Despite the success of HAART in reducing mortality, resistant strains continue to emerge in the clinic, underscoring the importance of developing next-generation drugs. Protein-protein interactions and human cellular cofactors represent the new targets of tomorrow in HIV research. The most relevant results obtained in the last few years by the two new strategies are described herein. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. The DNA-bending protein HMGB1 is a cellular cofactor of Sleeping Beauty transposition.

    PubMed

    Zayed, Hatem; Izsvák, Zsuzsanna; Khare, Dheeraj; Heinemann, Udo; Ivics, Zoltán

    2003-05-01

    Sleeping Beauty (SB) is the most active Tc1/ mariner-type transposon in vertebrates. SB contains two transposase-binding sites (DRs) at the end of each terminal inverted repeat (IR), a feature termed the IR/DR structure. We investigated the involvement of cellular proteins in the regulation of SB transposition. Here, we establish that the DNA-bending, high-mobility group protein, HMGB1 is a host-encoded cofactor of SB transposition. Transposition was severely reduced in mouse cells deficient in HMGB1. This effect was rescued by transient over-expression of HMGB1, and was partially complemented by HMGB2, but not with the HMGA1 protein. Over-expression of HMGB1 in wild-type mouse cells enhanced transposition, indicating that HMGB1 can be a limiting factor of transposition. SB transposase was found to interact with HMGB1 in vivo, suggesting that the transposase may recruit HMGB1 to transposon DNA. HMGB1 stimulated preferential binding of the transposase to the DR further from the cleavage site, and promoted bending of DNA fragments containing the transposon IR. We propose that the role of HMGB1 is to ensure that transposase-transposon complexes are first formed at the internal DRs, and subsequently to promote juxtaposition of functional sites in transposon DNA, thereby assisting the formation of synaptic complexes.

  7. Molybdate uptake by Agrobacterium tumefaciens correlates with the cellular molybdenum cofactor status.

    PubMed

    Hoffmann, Marie-Christine; Ali, Koral; Sonnenschein, Marleen; Robrahn, Laura; Strauss, Daria; Narberhaus, Franz; Masepohl, Bernd

    2016-09-01

    Many enzymes require the molybdenum cofactor, Moco. Under Mo-limiting conditions, the high-affinity ABC transporter ModABC permits molybdate uptake and Moco biosynthesis in bacteria. Under Mo-replete conditions, Escherichia coli represses modABC transcription by the one-component regulator, ModE, consisting of a DNA-binding and a molybdate-sensing domain. Instead of a full-length ModE protein, many bacteria have a shorter ModE protein, ModE(S) , consisting of a DNA-binding domain only. Here, we asked how such proteins sense the intracellular molybdenum status. We show that the Agrobacterium tumefaciens ModE(S) protein Atu2564 is essential for modABC repression. ModE(S) binds two Mo-boxes in the modA promoter as shown by electrophoretic mobility shift assays. Northern analysis revealed cotranscription of modE(S) with the upstream gene, atu2565, which was dispensable for ModE(S) activity. To identify genes controlling ModE(S) function, we performed transposon mutagenesis. Tn5 insertions resulting in derepressed modA transcription mapped to the atu2565-modE(S) operon and several Moco biosynthesis genes. We conclude that A. tumefaciens ModE(S) activity responds to Moco availability rather than to molybdate concentration directly, as is the case for E. coli ModE. Similar results in Sinorhizobium meliloti suggest that Moco dependence is a common feature of ModE(S) regulators.

  8. Global analysis of induced transcription factors and cofactors identifies Tfdp2 as an essential coregulator during terminal erythropoiesis

    PubMed Central

    Chen, Cynthia; Lodish, Harvey F.

    2014-01-01

    Key transcriptional regulators of terminal erythropoiesis, such as GATA1 and TAL1, have been well characterized, but transcription factors and cofactors and their expression modulations have not yet been explored on a global scale. Here we use global gene expression analysis to identify 28 transcription factors and 19 transcriptional cofactors induced during terminal erythroid differentiation and whose promoters are enriched for binding by GATA1 and TAL1. Utilizing protein-protein interaction databases to identify cofactors for each transcription factor, we pinpoint several co-induced pairs, of which E2f2 and its cofactor Tfdp2 were the most highly induced. TFDP2 is a critical cofactor required for proper cell cycle control and gene expression. GATA1 and TAL1 are bound to the regulatory regions of Tfdp2 and upregulate its expression, and knockdown of Tfdp2 results in significantly reduced rates of proliferation, as well as reduced upregulation of many erythroid-important genes. Loss of Tfdp2 also globally inhibits the normal downregulation of many E2F2 target genes, including those that regulate the cell cycle, causing cells to accumulate in S phase and resulting in increased erythrocyte size. Our findings highlight the importance of TFDP2 in coupling the erythroid cell cycle with terminal differentiation and validate this study as a resource for future work on elucidating the role of diverse transcription factors and coregulators in erythropoiesis. PMID:24607859

  9. Global analysis of induced transcription factors and cofactors identifies Tfdp2 as an essential coregulator during terminal erythropoiesis.

    PubMed

    Chen, Cynthia; Lodish, Harvey F

    2014-06-01

    Key transcriptional regulators of terminal erythropoiesis, such as GATA-binding factor 1 (GATA1) and T-cell acute lymphocytic leukemia protein 1 (TAL1), have been well characterized, but transcription factors and cofactors and their expression modulations have not yet been explored on a global scale. Here, we use global gene expression analysis to identify 28 transcription factors and 19 transcriptional cofactors induced during terminal erythroid differentiation whose promoters are enriched for binding by GATA1 and TAL1. Utilizing protein-protein interaction databases to identify cofactors for each transcription factor, we pinpoint several co-induced pairs, of which E2f2 and its cofactor transcription factor Dp-2 (Tfdp2) were the most highly induced. TFDP2 is a critical cofactor required for proper cell cycle control and gene expression. GATA1 and TAL1 are bound to the regulatory regions of Tfdp2 and upregulate its expression and knockdown of Tfdp2 results in significantly reduced rates of proliferation as well as reduced upregulation of many erythroid-important genes. Loss of Tfdp2 also globally inhibits the normal downregulation of many E2F2 target genes, including those that regulate the cell cycle, causing cells to accumulate in S phase and resulting in increased erythrocyte size. Our findings highlight the importance of TFDP2 in coupling the erythroid cell cycle with terminal differentiation and validate this study as a resource for future work on elucidating the role of diverse transcription factors and coregulators in erythropoiesis.

  10. Characterization of the HIV-1 RNA associated proteome identifies Matrin 3 as a nuclear cofactor of Rev function

    PubMed Central

    2011-01-01

    Background Central to the fully competent replication cycle of the human immunodeficiency virus type 1 (HIV-1) is the nuclear export of unspliced and partially spliced RNAs mediated by the Rev posttranscriptional activator and the Rev response element (RRE). Results Here, we introduce a novel method to explore the proteome associated with the nuclear HIV-1 RNAs. At the core of the method is the generation of cell lines harboring an integrated provirus carrying RNA binding sites for the MS2 bacteriophage protein. Flag-tagged MS2 is then used for affinity purification of the viral RNA. By this approach we found that the viral RNA is associated with the host nuclear matrix component MATR3 (Matrin 3) and that its modulation affected Rev activity. Knockdown of MATR3 suppressed Rev/RRE function in the export of unspliced HIV-1 RNAs. However, MATR3 was able to associate with Rev only through the presence of RRE-containing viral RNA. Conclusions In this work, we exploited a novel proteomic method to identify MATR3 as a cellular cofactor of Rev activity. MATR3 binds viral RNA and is required for the Rev/RRE mediated nuclear export of unspliced HIV-1 RNAs. PMID:21771346

  11. Identifying Cellular and Molecular Mechanisms for Magnetosensation

    PubMed Central

    Clites, Benjamin L.; Pierce, Jonathan T.

    2017-01-01

    Diverse animals ranging from worms and insects to birds and turtles perf orm impressive journeys using the magnetic field of the earth as a cue. Although major cellular and molecular mechanisms for sensing mechanical and chemical cues have been elucidated over the past three decades, the mechanisms that animals use to sense magnetic fields remain largely mysterious. Here we survey progress on the search for magnetosensory neurons and magnetosensitive molecules important for animal behaviors. Emphasis is placed on magnetosensation in insects and birds, as well as on the magnetosensitive neuron pair AFD in the nematode Caenorhabditis elegans. We also review conventional criteria used to define animal magnetoreceptors and suggest how approaches used to identify receptors for other sensory modalities may be adapted for magnetoreceptors. Finally, we discuss prospects for under-utilized and novel approaches to identify the elusive magnetoreceptors in animals. PMID:28772099

  12. PRIC295, a Nuclear Receptor Coactivator, Identified from PPARα-Interacting Cofactor Complex

    PubMed Central

    Pyper, Sean R.; Viswakarma, Navin; Jia, Yuzhi; Zhu, Yi-Jun; Fondell, Joseph D.; Reddy, Janardan K.

    2010-01-01

    The peroxisome proliferator-activated receptor-α (PPARα) plays a key role in lipid metabolism and energy combustion. Chronic activation of PPARα in rodents leads to the development of hepatocellular carcinomas. The ability of PPARα to induce expression of its target genes depends on Mediator, an evolutionarily conserved complex of cofactors and, in particular, the subunit 1 (Med1) of this complex. Here, we report the identification and characterization of PPARα-interacting cofactor (PRIC)-295 (PRIC295), a novel coactivator protein, and show that it interacts with the Med1 and Med24 subunits of the Mediator complex. PRIC295 contains 10 LXXLL signature motifs that facilitate nuclear receptor binding and interacts with PPARα and five other members of the nuclear receptor superfamily in a ligand-dependent manner. PRIC295 enhances the transactivation function of PPARα, PPARγ, and ERα. These data demonstrate that PRIC295 interacts with nuclear receptors such as PPARα and functions as a transcription coactivator under in vitro conditions and may play an important role in mediating the effects in vivo as a member of the PRIC complex with Med1 and Med24. PMID:20885938

  13. Impact of sustained RNAi-mediated suppression of cellular cofactor Tat-SF1 on HIV-1 replication in CD4+ T cells.

    PubMed

    Green, Victoria A; Arbuthnot, Patrick; Weinberg, Marc S

    2012-11-15

    Conventional anti-HIV drug regimens targeting viral enzymes are plagued by the emergence of drug resistance. There is interest in targeting HIV-dependency factors (HDFs), host proteins that the virus requires for replication, as drugs targeting their function may prove protective. Reporter cell lines provide a rapid and convenient method of identifying putative HDFs, but this approach may lead to misleading results and a failure to detect subtle detrimental effects on cells that result from HDF suppression. Thus, alternative methods for HDF validation are required. Cellular Tat-SF1 has long been ascribed a cofactor role in Tat-dependent transactivation of viral transcription elongation. Here we employ sustained RNAi-mediated suppression of Tat-SF1 to validate its requirement for HIV-1 replication in a CD4+ T cell-derived line and its potential as a therapeutic target. shRNA-mediated suppression of Tat-SF1 reduced HIV-1 replication and infectious particle production from TZM-bl reporter cells. This effect was not a result of increased apoptosis, loss of cell viability or an immune response. To validate its requirement for HIV-1 replication in a more relevant cell line, CD4+ SupT1 cell populations were generated that stably expressed shRNAs. HIV-1 replication was significantly reduced for two weeks (~65%) in cells with depleted Tat-SF1, although the inhibition of viral replication was moderate when compared to SupT1 cells expressing a shRNA targeting the integration cofactor LEDGF/p75. Tat-SF1 suppression was attenuated over time, resulting from decreased shRNA guide strand expression, suggesting that there is a selective pressure to restore Tat-SF1 levels. This study validates Tat-SF1 as an HDF in CD4+ T cell-derived SupT1 cells. However, our findings also suggest that Tat-SF1 is not a critical cofactor required for virus replication and its suppression may affect cell growth. Therefore, this study demonstrates the importance of examining HIV-1 replication kinetics

  14. Acidic residues important for substrate binding and cofactor reactivity in eukaryotic ornithine decarboxylase identified by alanine scanning mutagenesis.

    PubMed

    Osterman, A L; Kinch, L N; Grishin, N V; Phillips, M A

    1995-05-19

    Ornithine decarboxylases from Trypanosoma brucei, mouse, and Leishmania donovani share strict specificity for three basic amino acids, ornithine, lysine, and arginine. To identify residues involved in this substrate specificity and/or in the reaction chemistry, six conserved acidic resides (Asp-88, Glu-94, Asp-233, Glu-274, Asp-361, and Asp-364) were mutated to alanine in the T. brucei enzyme. Each mutation causes a substantial loss in enzyme efficiency. Most notably, mutation of Asp-361 increases the Km for ornithine by 2000-fold, with little effect on kcat, suggesting that this residue is an important substrate binding determinant. Mutation of the only strictly conserved acidic residue, Glu-274, decreases kcat 50-fold; however, substitution of N-methylpyridoxal-5'-phosphate for pyridoxal-5'-phosphate as the cofactor in the reaction restores the kcat of E274A to wild-type levels. These data demonstrate that Glu-274 interacts with the protonated pyridine nitrogen of the cofactor to enhance the electron withdrawing capability of the ring, analogous to Asp-222 in aspartate aminotransferase (Onuffer, J. J., and Kirsch, J. F. (1994) Protein Eng. 7, 413-424). Eukaryotic ornithine decarboxylase is a homodimer with two shared active sites. Residues 88, 94, 233, and 274 are contributed to each active site from the same subunit as Lys-69, while residues 361 and 364 are part of the Cys-360 subunit.

  15. Interactome Analysis of the Human Respiratory Syncytial Virus RNA Polymerase Complex Identifies Protein Chaperones as Important Cofactors That Promote L-Protein Stability and RNA Synthesis

    PubMed Central

    Munday, Diane C.; Wu, Weining; Smith, Nikki; Fix, Jenna; Noton, Sarah Louise; Galloux, Marie; Touzelet, Olivier; Armstrong, Stuart D.; Dawson, Jenna M.; Aljabr, Waleed; Easton, Andrew J.; Rameix-Welti, Marie-Anne; de Oliveira, Andressa Peres; Simabuco, Fernando M.; Ventura, Armando M.; Hughes, David J.; Barr, John N.; Fearns, Rachel; Digard, Paul

    2014-01-01

    ABSTRACT The human respiratory syncytial virus (HRSV) core viral RNA polymerase comprises the large polymerase protein (L) and its cofactor, the phosphoprotein (P), which associate with the viral ribonucleoprotein complex to replicate the genome and, together with the M2-1 protein, transcribe viral mRNAs. While cellular proteins have long been proposed to be involved in the synthesis of HRSV RNA by associating with the polymerase complex, their characterization has been hindered by the difficulty of purifying the viral polymerase from mammalian cell culture. In this study, enhanced green fluorescent protein (EGFP)-tagged L- and P-protein expression was coupled with high-affinity anti-GFP antibody-based immunoprecipitation and quantitative proteomics to identify cellular proteins that interacted with either the L- or the P-proteins when expressed as part of a biologically active viral RNP. Several core groups of cellular proteins were identified that interacted with each viral protein including, in both cases, protein chaperones. Ablation of chaperone activity by using small-molecule inhibitors confirmed previously reported studies which suggested that this class of proteins acted as positive viral factors. Inhibition of HSP90 chaperone function in the current study showed that HSP90 is critical for L-protein function and stability, whether in the presence or absence of the P-protein. Inhibition studies suggested that HSP70 also disrupts virus biology and might help the polymerase remodel the nucleocapsid to allow RNA synthesis to occur efficiently. This indicated a proviral role for protein chaperones in HRSV replication and demonstrates that the function of cellular proteins can be targeted as potential therapeutics to disrupt virus replication. IMPORTANCE Human respiratory syncytial virus (HRSV) represents a major health care and economic burden, being the main cause of severe respiratory infections in infants worldwide. No vaccine or effective therapy is

  16. Interactome analysis of the human respiratory syncytial virus RNA polymerase complex identifies protein chaperones as important cofactors that promote L-protein stability and RNA synthesis.

    PubMed

    Munday, Diane C; Wu, Weining; Smith, Nikki; Fix, Jenna; Noton, Sarah Louise; Galloux, Marie; Touzelet, Olivier; Armstrong, Stuart D; Dawson, Jenna M; Aljabr, Waleed; Easton, Andrew J; Rameix-Welti, Marie-Anne; de Oliveira, Andressa Peres; Simabuco, Fernando M; Ventura, Armando M; Hughes, David J; Barr, John N; Fearns, Rachel; Digard, Paul; Eléouët, Jean-François; Hiscox, Julian A

    2015-01-15

    The human respiratory syncytial virus (HRSV) core viral RNA polymerase comprises the large polymerase protein (L) and its cofactor, the phosphoprotein (P), which associate with the viral ribonucleoprotein complex to replicate the genome and, together with the M2-1 protein, transcribe viral mRNAs. While cellular proteins have long been proposed to be involved in the synthesis of HRSV RNA by associating with the polymerase complex, their characterization has been hindered by the difficulty of purifying the viral polymerase from mammalian cell culture. In this study, enhanced green fluorescent protein (EGFP)-tagged L- and P-protein expression was coupled with high-affinity anti-GFP antibody-based immunoprecipitation and quantitative proteomics to identify cellular proteins that interacted with either the L- or the P-proteins when expressed as part of a biologically active viral RNP. Several core groups of cellular proteins were identified that interacted with each viral protein including, in both cases, protein chaperones. Ablation of chaperone activity by using small-molecule inhibitors confirmed previously reported studies which suggested that this class of proteins acted as positive viral factors. Inhibition of HSP90 chaperone function in the current study showed that HSP90 is critical for L-protein function and stability, whether in the presence or absence of the P-protein. Inhibition studies suggested that HSP70 also disrupts virus biology and might help the polymerase remodel the nucleocapsid to allow RNA synthesis to occur efficiently. This indicated a proviral role for protein chaperones in HRSV replication and demonstrates that the function of cellular proteins can be targeted as potential therapeutics to disrupt virus replication. Human respiratory syncytial virus (HRSV) represents a major health care and economic burden, being the main cause of severe respiratory infections in infants worldwide. No vaccine or effective therapy is available. This study

  17. HIV-1 integrase modulates the interaction of the HIV-1 cellular cofactor LEDGF/p75 with chromatin.

    PubMed

    Astiazaran, Paulina; Bueno, Murilo Td; Morales, Elisa; Kugelman, Jeffrey R; Garcia-Rivera, Jose A; Llano, Manuel

    2011-04-21

    Chromatin binding plays a central role in the molecular mechanism of LEDGF/p75 in HIV-1 DNA integration. Conflicting results have been reported in regards to the relevance of the LEDGF/p75 chromatin binding element PWWP domain in its HIV-1 cofactor activity. Here we present evidence that re-expression of a LEDGF/p75 mutant lacking the PWWP domain (ΔPWWP) rescued HIV-1 infection in cells verified to express background levels of endogenous LEDGF/p75 that do not support efficient HIV-1 infection. The HIV-1 cofactor activity of LEDGF/p75 ΔPWWP was similar to that of LEDGF/p75 wild type (WT). A possible molecular explanation for the nonessential role of PWWP domain in the HIV-1 cofactor activity of LEDGF/p75 comes from the fact that coexpression of HIV-1 integrase significantly restored the impaired chromatin binding activity of LEDGF/p75 ΔPWWP. However, integrase failed to promote chromatin binding of a non-chromatin bound LEDGF/p75 mutant that lacks both the PWWP domain and the AT hook motifs (ΔPWWP/AT) and that exhibits negligible HIV-1 cofactor activity. The effect of integrase on the chromatin binding of LEDGF/p75 requires the direct interaction of these two proteins. An HIV-1 integrase mutant, unable to interact with LEDGF/p75, failed to enhance chromatin binding, whereas integrase wild type did not increase the chromatin binding strength of a LEDGF/p75 mutant lacking the integrase binding domain (ΔIBD). Our data reveal that the PWWP domain of LEDGF/p75 is not essential for its HIV-1 cofactor activity, possibly due to an integrase-mediated increase of the chromatin binding strength of this LEDGF/p75 mutant.

  18. A systematic approach to identify cellular auxetic materials

    NASA Astrophysics Data System (ADS)

    Körner, Carolin; Liebold-Ribeiro, Yvonne

    2015-02-01

    Auxetics are materials showing a negative Poisson’s ratio. This characteristic leads to unusual mechanical properties that make this an interesting class of materials. So far no systematic approach for generating auxetic cellular materials has been reported. In this contribution, we present a systematic approach to identifying auxetic cellular materials based on eigenmode analysis. The fundamental mechanism generating auxetic behavior is identified as rotation. With this knowledge, a variety of complex two-dimensional (2D) and three-dimensional (3D) auxetic structures based on simple unit cells can be identified.

  19. Identifying patterns from one-rule-firing cellular automata.

    PubMed

    Shin, Jae Kyun

    2011-01-01

    A new firing scheme for cellular automata in which only one rule is fired at a time produces myriad patterns. In addition to geometric patterns, natural patterns such as flowers and snow crystals were also generated. This study proposes an efficient method identifying the patterns using a minimal number of digits. Complexity of the generated patterns is discussed in terms of the shapes and colors of the patterns.

  20. Kaposi's sarcoma-associated herpesvirus ORF57 interacts with cellular RNA export cofactors RBM15 and OTT3 to promote expression of viral ORF59.

    PubMed

    Majerciak, Vladimir; Uranishi, Hiroaki; Kruhlak, Michael; Pilkington, Guy R; Massimelli, Maria Julia; Bear, Jenifer; Pavlakis, George N; Felber, Barbara K; Zheng, Zhi-Ming

    2011-02-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which promotes the accumulation of specific KSHV mRNA targets, including ORF59 mRNA. We report that the cellular export NXF1 cofactors RBM15 and OTT3 participate in ORF57-enhanced expression of KSHV ORF59. We also found that ectopic expression of RBM15 or OTT3 augments ORF59 production in the absence of ORF57. While RBM15 promotes the accumulation of ORF59 RNA predominantly in the nucleus compared to the levels in the cytoplasm, we found that ORF57 shifted the nucleocytoplasmic balance by increasing ORF59 RNA accumulation in the cytoplasm more than in the nucleus. By promoting the accumulation of cytoplasmic ORF59 RNA, ORF57 offsets the nuclear RNA accumulation mediated by RBM15 by preventing nuclear ORF59 RNA from hyperpolyadenylation. ORF57 interacts directly with the RBM15 C-terminal portion containing the SPOC domain to reduce RBM15 binding to ORF59 RNA. Although ORF57 homologs Epstein-Barr virus (EBV) EB2, herpes simplex virus (HSV) ICP27, varicella-zoster virus (VZV) IE4/ORF4, and cytomegalovirus (CMV) UL69 also interact with RBM15 and OTT3, EBV EB2, which also promotes ORF59 expression, does not function like KSHV ORF57 to efficiently prevent RBM15-mediated nuclear accumulation of ORF59 RNA and RBM15's association with polyadenylated RNAs. Collectively, our data provide novel insight elucidating a molecular mechanism by which ORF57 promotes the expression of viral intronless genes.

  1. Identifying and targeting determinants of melanoma cellular invasion

    PubMed Central

    Jayachandran, Aparna; Prithviraj, Prashanth; Lo, Pu-Han; Walkiewicz, Marzena; Anaka, Matthew; Woods, Briannyn L.; Tan, BeeShin

    2016-01-01

    Epithelial-to-mesenchymal transition is a critical process that increases the malignant potential of melanoma by facilitating invasion and dissemination of tumor cells. This study identified genes involved in the regulation of cellular invasion and evaluated whether they can be targeted to inhibit melanoma invasion. We identified Peroxidasin (PXDN), Netrin 4 (NTN4) and GLIS Family Zinc Finger 3 (GLIS3) genes consistently elevated in invasive mesenchymal-like melanoma cells. These genes and proteins were highly expressed in metastatic melanoma tumors, and gene silencing led to reduced melanoma invasion in vitro. Furthermore, migration of PXDN, NTN4 or GLIS3 siRNA transfected melanoma cells was inhibited following transplantation into the embryonic chicken neural tube compared to control siRNA transfected melanoma cells. Our study suggests that PXDN, NTN4 and GLIS3 play a functional role in promoting melanoma cellular invasion, and therapeutic approaches directed toward inhibiting the action of these proteins may reduce the incidence or progression of metastasis in melanoma patients. PMID:27172792

  2. Cofactor engineering for more efficient production of chemicals and biofuels.

    PubMed

    Wang, Meng; Chen, Biqiang; Fang, Yunming; Tan, Tianwei

    2017-09-20

    Cofactors are involved in numerous intracellular reactions and critically influence redox balance and cellular metabolism. Cofactor engineering can support and promote the biocatalysis process, even help driving thermodynamically unfavorable reactions forwards. To achieve efficient production of chemicals and biofuels, cofactor engineering strategies such as altering cofactor supply or modifying reactants' cofactor preference have been developed to maintain redox balance. This review focuses primarily on the effects of cofactor engineering on carbon and energy metabolism. Coupling carbon metabolism with cofactor engineering can promote large-scale production, and even offer possibilities for producing new products or converting new materials. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Genome-wide functional screening identifies CDC37 as a crucial HSP90-cofactor for KIT oncogenic expression in gastrointestinal stromal tumors.

    PubMed

    Mariño-Enríquez, A; Ou, W-B; Cowley, G; Luo, B; Jonker, A H; Mayeda, M; Okamoto, M; Eilers, G; Czaplinski, J T; Sicinska, E; Wang, Y; Taguchi, T; Demetri, G D; Root, D E; Fletcher, J A

    2014-04-03

    Most gastrointestinal stromal tumors (GISTs) contain KIT or PDGFRA kinase gain-of-function mutations, and therefore respond clinically to imatinib and other tyrosine kinase inhibitor (TKI) therapies. However, clinical progression subsequently results from selection of TKI-resistant clones, typically containing secondary mutations in the KIT kinase domain, which can be heterogeneous between and within GIST metastases in a given patient. TKI-resistant KIT oncoproteins require HSP90 chaperoning and are potently inactivated by HSP90 inhibitors, but clinical applications in GIST patients are constrained by the toxicity resulting from concomitant inactivation of various other HSP90 client proteins, beyond KIT and PDGFRA. To identify novel targets responsible for KIT oncoprotein function, we performed parallel genome-scale short hairpin RNA (shRNA)-mediated gene knockdowns in KIT-mutant GIST-T1 and GIST882. GIST cells were infected with a lentiviral shRNA pooled library targeting 11 194 human genes, and allowed to proliferate for 5-7 weeks, at which point assessment of relative hairpin abundance identified the HSP90 cofactor, CDC37, as one of the top six GIST-specific essential genes. Validations in treatment-naive (GIST-T1, GIST882) vs imatinib-resistant GISTs (GIST48, GIST430) demonstrated that: (1) CDC37 interacts with oncogenic KIT; (2) CDC37 regulates expression and activation of KIT and downstream signaling intermediates in GIST; and (3) unlike direct HSP90 inhibition, CDC37 knockdown accomplishes prolonged KIT inhibition (>20 days) in GIST. These studies highlight CDC37 as a key biologic vulnerability in both imatinib-sensitive and imatinib-resistant GIST. CDC37 targeting is expected to be selective for KIT/PDGFRA and a subset of other HSP90 clients, and thereby represents a promising strategy for inactivating the myriad KIT/PDGFRA oncoproteins in TKI-resistant GIST patients.

  4. Identifying decomposition products in extracts of cellular metabolites.

    PubMed

    Kimball, Elizabeth; Rabinowitz, Joshua D

    2006-11-15

    Most methods of analyzing intracellular metabolites require extraction of metabolites from the cells. A concern in these methods is underestimation of metabolite levels due to incomplete extraction. In comparing extraction methods, then, it would seem that the best method for extracting a particular metabolite is the one that gives the largest yield. In extracting Escherichia coli with different methanol:water mixtures, we observed that >or=50% water gave an increased yield of nucleosides and bases compared with cellular metabolites. For extraction of E. coli with methanol:water, cold temperature and a high methanol fraction minimize artifacts due to metabolite decomposition.

  5. Fluorinated vitamin b(12) analogs are cofactors of corrinoid-dependent enzymes: a f-labeled nuclear magnetic resonance probe for identifying corrinoid-protein interactions.

    PubMed

    Stupperich, E; Eisinger, H J; Kerssebaum, R; Nexø, E

    1993-02-01

    The homoacetogenic bacterium Sporomusa ovata synthesized the vitamin B(12) analog phenolyl cobamide or 4-fluorophenolyl cobamide when the methanol medium of growing cells was supplemented with 10 mM phenol or 5 mM 4-fluorophenol. Phenol and, presumably, 4-fluorophenol were specifically incorporated into these cobamides, since phenol was not metabolized significantly into amino acids or into acetic acid, the product of the catabolism. The phenol-containing cobamides contributed up to 90% of the protein-bound cobamides of the 1,300 to 1,900 nmol of corrinoid per g of dry cell material formed. Fluorine-19 nuclear magnetic resonance spectroscopy of 4-fluorophenolyl cobamide exhibited a resonance near 30 ppm. An additional signal emerged at 25 ppm when 4-fluorophenolyl cobamide was investigated as the cofactor of a corrinoid-dependent protein. The two resonances indicated distinct cofactor arrangements within the protein's active site. A 5-ppm high-field shift change suggested van der Waal's interactions between the fluorinated nucleotide of the cofactor and adjacent amino acid residues of the enzyme. Similarly, Propionibacterium freudenreichii and Methanobacterium thermoautotrophicum synthesized 5-fluorobenzimidazolyl cobamide. The human corrinoid binders intrinsic factor, transcobalamin, and haptocorrin recognized this corrinoid like vitamin B(12). Hence, it is possible to use F-labeled nuclear magnetic resonance spectroscopy for analyses of protein-bound cobamides.

  6. Fluorinated Vitamin B12 Analogs Are Cofactors of Corrinoid-Dependent Enzymes: a 19F-Labeled Nuclear Magnetic Resonance Probe for Identifying Corrinoid-Protein Interactions

    PubMed Central

    Stupperich, Erhard; Eisinger, Hans-Jürgen; Kerssebaum, Rainer; Nexø, Ebba

    1993-01-01

    The homoacetogenic bacterium Sporomusa ovata synthesized the vitamin B12 analog phenolyl cobamide or 4-fluorophenolyl cobamide when the methanol medium of growing cells was supplemented with 10 mM phenol or 5 mM 4-fluorophenol. Phenol and, presumably, 4-fluorophenol were specifically incorporated into these cobamides, since phenol was not metabolized significantly into amino acids or into acetic acid, the product of the catabolism. The phenol-containing cobamides contributed up to 90% of the protein-bound cobamides of the 1,300 to 1,900 nmol of corrinoid per g of dry cell material formed. Fluorine-19 nuclear magnetic resonance spectroscopy of 4-fluorophenolyl cobamide exhibited a resonance near 30 ppm. An additional signal emerged at 25 ppm when 4-fluorophenolyl cobamide was investigated as the cofactor of a corrinoid-dependent protein. The two resonances indicated distinct cofactor arrangements within the protein's active site. A 5-ppm high-field shift change suggested van der Waal's interactions between the fluorinated nucleotide of the cofactor and adjacent amino acid residues of the enzyme. Similarly, Propionibacterium freudenreichii and Methanobacterium thermoautotrophicum synthesized 5-fluorobenzimidazolyl cobamide. The human corrinoid binders intrinsic factor, transcobalamin, and haptocorrin recognized this corrinoid like vitamin B12. Hence, it is possible to use 19F-labeled nuclear magnetic resonance spectroscopy for analyses of protein-bound cobamides. PMID:16348877

  7. Metal and cofactor insertion.

    PubMed

    Mendel, Ralf R; Smith, Alison G; Marquet, Andree; Warren, Martin J

    2007-10-01

    Cells require metal ions as cofactors for the assembly of metalloproteins. Principally one has to distinguish between metal ions that are directly incorporated into their cognate sites on proteins and those metal ions that have to become part of prosthetic groups, cofactors or complexes prior to insertion of theses moieties into target proteins. Molybdenum is only active as part of the molybdenum cofactor, iron can be part of diverse Fe-S clusters or of the heme group, while copper ions are directly delivered to their targets. We will focus in greater detail on molybdenum metabolism because molybdenum metabolism is a good example for demonstrating the role and the network of metals in metabolism: each of the three steps in the pathway of molybdenum cofactor formation depends on a different metal (iron, copper, molybdenum) and also the enzymes finally harbouring the molybdenum cofactor need additional metal-containing groups to function (iron sulfur-clusters, heme-iron).

  8. Protein cofactor competition regulates the action of a multifunctional RNA helicase in different pathways

    PubMed Central

    Heininger, Annika U.; Hackert, Philipp; Andreou, Alexandra Z.; Boon, Kum-Loong; Memet, Indira; Prior, Mira; Clancy, Anne; Schmidt, Bernhard; Urlaub, Henning; Schleiff, Enrico; Sloan, Katherine E.; Deckers, Markus; Lührmann, Reinhard; Enderlein, Jörg; Klostermeier, Dagmar; Rehling, Peter; Bohnsack, Markus T.

    2016-01-01

    ABSTRACT A rapidly increasing number of RNA helicases are implicated in several distinct cellular processes, however, the modes of regulation of multifunctional RNA helicases and their recruitment to different target complexes have remained unknown. Here, we show that the distribution of the multifunctional DEAH-box RNA helicase Prp43 between its diverse cellular functions can be regulated by the interplay of its G-patch protein cofactors. We identify the orphan G-patch protein Cmg1 (YLR271W) as a novel cofactor of Prp43 and show that it stimulates the RNA binding and ATPase activity of the helicase. Interestingly, Cmg1 localizes to the cytoplasm and to the intermembrane space of mitochondria and its overexpression promotes apoptosis. Furthermore, our data reveal that different G-patch protein cofactors compete for interaction with Prp43. Changes in the expression levels of Prp43-interacting G-patch proteins modulate the cellular localization of Prp43 and G-patch protein overexpression causes accumulation of the helicase in the cytoplasm or nucleoplasm. Overexpression of several G-patch proteins also leads to defects in ribosome biogenesis that are consistent with withdrawal of the helicase from this pathway. Together, these findings suggest that the availability of cofactors and the sequestering of the helicase are means to regulate the activity of multifunctional RNA helicases and their distribution between different cellular processes. PMID:26821976

  9. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying the peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  10. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  11. The Molybdenum Cofactor*

    PubMed Central

    Mendel, Ralf R.

    2013-01-01

    The transition element molybdenum needs to be complexed by a special cofactor to gain catalytic activity. Molybdenum is bound to a unique pterin, thus forming the molybdenum cofactor (Moco), which, in different variants, is the active compound at the catalytic site of all molybdenum-containing enzymes in nature, except bacterial molybdenum nitrogenase. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also require iron, ATP, and copper. After its synthesis, Moco is distributed, involving Moco-binding proteins. A deficiency in the biosynthesis of Moco has lethal consequences for the respective organisms. PMID:23539623

  12. The molybdenum cofactor.

    PubMed

    Mendel, Ralf R

    2013-05-10

    The transition element molybdenum needs to be complexed by a special cofactor to gain catalytic activity. Molybdenum is bound to a unique pterin, thus forming the molybdenum cofactor (Moco), which, in different variants, is the active compound at the catalytic site of all molybdenum-containing enzymes in nature, except bacterial molybdenum nitrogenase. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also require iron, ATP, and copper. After its synthesis, Moco is distributed, involving Moco-binding proteins. A deficiency in the biosynthesis of Moco has lethal consequences for the respective organisms.

  13. Cell Type-Specific Expression Analysis to Identify Putative Cellular Mechanisms for Neurogenetic Disorders

    PubMed Central

    Xu, Xiaoxiao; Wells, Alan B.; O'Brien, David R.; Nehorai, Arye

    2014-01-01

    Recent advances have substantially increased the number of genes that are statistically associated with complex genetic disorders of the CNS such as autism and schizophrenia. It is now clear that there will likely be hundreds of distinct loci contributing to these disorders, underscoring a remarkable genetic heterogeneity. It is unclear whether this genetic heterogeneity indicates an equal heterogeneity of cellular mechanisms for these diseases. The commonality of symptoms across patients suggests there could be a functional convergence downstream of these loci upon a limited number of cell types or circuits that mediate the affected behaviors. One possible mechanism for this convergence would be the selective expression of at least a subset of these genes in the cell types that comprise these circuits. Using profiling data from mice and humans, we have developed and validated an approach, cell type-specific expression analysis, for identifying candidate cell populations likely to be disrupted across sets of patients with distinct genetic lesions. Using human genetics data and postmortem gene expression data, our approach can correctly identify the cell types for disorders of known cellular etiology, including narcolepsy and retinopathies. Applying this approach to autism, a disease where the cellular mechanism is unclear, indicates there may be multiple cellular routes to this disorder. Our approach may be useful for identifying common cellular mechanisms arising from distinct genetic lesions. PMID:24453331

  14. Cellular adhesome screen identifies critical modulators of focal adhesion dynamics, cellular traction forces and cell migration behaviour.

    PubMed

    Fokkelman, Michiel; Balcıoğlu, Hayri E; Klip, Janna E; Yan, Kuan; Verbeek, Fons J; Danen, Erik H J; van de Water, Bob

    2016-08-17

    Cancer cells migrate from the primary tumour into surrounding tissue in order to form metastasis. Cell migration is a highly complex process, which requires continuous remodelling and re-organization of the cytoskeleton and cell-matrix adhesions. Here, we aimed to identify genes controlling aspects of tumour cell migration, including the dynamic organization of cell-matrix adhesions and cellular traction forces. In a siRNA screen targeting most cell adhesion-related genes we identified 200+ genes that regulate size and/or dynamics of cell-matrix adhesions in MCF7 breast cancer cells. In a subsequent secondary screen, the 64 most effective genes were evaluated for growth factor-induced cell migration and validated by tertiary RNAi pool deconvolution experiments. Four validated hits showed significantly enlarged adhesions accompanied by reduced cell migration upon siRNA-mediated knockdown. Furthermore, loss of PPP1R12B, HIPK3 or RAC2 caused cells to exert higher traction forces, as determined by traction force microscopy with elastomeric micropillar post arrays, and led to considerably reduced force turnover. Altogether, we identified genes that co-regulate cell-matrix adhesion dynamics and traction force turnover, thereby modulating overall motility behaviour.

  15. Cellular adhesome screen identifies critical modulators of focal adhesion dynamics, cellular traction forces and cell migration behaviour

    PubMed Central

    Fokkelman, Michiel; Balcıoğlu, Hayri E.; Klip, Janna E.; Yan, Kuan; Verbeek, Fons J.; Danen, Erik H. J.; van de Water, Bob

    2016-01-01

    Cancer cells migrate from the primary tumour into surrounding tissue in order to form metastasis. Cell migration is a highly complex process, which requires continuous remodelling and re-organization of the cytoskeleton and cell-matrix adhesions. Here, we aimed to identify genes controlling aspects of tumour cell migration, including the dynamic organization of cell-matrix adhesions and cellular traction forces. In a siRNA screen targeting most cell adhesion-related genes we identified 200+ genes that regulate size and/or dynamics of cell-matrix adhesions in MCF7 breast cancer cells. In a subsequent secondary screen, the 64 most effective genes were evaluated for growth factor-induced cell migration and validated by tertiary RNAi pool deconvolution experiments. Four validated hits showed significantly enlarged adhesions accompanied by reduced cell migration upon siRNA-mediated knockdown. Furthermore, loss of PPP1R12B, HIPK3 or RAC2 caused cells to exert higher traction forces, as determined by traction force microscopy with elastomeric micropillar post arrays, and led to considerably reduced force turnover. Altogether, we identified genes that co-regulate cell-matrix adhesion dynamics and traction force turnover, thereby modulating overall motility behaviour. PMID:27531518

  16. Transcriptome analyses identify key cellular factors associated with HIV-1 associated neuropathogenesis in infected men

    PubMed Central

    Venkatachari, Narasimhan J.; Jain, Siddhartha; Walker, Leah; Bivalkar-Mehla, Shalmali; Chattopadhyay, Ansuman; Bar-Joseph, Ziv; Rinaldo, Charles; Ragin, Ann; Seaberg, Eric; Levine, Andrew; Becker, James; Martin, Eileen; Sacktor, Ned; Ayyavoo, Velpandi

    2017-01-01

    Objective HIV-1 viral proteins and host inflammatory factors have a direct role in neuronal toxicity in in vitro, however, the contribution of these factors in vivo in HIV-1 associated neurocognitive disorder (HAND) is not fully understood. We applied novel Systems Biology approaches to identify specific cellular and viral factors and their related pathways that are associated with different stages of HAND. Design A cross-sectional study of individuals enrolled in the Multicenter AIDS Cohort Study (MACS) including HIV-1 seronegative (N=36) and HIV-1 seropositive individuals without neurocognitive symptoms (N=16), or with mild neurocognitive disorder (MND) (N=8) or HIV-associated dementia (HAD) (N=16). Methods A systematic evaluation of global transcriptome of peripheral blood mononuclear cells (PBMCs) obtained from HIV-1 seronegative individuals and from HIV-1 positive men without neurocognitive symptoms, or MND or HAD was performed. Results MND and HAD were associated with specific changes in mRNA transcripts and miRNAs in PBMCs. Comparison of upstream regulators and TimePath analyses identified specific cellular factors associated with MND and HAD, while HIV-1 viral proteins played a greater role in HAD. Additionally, expression of specific microRNAs – miR-let-7a, miR-124, miR-15a and others were found to correlate with mRNA gene expression and may have a potential protective role in asymptomatic HIV-1 seropositive individuals by regulating cellular signal transduction pathways downstream of chemokines and cytokines. Conclusions These results identify signature transcriptome changes in PBMCs associated with stages of HAND and shed light on the potential contribution of host cellular factors and viral proteins in HAND development. PMID:28005686

  17. A chemoproteomic method for identifying cellular targets of covalent kinase inhibitors

    PubMed Central

    Chen, Ying-Chu; Zhang, Chao

    2016-01-01

    Protein kinases are attractive drug targets for numerous human diseases including cancers, diabetes and neurodegeneration. A number of kinase inhibitors that covalently target a cysteine residue in their target kinases have recently entered use in the cancer clinic. Despite the advantages of covalent kinases inhibitors, their inherent reactivity can lead to non-specific binding to other cellular proteins and cause off- target effects in cells. It is thus essential to determine the identity of these off targets in order to fully account for the phenotype and to improve the selectivity and efficacy of covalent inhibitors. Herein we present a detailed protocol for a chemoproteomic method to enrich and identify cellular targets of covalent kinase inhibitors. PMID:27551330

  18. Identifying cellular targets of small-molecule probes and drugs with biochemical enrichment and SILAC.

    PubMed

    Ong, Shao-En; Li, Xiaoyu; Schenone, Monica; Schreiber, Stuart L; Carr, Steven A

    2012-01-01

    Sequencing of the human genome in the last decade has not yet led to a concomitant increase in the numbers of novel drug targets. While the pharmaceutical industry has invested heavily in improving drugs for existing protein targets, it has not tended toward a similar investment in experimental approaches to identify cellular targets of drugs. It is striking that the targets of numerous widely used FDA-approved drugs remain unknown. The development of robust, unbiased methods for target identification would greatly enhance our understanding the mechanisms-of-action of small molecules. Cell-based phenotypic screens followed by unbiased target identification have the potential to identify novel combinations of small molecules and their protein targets, shed light on drug polypharmacology, and enable unbiased screening approaches to drug discovery. Classical biochemical enrichment with immobilized small molecules has been used for over four decades but has been limited by issues concerning specificity and sensitivity. The application of mass spectrometry-based quantitative proteomics in combination with these affinity reagents has proven to be especially useful in addressing these common issues in affinity purification experiments. We describe the use of SILAC in identifying proteins that bind small-molecule probes and drugs in a cellular context.

  19. A cellular model of inflammation for identifying TNF-α synthesis inhibitors

    PubMed Central

    Tweedie, David; Luo, Weiming; Short, Ryan G.; Brossi, Arnold; Holloway, Harold W.; Li, Yazhou; Yu, Qian-sheng; Greig, Nigel H.

    2009-01-01

    Neuroinflammation is a common facet of both acute and chronic neurodegenerative conditions, exemplified by stroke and by Alzheimer’s and Parkinson’s disease, and the presence of elevated levels of the proinflammatory cytokine, tumor necrosis factor-α (TNF-α) has been documented in each. Although initial TNF-α generation is associated with a protective compensatory response, its unregulated chronic elevation is generally detrimental and can drive the disease process. In such circumstances, therapeutic strategies that can both gain access to the brain and target the production of TNF-α are predicted to be of clinical benefit. An in vitro mouse macrophage-like cellular screen, utilizing RAW 264.7 cells, was hence developed to identify novel TNF-α lowering agents incorporating lipophilic physicochemical characteristics predicted to allow penetration of the blood-brain barrier. Cultured RAW 264.7 cells exposed to lipopolysaccharide (LPS) induced a rapid, marked and concentration-dependent cellular release of TNF-α into the cell culture media, which was readily detected by Enzyme Linked ImmunoSorbent Assay (ELISA). The effects of four characterized thalidomide-based TNF-α lowering agents were assessed alongside 10 novel uncharacterized compounds synthesized on the same backbone. One of these new analogs possessed activity of sufficient magnitude to warrant further investigation. Activity determined in the cellular model translated to an in vivo rodent model of acute LPS-induced TNF-α elevation. The utility of the TNF-α cellular assay lies in its simplicity and robust nature, providing a tool for initial pharmacological screening to allow for the rapid identification novel TNF-α lowering agents. PMID:19583982

  20. Cervical cancer: is herpes simplex virus type II a cofactor?

    PubMed Central

    Jones, C

    1995-01-01

    In many ways, cervical cancer behaves as a sexually transmitted disease. The major risk factors are multiple sexual partners and early onset of sexual activity. Although high-risk types of human papillomaviruses (HPV) play an important role in the development of nearly all cases of cervical cancer, other sexually transmitted infectious agents may be cofactors. Herpes simplex virus type 2 (HSV-2) is transmitted primarily by sexual contact and therefore has been implicated as a risk factor. Several independent studies suggest that HSV-2 infections correlate with a higher than normal incidence of cervical cancer. In contrast, other epidemiological studies have concluded that infection with HSV-2 is not a major risk factor. Two separate transforming domains have been identified within the HSV-2 genome, but continued viral gene expression apparently is not necessary for neoplastic transformation. HSV infections lead to unscheduled cellular DNA synthesis, chromosomal amplifications, and mutations. These observations suggest that HSV-2 is not a typical DNA tumor virus. It is hypothesized that persistent or abortive infections induce permanent genetic alterations that interfere with differentiation of cervical epithelium and subsequently induce abnormal proliferation. Thus, HSV-2 may be a cofactor in some but not all cases of cervical cancer. PMID:8665469

  1. Insect Cell-Derived Cofactors Become Fully Functional after Proteinase K and Heat Treatment for High-Fidelity Amplification of Glycosylphosphatidylinositol-Anchored Recombinant Scrapie and BSE Prion Proteins

    PubMed Central

    Imamura, Morikazu; Kato, Nobuko; Okada, Hiroyuki; Yoshioka, Miyako; Iwamaru, Yoshifumi; Shimizu, Yoshihisa; Mohri, Shirou; Yokoyama, Takashi; Murayama, Yuichi

    2013-01-01

    The central event in prion infection is the conformational conversion of host-encoded cellular prion protein (PrPC) into the pathogenic isoform (PrPSc). Diverse mammalian species possess the cofactors required for in vitro replication of PrPSc by protein-misfolding cyclic amplification (PMCA), but lower organisms, such as bacteria, yeasts, and insects, reportedly lack the essential cofactors. Various cellular components, such as RNA, lipids, and other identified cofactor molecules, are commonly distributed in both eukaryotes and prokaryotes, but the reasons for the absence of cofactor activity in lower organisms remain to be elucidated. Previously, we reported that brain-derived factors were necessary for the in vitro replication of glycosylphosphatidylinositol-anchored baculovirus-derived recombinant PrP (Bac-PrP). Here, we demonstrate that following protease digestion and heat treatment, insect cell lysates had the functional cofactor activity required for Bac-PrP replication by PMCA. Mammalian PrPSc seeds and Bac-PrPSc generated by PMCA using Bac-PrP and insect cell-derived cofactors showed similar pathogenicity and produced very similar lesions in the brains of inoculated mice. These results suggested that the essential cofactors required for the high-fidelity replication of mammalian PrPSc were present in the insect cells but that the cofactor activity was masked or inhibited in the native state. We suggest that not only RNA, but also DNA, are the key components of PMCA, although other cellular factors were necessary for the expression of the cofactor activity of nucleic acids. PMCA using only insect cell-derived substances (iPMCA) was highly useful for the ultrasensitive detection of PrPSc of some prion strains. PMID:24367521

  2. Identifying the ERAD ubiquitin E3 ligases for viral and cellular targeting of MHC class I.

    PubMed

    van den Boomen, D J H; Lehner, P J

    2015-12-01

    The human cytomegalovirus (HCMV) US2 and US11 gene products hijack mammalian ER-associated degradation (ERAD) to induce rapid degradation of major histocompatibility class I (MHC-I) molecules. The rate-limiting step in this pathway is thought to be the polyubiquitination of MHC-I by distinct host ERAD E3 ubiquitin ligases. TRC8 was identified as the ligase responsible for US2-mediated MHC-I degradation and shown to be required for the cleavage-dependent degradation of some tail-anchored proteins. In addition to MHC-I, plasma membrane profiling identified further immune receptors, which are also substrates for the US2/TRC8 complex. These include at least six α integrins, the coagulation factor thrombomodulin and the NK cell ligand CD112. US2's use of specific HCMV-encoded adaptors makes it an adaptable viral degradation hub. US11-mediated degradation is MHC-I-specific and genetic screens have identified TMEM129, an uncharacterised RING-C2 E3 ligase, as responsible for US11-mediated degradation. In a unique auto-regulatory loop, US11 readily responds to changes in cellular expression of MHC-I. Free US11 either rebinds more MHC-I or is itself degraded by the HRD1/SEL1L E3 ligase complex. While virally encoded US2 and US11 appropriate mammalian ERAD, the MHC-I complex also undergoes stringent cellular quality control and misfolded MHC-I is degraded by the HRD1/SEL1L complex. We discuss the identification and central role of E3 ubiquitin ligases in ER quality control and viral degradation of the MHC-I chain. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  3. Biology of the molybdenum cofactor.

    PubMed

    Mendel, Ralf R

    2007-01-01

    The transition element molybdenum (Mo) is an essential micronutrient for plants where it is needed as a catalytically active metal during enzyme catalysis. Four plant enzymes depend on molybdenum: nitrate reductase, sulphite oxidase, xanthine dehydrogenase, and aldehyde oxidase. However, in order to gain biological activity and fulfil its function in enzymes, molybdenum has to be complexed by a pterin compound thus forming the molybdenum cofactor. In this article, the path of molybdenum from its uptake into the cell, via formation of the molybdenum cofactor and its storage, to the final modification of the molybdenum cofactor and its insertion into apo-metalloenzymes will be reviewed.

  4. The use of cellular diagnostics for identifying sub-lethal stress in reef corals.

    PubMed

    Downs, Craig A; Ostrander, Gary K; Rougee, Luc; Rongo, Teina; Knutson, Sean; Williams, David E; Mendiola, Wendy; Holbrook, Jackalyn; Richmond, Robert H

    2012-04-01

    Coral reefs throughout the world are exhibiting documented declines in coral cover and species diversity, which have been linked to anthropogenic stressors including land-based sources of pollution. Reductions in coastal water and substratum quality are affecting coral survivorship, reproduction and recruitment, and hence, the persistence of coral reefs. One major obstacle in effectively addressing these declines is the lack of tools that can identify cause-and-effect relationships between stressors and specific coral reef losses, while a second problem is the inability to measure the efficacy of mitigation efforts in a timely fashion. We examined corals from six coral reefs on Guam, Mariana Islands, which were being affected by different environmental stressors (e.g. PAH's, pesticides, PCB's and sedimentation). Cellular diagnostic analysis differentiated the cellular-physiological condition of these corals. Examination of protein expression provided insight into their homeostatic responses to chemical and physical stressors in exposed corals prior to outright mortality, providing improved opportunities for developing locally-based management responses. This approach adds critically needed tools for addressing the effects of multiple stressors on corals and will allow researchers to move beyond present assessment and monitoring techniques that simply document the loss of coral abundance and diversity.

  5. Identifying the cellular mechanisms of symbiont-induced epithelial morphogenesis in the squid-Vibrio association.

    PubMed

    Koropatnick, Tanya; Goodson, Michael S; Heath-Heckman, Elizabeth A C; McFall-Ngai, Margaret

    2014-02-01

    The symbiotic association between the Hawaiian bobtail squid Euprymna scolopes and the luminous marine bacterium Vibrio fischeri provides a unique opportunity to study epithelial morphogenesis. Shortly after hatching, the squid host harvests bacteria from the seawater using currents created by two elaborate fields of ciliated epithelia on the surface of the juvenile light organ. After light organ colonization, the symbiont population signals the gradual loss of the ciliated epithelia through apoptosis of the cells, which culminates in the complete regression of these tissues. Whereas aspects of this process have been studied at the morphological, biochemical, and molecular levels, no in-depth analysis of the cellular events has been reported. Here we describe the cellular structure of the epithelial field and present evidence that the symbiosis-induced regression occurs in two steps. Using confocal microscopic analyses, we observed an initial epithelial remodeling, which serves to disable the function of the harvesting apparatus, followed by a protracted regression involving actin rearrangements and epithelial cell extrusion. We identified a metal-dependent gelatinolytic activity in the symbiont-induced morphogenic epithelial fields, suggesting the involvement of Zn-dependent matrix metalloproteinase(s) (MMP) in light organ morphogenesis. These data show that the bacterial symbionts not only induce apoptosis of the field, but also change the form, function, and biochemistry of the cells as part of the morphogenic program.

  6. Transposon mutagenesis identifies genes and cellular processes driving epithelial-mesenchymal transition in hepatocellular carcinoma

    PubMed Central

    Kodama, Takahiro; Newberg, Justin Y.; Kodama, Michiko; Rangel, Roberto; Yoshihara, Kosuke; Tien, Jean C.; Parsons, Pamela H.; Wu, Hao; Finegold, Milton J.; Copeland, Neal G.; Jenkins, Nancy A.

    2016-01-01

    Epithelial-mesenchymal transition (EMT) is thought to contribute to metastasis and chemoresistance in patients with hepatocellular carcinoma (HCC), leading to their poor prognosis. The genes driving EMT in HCC are not yet fully understood, however. Here, we show that mobilization of Sleeping Beauty (SB) transposons in immortalized mouse hepatoblasts induces mesenchymal liver tumors on transplantation to nude mice. These tumors show significant down-regulation of epithelial markers, along with up-regulation of mesenchymal markers and EMT-related transcription factors (EMT-TFs). Sequencing of transposon insertion sites from tumors identified 233 candidate cancer genes (CCGs) that were enriched for genes and cellular processes driving EMT. Subsequent trunk driver analysis identified 23 CCGs that are predicted to function early in tumorigenesis and whose mutation or alteration in patients with HCC is correlated with poor patient survival. Validation of the top trunk drivers identified in the screen, including MET (MET proto-oncogene, receptor tyrosine kinase), GRB2-associated binding protein 1 (GAB1), HECT, UBA, and WWE domain containing 1 (HUWE1), lysine-specific demethylase 6A (KDM6A), and protein-tyrosine phosphatase, nonreceptor-type 12 (PTPN12), showed that deregulation of these genes activates an EMT program in human HCC cells that enhances tumor cell migration. Finally, deregulation of these genes in human HCC was found to confer sorafenib resistance through apoptotic tolerance and reduced proliferation, consistent with recent studies showing that EMT contributes to the chemoresistance of tumor cells. Our unique cell-based transposon mutagenesis screen appears to be an excellent resource for discovering genes involved in EMT in human HCC and potentially for identifying new drug targets. PMID:27247392

  7. Proteomic Analysis of Kveim Reagent Identifies Targets of Cellular Immunity in Sarcoidosis.

    PubMed

    Eberhardt, Christian; Thillai, Muhunthan; Parker, Robert; Siddiqui, Nazneen; Potiphar, Lee; Goldin, Rob; Timms, John F; Wells, Athol U; Kon, Onn M; Wickremasinghe, Melissa; Mitchell, Donald; Weeks, Mark E; Lalvani, Ajit

    2017-01-01

    . Stimulation with both Kveim reagent and vimentin induces a specific pro-inflammatory cytokine secretion from sarcoidosis PBMCs. Further investigation of cellular immune responses to Kveim-specific proteins may identify novel biomarkers to assist the diagnosis of sarcoidosis.

  8. Proteomic Analysis of Kveim Reagent Identifies Targets of Cellular Immunity in Sarcoidosis

    PubMed Central

    Parker, Robert; Siddiqui, Nazneen; Potiphar, Lee; Goldin, Rob; Timms, John F.; Wells, Athol U.; Kon, Onn M.; Wickremasinghe, Melissa; Mitchell, Donald; Weeks, Mark E.; Lalvani, Ajit

    2017-01-01

    or alpha-actinin-4. Conclusions Stimulation with both Kveim reagent and vimentin induces a specific pro-inflammatory cytokine secretion from sarcoidosis PBMCs. Further investigation of cellular immune responses to Kveim-specific proteins may identify novel biomarkers to assist the diagnosis of sarcoidosis. PMID:28114394

  9. The general definition of the p97/valosin-containing protein (VCP)-interacting motif (VIM) delineates a new family of p97 cofactors.

    PubMed

    Stapf, Christopher; Cartwright, Edward; Bycroft, Mark; Hofmann, Kay; Buchberger, Alexander

    2011-11-04

    Cellular functions of the essential, ubiquitin-selective AAA ATPase p97/valosin-containing protein (VCP) are controlled by regulatory cofactors determining substrate specificity and fate. Most cofactors bind p97 through a ubiquitin regulatory X (UBX) or UBX-like domain or linear sequence motifs, including the hitherto ill defined p97/VCP-interacting motif (VIM). Here, we present the new, minimal consensus sequence RX(5)AAX(2)R as a general definition of the VIM that unites a novel family of known and putative p97 cofactors, among them UBXD1 and ZNF744/ANKZF1. We demonstrate that this minimal VIM consensus sequence is necessary and sufficient for p97 binding. Using NMR chemical shift mapping, we identified several residues of the p97 N-terminal domain (N domain) that are critical for VIM binding. Importantly, we show that cellular stress resistance conferred by the yeast VIM-containing cofactor Vms1 depends on the physical interaction between its VIM and the critical N domain residues of the yeast p97 homolog, Cdc48. Thus, the VIM-N domain interaction characterized in this study is required for the physiological function of Vms1 and most likely other members of the newly defined VIM family of cofactors.

  10. Using exploratory regression to identify optimal driving factors for cellular automaton modeling of land use change.

    PubMed

    Feng, Yongjiu; Tong, Xiaohua

    2017-09-22

    Defining transition rules is an important issue in cellular automaton (CA)-based land use modeling because these models incorporate highly correlated driving factors. Multicollinearity among correlated driving factors may produce negative effects that must be eliminated from the modeling. Using exploratory regression under pre-defined criteria, we identified all possible combinations of factors from the candidate factors affecting land use change. Three combinations that incorporate five driving factors meeting pre-defined criteria were assessed. With the selected combinations of factors, three logistic regression-based CA models were built to simulate dynamic land use change in Shanghai, China, from 2000 to 2015. For comparative purposes, a CA model with all candidate factors was also applied to simulate the land use change. Simulations using three CA models with multicollinearity eliminated performed better (with accuracy improvements about 3.6%) than the model incorporating all candidate factors. Our results showed that not all candidate factors are necessary for accurate CA modeling and the simulations were not sensitive to changes in statistically non-significant driving factors. We conclude that exploratory regression is an effective method to search for the optimal combinations of driving factors, leading to better land use change models that are devoid of multicollinearity. We suggest identification of dominant factors and elimination of multicollinearity before building land change models, making it possible to simulate more realistic outcomes.

  11. A Versatile Transreplication-Based System To Identify Cellular Proteins Involved in Geminivirus Replication

    PubMed Central

    Morilla, Gabriel; Castillo, Araceli G.; Preiss, Werner; Jeske, Holger; Bejarano, Eduardo R.

    2006-01-01

    A versatile green fluorescent protein (GFP) expression cassette containing the replication origins of the monopartite begomovirus Tomato yellow leaf curl Sardinia virus (TYLCSV) is described. Transgenic Nicotiana benthamiana plants containing one copy of the cassette stably integrated into their genome were superinfected with TYLCSV, which mobilized and replicated the cassette as an episomal replicon. The expression of the reporter gene (the GFP gene) was thereby modified. Whereas GFP fluorescence was dimmed in the intercostal areas, an increase of green fluorescence in veins of all leaves placed above the inoculation site, as well as in transport tissues of roots and stems, was observed. The release of episomal trans replicons from the transgene and the increase in GFP expression were dependent on the cognate geminiviral replication-associated protein (Rep) and required interaction between Rep and the intergenic region of TYLCSV. This expression system is able to monitor the replication status of TYLCSV in plants, as induction of GFP expression is only produced in those tissues where Rep is present. To further confirm this notion, the expression of a host factor required for geminivirus replication, the proliferating cellular nuclear antigen (PCNA) was transiently silenced. Inhibition of PCNA prevented GFP induction in veins and reduced viral DNA. We propose that these plants could be widely used to easily identify host factors required for geminivirus replication by virus-induced gene silencing. PMID:16537630

  12. Yeast Reporter Assay to Identify Cellular Components of Ricin Toxin A Chain Trafficking

    PubMed Central

    Becker, Björn; Schnöder, Tina; Schmitt, Manfred J.

    2016-01-01

    RTA, the catalytic A-subunit of the ribosome inactivating A/B toxin ricin, inhibits eukaryotic protein biosynthesis by depurination of 28S rRNA. Although cell surface binding of ricin holotoxin is mainly mediated through its B-subunit (RTB), sole application of RTA is also toxic, albeit to a significantly lower extent, suggesting alternative pathways for toxin uptake and transport. Since ricin toxin trafficking in mammalian cells is still not fully understood, we developed a GFP-based reporter assay in yeast that allows rapid identification of cellular components required for RTA uptake and subsequent transport through a target cell. We hereby show that Ypt6p, Sft2p and GARP-complex components play an important role in RTA transport, while neither the retromer complex nor COPIB vesicles are part of the transport machinery. Analyses of yeast knock-out mutants with chromosomal deletion in genes whose products regulate ADP-ribosylation factor GTPases (Arf-GTPases) and/or retrograde Golgi-to-ER (endoplasmic reticulum) transport identified Sso1p, Snc1p, Rer1p, Sec22p, Erv46p, Gea1p and Glo3p as novel components in RTA transport, suggesting the developed reporter assay as a powerful tool to dissect the multistep processes of host cell intoxication in yeast. PMID:27929418

  13. A functional screen for copper homeostasis genes identifies a pharmacologically tractable cellular system.

    PubMed

    Schlecht, Ulrich; Suresh, Sundari; Xu, Weihong; Aparicio, Ana Maria; Chu, Angela; Proctor, Michael J; Davis, Ronald W; Scharfe, Curt; St Onge, Robert P

    2014-04-05

    Copper is essential for the survival of aerobic organisms. If copper is not properly regulated in the body however, it can be extremely cytotoxic and genetic mutations that compromise copper homeostasis result in severe clinical phenotypes. Understanding how cells maintain optimal copper levels is therefore highly relevant to human health. We found that addition of copper (Cu) to culture medium leads to increased respiratory growth of yeast, a phenotype which we then systematically and quantitatively measured in 5050 homozygous diploid deletion strains. Cu's positive effect on respiratory growth was quantitatively reduced in deletion strains representing 73 different genes, the function of which identify increased iron uptake as a cause of the increase in growth rate. Conversely, these effects were enhanced in strains representing 93 genes. Many of these strains exhibited respiratory defects that were specifically rescued by supplementing the growth medium with Cu. Among the genes identified are known and direct regulators of copper homeostasis, genes required to maintain low vacuolar pH, and genes where evidence supporting a functional link with Cu has been heretofore lacking. Roughly half of the genes are conserved in man, and several of these are associated with Mendelian disorders, including the Cu-imbalance syndromes Menkes and Wilson's disease. We additionally demonstrate that pharmacological agents, including the approved drug disulfiram, can rescue Cu-deficiencies of both environmental and genetic origin. A functional screen in yeast has expanded the list of genes required for Cu-dependent fitness, revealing a complex cellular system with implications for human health. Respiratory fitness defects arising from perturbations in this system can be corrected with pharmacological agents that increase intracellular copper concentrations.

  14. A functional screen for copper homeostasis genes identifies a pharmacologically tractable cellular system

    PubMed Central

    2014-01-01

    Background Copper is essential for the survival of aerobic organisms. If copper is not properly regulated in the body however, it can be extremely cytotoxic and genetic mutations that compromise copper homeostasis result in severe clinical phenotypes. Understanding how cells maintain optimal copper levels is therefore highly relevant to human health. Results We found that addition of copper (Cu) to culture medium leads to increased respiratory growth of yeast, a phenotype which we then systematically and quantitatively measured in 5050 homozygous diploid deletion strains. Cu’s positive effect on respiratory growth was quantitatively reduced in deletion strains representing 73 different genes, the function of which identify increased iron uptake as a cause of the increase in growth rate. Conversely, these effects were enhanced in strains representing 93 genes. Many of these strains exhibited respiratory defects that were specifically rescued by supplementing the growth medium with Cu. Among the genes identified are known and direct regulators of copper homeostasis, genes required to maintain low vacuolar pH, and genes where evidence supporting a functional link with Cu has been heretofore lacking. Roughly half of the genes are conserved in man, and several of these are associated with Mendelian disorders, including the Cu-imbalance syndromes Menkes and Wilson’s disease. We additionally demonstrate that pharmacological agents, including the approved drug disulfiram, can rescue Cu-deficiencies of both environmental and genetic origin. Conclusions A functional screen in yeast has expanded the list of genes required for Cu-dependent fitness, revealing a complex cellular system with implications for human health. Respiratory fitness defects arising from perturbations in this system can be corrected with pharmacological agents that increase intracellular copper concentrations. PMID:24708151

  15. Knock-In Mice with NOP-eGFP Receptors Identify Receptor Cellular and Regional Localization

    PubMed Central

    Ozawa, Akihiko; Brunori, Gloria; Mercatelli, Daniela; Wu, Jinhua; Cippitelli, Andrea; Zou, Bende; Xie, Xinmin (Simon); Williams, Melissa; Zaveri, Nurulain T.; Low, Sarah; Scherrer, Grégory; Kieffer, Brigitte L.

    2015-01-01

    The nociceptin/orphanin FQ (NOP) receptor, the fourth member of the opioid receptor family, is involved in many processes common to the opioid receptors including pain and drug abuse. To better characterize receptor location and trafficking, knock-in mice were created by inserting the gene encoding enhanced green fluorescent protein (eGFP) into the NOP receptor gene (Oprl1) and producing mice expressing a functional NOP-eGFP C-terminal fusion in place of the native NOP receptor. The NOP-eGFP receptor was present in brain of homozygous knock-in animals in concentrations somewhat higher than in wild-type mice and was functional when tested for stimulation of [35S]GTPγS binding in vitro and in patch-clamp electrophysiology in dorsal root ganglia (DRG) neurons and hippocampal slices. Inhibition of morphine analgesia was equivalent when tested in knock-in and wild-type mice. Imaging revealed detailed neuroanatomy in brain, spinal cord, and DRG and was generally consistent with in vitro autoradiographic imaging of receptor location. Multicolor immunohistochemistry identified cells coexpressing various spinal cord and DRG cellular markers, as well as coexpression with μ-opioid receptors in DRG and brain regions. Both in tissue slices and primary cultures, the NOP-eGFP receptors appear throughout the cell body and in processes. These knock-in mice have NOP receptors that function both in vitro and in vivo and appear to be an exceptional tool to study receptor neuroanatomy and correlate with NOP receptor function. SIGNIFICANCE STATEMENT The NOP receptor, the fourth member of the opioid receptor family, is involved in pain, drug abuse, and a number of other CNS processes. The regional and cellular distribution has been difficult to determine due to lack of validated antibodies for immunohistochemical analysis. To provide a new tool for the investigation of receptor localization, we have produced knock-in mice with a fluorescent-tagged NOP receptor in place of the native

  16. Genome Wide Expression Profiling during Spinal Cord Regeneration Identifies Comprehensive Cellular Responses in Zebrafish

    PubMed Central

    Hui, Subhra Prakash; Sengupta, Dhriti; Lee, Serene Gek Ping; Sen, Triparna; Kundu, Sudip; Mathavan, Sinnakaruppan; Ghosh, Sukla

    2014-01-01

    Background Among the vertebrates, teleost and urodele amphibians are capable of regenerating their central nervous system. We have used zebrafish as a model to study spinal cord injury and regeneration. Relatively little is known about the molecular mechanisms underlying spinal cord regeneration and information based on high density oligonucleotide microarray was not available. We have used a high density microarray to profile the temporal transcriptome dynamics during the entire phenomenon. Results A total of 3842 genes expressed differentially with significant fold changes during spinal cord regeneration. Cluster analysis revealed event specific dynamic expression of genes related to inflammation, cell death, cell migration, cell proliferation, neurogenesis, neural patterning and axonal regrowth. Spatio-temporal analysis of stat3 expression suggested its possible function in controlling inflammation and cell proliferation. Genes involved in neurogenesis and their dorso-ventral patterning (sox2 and dbx2) are differentially expressed. Injury induced cell proliferation is controlled by many cell cycle regulators and some are commonly expressed in regenerating fin, heart and retina. Expression pattern of certain pathway genes are identified for the first time during regeneration of spinal cord. Several genes involved in PNS regeneration in mammals like stat3, socs3, atf3, mmp9 and sox11 are upregulated in zebrafish SCI thus creating PNS like environment after injury. Conclusion Our study provides a comprehensive genetic blue print of diverse cellular response(s) during regeneration of zebrafish spinal cord. The data highlights the importance of different event specific gene expression that could be better understood and manipulated further to induce successful regeneration in mammals. PMID:24465396

  17. Protein Quantitative Trait Loci Identify Novel Candidates Modulating Cellular Response to Chemotherapy

    PubMed Central

    Gorsic, Lidija K.; Antao, Nirav N.; Wong, Shan S.; Chung, Sophie H.; Gill, Daniel F.; Im, Hae K.; Myers, Jamie L.; White, Kevin P.; Jones, Richard Baker; Dolan, M. Eileen

    2014-01-01

    Annotating and interpreting the results of genome-wide association studies (GWAS) remains challenging. Assigning function to genetic variants as expression quantitative trait loci is an expanding and useful approach, but focuses exclusively on mRNA rather than protein levels. Many variants remain without annotation. To address this problem, we measured the steady state abundance of 441 human signaling and transcription factor proteins from 68 Yoruba HapMap lymphoblastoid cell lines to identify novel relationships between inter-individual protein levels, genetic variants, and sensitivity to chemotherapeutic agents. Proteins were measured using micro-western and reverse phase protein arrays from three independent cell line thaws to permit mixed effect modeling of protein biological replicates. We observed enrichment of protein quantitative trait loci (pQTLs) for cellular sensitivity to two commonly used chemotherapeutics: cisplatin and paclitaxel. We functionally validated the target protein of a genome-wide significant trans-pQTL for its relevance in paclitaxel-induced apoptosis. GWAS overlap results of drug-induced apoptosis and cytotoxicity for paclitaxel and cisplatin revealed unique SNPs associated with the pharmacologic traits (at p<0.001). Interestingly, GWAS SNPs from various regions of the genome implicated the same target protein (p<0.0001) that correlated with drug induced cytotoxicity or apoptosis (p≤0.05). Two genes were functionally validated for association with drug response using siRNA: SMC1A with cisplatin response and ZNF569 with paclitaxel response. This work allows pharmacogenomic discovery to progress from the transcriptome to the proteome and offers potential for identification of new therapeutic targets. This approach, linking targeted proteomic data to variation in pharmacologic response, can be generalized to other studies evaluating genotype-phenotype relationships and provide insight into chemotherapeutic mechanisms. PMID:24699359

  18. Intracellular Trafficking of the Pyridoxal Cofactor. Implications for Health and Metabolic Disease*

    PubMed Central

    Whittaker, James W.

    2016-01-01

    The importance of the vitamin B6-derived pyridoxal cofactor for human health has been established through more than 70 years of intensive biochemical research, revealing its fundamental roles in metabolism. B6 deficiency, resulting from nutritional limitation or impaired uptake from dietary sources, is associated with epilepsy, neuromuscular disease and neurodegeneration. Hereditary disorders of B6 processing are also known, and genetic defects in pathways involved in transport of B6 into the cell and its transformation to the pyridoxal-5′-phosphate enzyme cofactor can contribute to cardiovascular disease by interfering with homocysteine metabolism and the biosynthesis of vasomodulatory polyamines. Compared to the processes involved in cellular uptake and processing of the B6 vitamers, trafficking of the PLP cofactor across intracellular membranes is very poorly understood, even though the availability of PLP within subcellular compartments (particularly the mitochondrion) may have important health implications. The aim of this review is to concisely summarize the state of current knowledge of intracellular trafficking of PLP and to identify key directions for future research. PMID:26619753

  19. Quantitative phase-digital holographic microscopy: a new imaging modality to identify original cellular biomarkers of diseases

    NASA Astrophysics Data System (ADS)

    Marquet, P.; Rothenfusser, K.; Rappaz, B.; Depeursinge, C.; Jourdain, P.; Magistretti, P. J.

    2016-03-01

    Quantitative phase microscopy (QPM) has recently emerged as a powerful label-free technique in the field of living cell imaging allowing to non-invasively measure with a nanometric axial sensitivity cell structure and dynamics. Since the phase retardation of a light wave when transmitted through the observed cells, namely the quantitative phase signal (QPS), is sensitive to both cellular thickness and intracellular refractive index related to the cellular content, its accurate analysis allows to derive various cell parameters and monitor specific cell processes, which are very likely to identify new cell biomarkers. Specifically, quantitative phase-digital holographic microscopy (QP-DHM), thanks to its numerical flexibility facilitating parallelization and automation processes, represents an appealing imaging modality to both identify original cellular biomarkers of diseases as well to explore the underlying pathophysiological processes.

  20. Experimental approaches to identify cellular G-quadruplex structures and functions.

    PubMed

    Di Antonio, Marco; Rodriguez, Raphaël; Balasubramanian, Shankar

    2012-05-01

    Guanine-rich nucleic acids can fold into non-canonical DNA secondary structures called G-quadruplexes. The formation of these structures can interfere with the biology that is crucial to sustain cellular homeostases and metabolism via mechanisms that include transcription, translation, splicing, telomere maintenance and DNA recombination. Thus, due to their implication in several biological processes and possible role promoting genomic instability, G-quadruplex forming sequences have emerged as potential therapeutic targets. There has been a growing interest in the development of synthetic molecules and biomolecules for sensing G-quadruplex structures in cellular DNA. In this review, we summarise and discuss recent methods developed for cellular imaging of G-quadruplexes, and the application of experimental genomic approaches to detect G-quadruplexes throughout genomic DNA. In particular, we will discuss the use of engineered small molecules and natural proteins to enable pull-down, ChIP-Seq, ChIP-chip and fluorescence imaging of G-quadruplex structures in cellular DNA.

  1. PBX proteins: much more than Hox cofactors.

    PubMed

    Laurent, Audrey; Bihan, Réjane; Omilli, Francis; Deschamps, Stéphane; Pellerin, Isabelle

    2008-01-01

    Pre-B cell leukaemia transcription factors (PBXs) were originally identified as Hox cofactors, acting within transcriptional regulation complexes to regulate genetic programs during development. Increasing amount of evidence revealed that PBX function is not restricted to a partnership with Hox or homeodomain proteins. Indeed, PBXs are expressed throughout murine embryonic development and are involved in several developmental pathways including Hox-independent mechanisms. This review summarizes what is known about PBX partnerships and proposes to position PBXs as central developmental factors whose role consists of integrating transduction signals, in order to regulate gene expression programs during development.

  2. Genome-wide association study using cellular traits identifies a new regulator of root development in Arabidopsis.

    PubMed

    Meijón, Mónica; Satbhai, Santosh B; Tsuchimatsu, Takashi; Busch, Wolfgang

    2014-01-01

    With the increased availability of high-resolution sequence information, genome-wide association (GWA) studies have become feasible in a number of species. The vast majority of these studies are conducted in human populations, where it is difficult to provide strong evidence for the functional involvement of unknown genes that are identified using GWA. Here we used the model organism Arabidopsis thaliana to combine high-throughput confocal microscopy imaging of traits at the cellular level, GWA and expression analyses to identify genomic regions that are associated with developmental cell-type traits. We identify and characterize a new F-box gene, KUK, that regulates meristem and cell length. We further show that polymorphisms in the coding sequence are the major causes of KUK allele-dependent natural variation in root development. This work demonstrates the feasibility of GWA using cellular traits to identify causal genes for basic biological processes such as development.

  3. Live Cell Discovery of Microbial Vitamin Transport and Enzyme-Cofactor Interactions

    SciTech Connect

    Anderson, Lindsey N.; Koech, Phillip K.; Plymale, Andrew E.; Landorf, Elizabeth V.; Konopka, Allan; Collart, Frank; Lipton, Mary S.; Romine, Margaret F.; Wright, Aaron T.

    2016-02-02

    The rapid completion of microbial genomes is inducing a conundrum in functional gene discovery. Novel methods are critically needed to shorten the gap between characterizing a microbial genome and experimentally validating bioinformatically-predicted functions. Of particular importance are transport mechanisms, used to shuttle nutrients and metabolites across cell mem-branes, such as B vitamins, which are indispensable to metabolic reactions crucial to the survival of diverse microbes ranging from members of environmental microbial communities to human pathogens. Methods to accurately assign function and specificity for a wide range of experimentally unidentified and/or predicted membrane-embedded transport proteins, and characterization of intra-cellular enzyme-cofactor/nutrient associations are needed to enable a significantly improved understanding of microbial biochemis-try and physiology, how microbes associate with others, and how they sense and respond to environmental perturbations. Chemical probes derived from B vitamins B1, B2, and B7 have allowed us to experimentally address the aforementioned needs by identifying B vitamin transporters and intracellular protein-cofactor associations through live cell labeling of the filamentous anoxygenic pho-toheterotroph, Chloroflexus aurantiacus J-10-fl, known for both B vitamin biosynthesis and environmental salvage. Our probes provide a unique opportunity to directly link cellular activity and protein function back to ecosystem and/or host dynamics by iden-tifying B vitamin transport and disposition mechanisms required for survival.

  4. Metabolic Regulation of Histone Acetyltransferases by Endogenous Acyl-CoA Cofactors

    PubMed Central

    Guasch, Laura; Nicklaus, Marc C.; Meier, Jordan L.

    2015-01-01

    SUMMARY The finding that chromatin modifications are sensitive to changes in cellular cofactor levels potentially links altered tumor cell metabolism and gene expression. However, the specific enzymes and metabolites that connect these two processes remain obscure. Characterizing these metabolic-epigenetic axes is critical to understanding how metabolism supports signaling in cancer, and developing therapeutic strategies to disrupt this process. Here, we describe a chemical approach to define the metabolic regulation of lysine acetyltransferase (KAT) enzymes. Using a novel chemoproteomic probe, we identify a previously unreported interaction between fatty acyl-CoAs and KAT enzymes. Further analysis reveals that palmitoyl-CoA is a potent inhibitor of KAT activity and that fatty acyl-CoA precursors reduce cellular acetylation levels. These studies implicate fatty acyl-CoAs as endogenous regulators of histone acetylation, and suggest novel strategies for the investigation and metabolic modulation of epigenetic signaling. PMID:26190825

  5. Chemical Genomics Identifies the PERK-Mediated Unfolded Protein Stress Response as a Cellular Target for Influenza Virus Inhibition

    PubMed Central

    Landeras-Bueno, Sara; Fernández, Yolanda; Falcón, Ana; Oliveros, Juan Carlos

    2016-01-01

    ABSTRACT Influenza A viruses generate annual epidemics and occasional pandemics of respiratory disease with important consequences for human health and the economy. Therefore, a large effort has been devoted to the development of new anti-influenza virus drugs directed to viral targets, as well as to the identification of cellular targets amenable to anti-influenza virus therapy. Here we have addressed the identification of such potential cellular targets by screening collections of drugs approved for human use. We reasoned that screening with a green fluorescent protein-based recombinant replicon system would identify cellular targets involved in virus transcription/replication and/or gene expression and hence address an early stage of virus infection. By using such a strategy, we identified Montelukast (MK) as an inhibitor of virus multiplication. MK inhibited virus gene expression but did not alter viral RNA synthesis in vitro or viral RNA accumulation in vivo. The low selectivity index of MK prevented its use as an antiviral, but it was sufficient to identify a new cellular pathway suitable for anti-influenza virus intervention. By deep sequencing of RNA isolated from mock- and virus-infected human cells, treated with MK or left untreated, we showed that it stimulates the PERK-mediated unfolded protein stress response. The phosphorylation of PERK was partly inhibited in virus-infected cells but stimulated in MK-treated cells. Accordingly, pharmacological inhibition of PERK phosphorylation led to increased viral gene expression, while inhibition of PERK phosphatase reduced viral protein synthesis. These results suggest the PERK-mediated unfolded protein response as a potential cellular target to modulate influenza virus infection. PMID:27094326

  6. High content analysis at single cell level identifies different cellular responses dependent on nanomaterial concentrations.

    PubMed

    Manshian, Bella B; Munck, Sebastian; Agostinis, Patrizia; Himmelreich, Uwe; Soenen, Stefaan J

    2015-09-08

    A mechanistic understanding of nanomaterial (NM) interaction with biological environments is pivotal for the safe transition from basic science to applied nanomedicine. NM exposure results in varying levels of internalized NM in different neighboring cells, due to variances in cell size, cell cycle phase and NM agglomeration. Using high-content analysis, we investigated the cytotoxic effects of fluorescent quantum dots on cultured cells, where all effects were correlated with the concentration of NMs at the single cell level. Upon binning the single cell data into different categories related to NM concentration, this study demonstrates, for the first time, that quantum dots activate both cytoprotective and cytotoxic mechanisms, resulting in a zero net result on the overall cell population, yet with significant effects in cells with higher cellular NM levels. Our results suggest that future NM cytotoxicity studies should correlate NM toxicity with cellular NM numbers on the single cell level, as conflicting mechanisms in particular cell subpopulations are commonly overlooked using classical toxicological methods.

  7. Sparse feature selection methods identify unexpected global cellular response to strontium-containing materials.

    PubMed

    Autefage, Hélène; Gentleman, Eileen; Littmann, Elena; Hedegaard, Martin A B; Von Erlach, Thomas; O'Donnell, Matthew; Burden, Frank R; Winkler, David A; Stevens, Molly M

    2015-04-07

    Despite the increasing sophistication of biomaterials design and functional characterization studies, little is known regarding cells' global response to biomaterials. Here, we combined nontargeted holistic biological and physical science techniques to evaluate how simple strontium ion incorporation within the well-described biomaterial 45S5 bioactive glass (BG) influences the global response of human mesenchymal stem cells. Our objective analyses of whole gene-expression profiles, confirmed by standard molecular biology techniques, revealed that strontium-substituted BG up-regulated the isoprenoid pathway, suggesting an influence on both sterol metabolite synthesis and protein prenylation processes. This up-regulation was accompanied by increases in cellular and membrane cholesterol and lipid raft contents as determined by Raman spectroscopy mapping and total internal reflection fluorescence microscopy analyses and by an increase in cellular content of phosphorylated myosin II light chain. Our unexpected findings of this strong metabolic pathway regulation as a response to biomaterial composition highlight the benefits of discovery-driven nonreductionist approaches to gain a deeper understanding of global cell-material interactions and suggest alternative research routes for evaluating biomaterials to improve their design.

  8. Defining efficient enzyme-cofactor pairs for bioorthogonal profiling of protein methylation

    SciTech Connect

    Islam, Kabirul; Chen, Yuling; Wu, Hong; Bothwell, Ian R.; Blum, Gil J.; Zeng, Hong; Dong, Aiping; Zheng, Weihong; Min, Jinrong; Deng, Haiteng; Luo, Minkui

    2013-11-18

    Protein methyltransferase (PMT)-mediated posttranslational modification of histone and nonhistone substrates modulates stability, localization, and interacting partners of target proteins in diverse cellular contexts. These events play critical roles in normal biological processes and are frequently deregulated in human diseases. In the course of identifying substrates of individual PMTs, bioorthogonal profiling of protein methylation (BPPM) has demonstrated its merits. In this approach, specific PMTs are engineered to process S-adenosyl-L-methionine (SAM) analogs as cofactor surrogates and label their substrates with distinct chemical modifications for target elucidation. Despite the proof-of-concept advancement of BPPM, few efforts have been made to explore its generality. With two cancer-relevant PMTs, EuHMT1 (GLP1/KMT1D) and EuHMT2 (G9a/KMT1C), as models, we defined the key structural features of engineered PMTs and matched SAM analogs that can render the orthogonal enzyme–cofactor pairs for efficient catalysis. Here we have demonstrated that the presence of sulfonium-β-sp2 carbon and flexible, medium-sized sulfonium-δ-substituents are crucial for SAM analogs as BPPM reagents. The bulky cofactors can be accommodated by tailoring the conserved Y1211/Y1154 residues and nearby hydrophobic cavities of EuHMT1/2. Profiling proteome-wide substrates with BPPM allowed identification of >500 targets of EuHMT1/2 with representative targets validated using native EuHMT1/2 and SAM. This finding indicates that EuHMT1/2 may regulate many cellular events previously unrecognized to be modulated by methylation. The present work, therefore, paves the way to a broader application of the BPPM technology to profile methylomes of diverse PMTs and elucidate their downstream functions.

  9. Identifying the Cellular Targets of Drug Action in the Central Nervous System Following Corticosteroid Therapy

    PubMed Central

    2013-01-01

    Corticosteroid (CS) therapy is used widely in the treatment of a range of pathologies, but can delay production of myelin, the insulating sheath around central nervous system nerve fibers. The cellular targets of CS action are not fully understood, that is, “direct” action on cells involved in myelin genesis [oligodendrocytes and their progenitors the oligodendrocyte precursor cells (OPCs)] versus “indirect” action on other neural cells. We evaluated the effects of the widely used CS dexamethasone (DEX) on purified OPCs and oligodendrocytes, employing complementary histological and transcriptional analyses. Histological assessments showed no DEX effects on OPC proliferation or oligodendrocyte genesis/maturation (key processes underpinning myelin genesis). Immunostaining and RT-PCR analyses show that both cell types express glucocorticoid receptor (GR; the target for DEX action), ruling out receptor expression as a causal factor in the lack of DEX-responsiveness. GRs function as ligand-activated transcription factors, so we simultaneously analyzed DEX-induced transcriptional responses using microarray analyses; these substantiated the histological findings, with limited gene expression changes in DEX-treated OPCs and oligodendrocytes. With identical treatment, microglial cells showed profound and global changes post-DEX addition; an unexpected finding was the identification of the transcription factor Olig1, a master regulator of myelination, as a DEX responsive gene in microglia. Our data indicate that CS-induced myelination delays are unlikely to be due to direct drug action on OPCs or oligodendrocytes, and may occur secondary to alterations in other neural cells, such as the immune component. To the best of our knowledge, this is the first comparative molecular and cellular analysis of CS effects in glial cells, to investigate the targets of this major class of anti-inflammatory drugs as a basis for myelination deficits. PMID:24147833

  10. Systems Biology-Based Investigation of Cellular Antiviral Drug Targets Identified by Gene-Trap Insertional Mutagenesis.

    PubMed

    Cheng, Feixiong; Murray, James L; Zhao, Junfei; Sheng, Jinsong; Zhao, Zhongming; Rubin, Donald H

    2016-09-01

    Viruses require host cellular factors for successful replication. A comprehensive systems-level investigation of the virus-host interactome is critical for understanding the roles of host factors with the end goal of discovering new druggable antiviral targets. Gene-trap insertional mutagenesis is a high-throughput forward genetics approach to randomly disrupt (trap) host genes and discover host genes that are essential for viral replication, but not for host cell survival. In this study, we used libraries of randomly mutagenized cells to discover cellular genes that are essential for the replication of 10 distinct cytotoxic mammalian viruses, 1 gram-negative bacterium, and 5 toxins. We herein reported 712 candidate cellular genes, characterizing distinct topological network and evolutionary signatures, and occupying central hubs in the human interactome. Cell cycle phase-specific network analysis showed that host cell cycle programs played critical roles during viral replication (e.g. MYC and TAF4 regulating G0/1 phase). Moreover, the viral perturbation of host cellular networks reflected disease etiology in that host genes (e.g. CTCF, RHOA, and CDKN1B) identified were frequently essential and significantly associated with Mendelian and orphan diseases, or somatic mutations in cancer. Computational drug repositioning framework via incorporating drug-gene signatures from the Connectivity Map into the virus-host interactome identified 110 putative druggable antiviral targets and prioritized several existing drugs (e.g. ajmaline) that may be potential for antiviral indication (e.g. anti-Ebola). In summary, this work provides a powerful methodology with a tight integration of gene-trap insertional mutagenesis testing and systems biology to identify new antiviral targets and drugs for the development of broadly acting and targeted clinical antiviral therapeutics.

  11. Systems Biology-Based Investigation of Cellular Antiviral Drug Targets Identified by Gene-Trap Insertional Mutagenesis

    PubMed Central

    Zhao, Junfei; Sheng, Jinsong; Rubin, Donald H.

    2016-01-01

    Viruses require host cellular factors for successful replication. A comprehensive systems-level investigation of the virus-host interactome is critical for understanding the roles of host factors with the end goal of discovering new druggable antiviral targets. Gene-trap insertional mutagenesis is a high-throughput forward genetics approach to randomly disrupt (trap) host genes and discover host genes that are essential for viral replication, but not for host cell survival. In this study, we used libraries of randomly mutagenized cells to discover cellular genes that are essential for the replication of 10 distinct cytotoxic mammalian viruses, 1 gram-negative bacterium, and 5 toxins. We herein reported 712 candidate cellular genes, characterizing distinct topological network and evolutionary signatures, and occupying central hubs in the human interactome. Cell cycle phase-specific network analysis showed that host cell cycle programs played critical roles during viral replication (e.g. MYC and TAF4 regulating G0/1 phase). Moreover, the viral perturbation of host cellular networks reflected disease etiology in that host genes (e.g. CTCF, RHOA, and CDKN1B) identified were frequently essential and significantly associated with Mendelian and orphan diseases, or somatic mutations in cancer. Computational drug repositioning framework via incorporating drug-gene signatures from the Connectivity Map into the virus-host interactome identified 110 putative druggable antiviral targets and prioritized several existing drugs (e.g. ajmaline) that may be potential for antiviral indication (e.g. anti-Ebola). In summary, this work provides a powerful methodology with a tight integration of gene-trap insertional mutagenesis testing and systems biology to identify new antiviral targets and drugs for the development of broadly acting and targeted clinical antiviral therapeutics. PMID:27632082

  12. Neurotrapping: Cellular Screens to Identify the Neural Substrates of Behavior in Drosophila

    PubMed Central

    White, Benjamin H.; Peabody, Nathan C.

    2009-01-01

    The availability of new tools for manipulating neuronal activity, coupled with the development of increasingly sophisticated techniques for targeting these tools to subsets of cells in living, behaving animals, is permitting neuroscientists to tease apart brain circuits by a method akin to classical mutagenesis. Just as mutagenesis can be used to introduce changes into an organism's DNA to identify the genes required for a given biological process, changes in activity can be introduced into the nervous system to identify the cells required for a given behavior. If the changes are introduced randomly, the cells can be identified without any prior knowledge of their properties. This strategy, which we refer to here as “neurotrapping,” has been implemented most effectively in Drosophila, where transgenes capable of either suppressing or stimulating neuronal activity can be reproducibly targeted to arbitrary subsets of neurons using so-called “enhancer-trap” techniques. By screening large numbers of enhancer-trap lines, experimenters have been able to identify groups of neurons which, when suppressed (or, in some cases, activated), alter a specific behavior. Parsing these groups of neurons to identify the minimal subset required for generating a behavior has proved difficult, but emerging tools that permit refined transgene targeting are increasing the resolution of the screening techniques. Some of the most recent neurotrapping screens have identified physiological substrates of behavior at the single neuron level. PMID:19949456

  13. The Arabidopsis thaliana hypocotyl, a model to identify and study control mechanisms of cellular expansion.

    PubMed

    Boron, Agnieszka Karolina; Vissenberg, Kris

    2014-05-01

    Developmental biology studies in general benefit from model organisms that are well characterized. Arabidopsis thaliana fulfills this criterion and represents one of the best experimental systems to study developmental processes in higher plants. Light is a crucial factor that drives photosynthesis, but that also regulates plant morphogenesis. As the hypocotyl is completely embryonic of origin, its growth occurs solely by expansion of the cells and this process is strongly dependent on the light conditions. In this review, we provide evidence that the hypocotyl serves as ideal model object to study cell expansion mechanisms and its regulation. We focus on the regulation of hypocotyl development by light and highlight the key modulating proteins in this signaling cascade. Downstream of light-signaling, cellular expansion is greatly dependent on specific cell wall depositions, which is related to cortical microtubular (re)arrangements and on composition and/or extensibility of the cell wall. We discuss possible further experimental approaches to broaden our knowledge on hypocotyl development, which will give an outlook on the probable evolution of the field.

  14. Impact of Resolution on Simulation of Closed Mesoscale Cellular Convection Identified by Dynamically Guided Watershed Segmentation

    SciTech Connect

    Martini, Matus N.; Gustafson, William I.; Yang, Qing; Xiao, Heng

    2014-11-18

    Organized mesoscale cellular convection (MCC) is a common feature of marine stratocumulus that forms in response to a balance between mesoscale dynamics and smaller scale processes such as cloud radiative cooling and microphysics. We use the Weather Research and Forecasting model with chemistry (WRF-Chem) and fully coupled cloud-aerosol interactions to simulate marine low clouds during the VOCALS-REx campaign over the southeast Pacific. A suite of experiments with 3- and 9-km grid spacing indicates resolution-dependent behavior. The simulations with finer grid spacing have smaller liquid water paths and cloud fractions, while cloud tops are higher. The observed diurnal cycle is reasonably well simulated. To isolate organized MCC characteristics we develop a new automated method, which uses a variation of the watershed segmentation technique that combines the detection of cloud boundaries with a test for coincident vertical velocity characteristics. This ensures that the detected cloud fields are dynamically consistent for closed MCC, the most common MCC type over the VOCALS-REx region. We demonstrate that the 3-km simulation is able to reproduce the scaling between horizontal cell size and boundary layer height seen in satellite observations. However, the 9-km simulation is unable to resolve smaller circulations corresponding to shallower boundary layers, instead producing invariant MCC horizontal scale for all simulated boundary layers depths. The results imply that climate models with grid spacing of roughly 3 km or smaller may be needed to properly simulate the MCC structure in the marine stratocumulus regions.

  15. A genetic modifier screen identifies multiple genes that interact with Drosophila Rap/Fzr and suggests novel cellular roles.

    PubMed

    Kaplow, Margarita E; Mannava, Laura J; Pimentel, Angel C; Fermin, Hector A; Hyatt, Vanetta J; Lee, John J; Venkatesh, Tadmiri R

    2007-01-01

    In the developing Drosophila eye, Rap/Fzr plays a critical role in neural patterning by regulating the timely exit of precursor cells. Rap/Fzr (Retina aberrant in pattern/Fizzy related) is an activator of the E3 Ubiquitin ligase, the APC (Anaphase Promoting Complex-cyclosome) that facilitates the stage specific proteolytic destruction of mitotic regulators, such as cyclins and cyclin-dependent kinases. To identify novel functional roles of Rap/Fzr, we conducted an F(1) genetic modifier screen to identify genes which interact with the partial-loss-function mutations in rap/fzr. We screened 2741 single P-element, lethal insertion lines and piggyBac lines on the second and third chromosome for dominant enhancers and suppressors of the rough eye phenotype of rap/fzr. From this screen, we have identified 40 genes that exhibit dosage-sensitive interactions with rap/fzr; of these, 31 have previously characterized cellular functions. Seven of the modifiers identified in this study are regulators of cell cycle progression with previously known interactions with rap/fzr. Among the remaining modifiers, 27 encode proteins involved in other cellular functions not directly related to cell-cycle progression. The newly identified variants fall into at least three groups based on their previously known cellular functions: transcriptional regulation, regulated proteolysis, and signal transduction. These results suggest that, in addition to cell cycle regulation, rap/fzr regulates ubiquitin-ligase-mediated protein degradation in the developing nervous system as well as in other tissues.

  16. Emerging roles for tubulin folding cofactors at the centrosome

    PubMed Central

    Fanarraga, Mónica López; Carranza, Gerardo; Castaño, Raquel; Jiménez, Victoria; Villegas, Juan Carlos

    2010-01-01

    Despite its fundamental role in centrosome biology, procentriole formation, both in the canonical and in the de novo replication pathways, remains poorly understood, and the molecular components that are involved in human cells are not well established. We found that one of the tubulin cofactors, TBCD, is localized at centrosomes and the midbody, and is required for spindle organization, cell abscission, centriole formation and ciliogenesis. Our studies have established a molecular link between the centriole and the midbody, demonstrating that this cofactor is also necessary for microtubule retraction during cell abscission. TBCD is the first centriolar protein identified that plays a role in the assembly of both “centriolar rosettes” during early ciliogenesis, and at the procentriole budding site by S/G2, a discovery that directly implicates tubulin cofactors in the cell division, cell migration and cell signaling research fields. PMID:20798813

  17. Protein-protein interaction networks identify targets which rescue the MPP+ cellular model of Parkinson’s disease

    NASA Astrophysics Data System (ADS)

    Keane, Harriet; Ryan, Brent J.; Jackson, Brendan; Whitmore, Alan; Wade-Martins, Richard

    2015-11-01

    Neurodegenerative diseases are complex multifactorial disorders characterised by the interplay of many dysregulated physiological processes. As an exemplar, Parkinson’s disease (PD) involves multiple perturbed cellular functions, including mitochondrial dysfunction and autophagic dysregulation in preferentially-sensitive dopamine neurons, a selective pathophysiology recapitulated in vitro using the neurotoxin MPP+. Here we explore a network science approach for the selection of therapeutic protein targets in the cellular MPP+ model. We hypothesised that analysis of protein-protein interaction networks modelling MPP+ toxicity could identify proteins critical for mediating MPP+ toxicity. Analysis of protein-protein interaction networks constructed to model the interplay of mitochondrial dysfunction and autophagic dysregulation (key aspects of MPP+ toxicity) enabled us to identify four proteins predicted to be key for MPP+ toxicity (P62, GABARAP, GBRL1 and GBRL2). Combined, but not individual, knockdown of these proteins increased cellular susceptibility to MPP+ toxicity. Conversely, combined, but not individual, over-expression of the network targets provided rescue of MPP+ toxicity associated with the formation of autophagosome-like structures. We also found that modulation of two distinct proteins in the protein-protein interaction network was necessary and sufficient to mitigate neurotoxicity. Together, these findings validate our network science approach to multi-target identification in complex neurological diseases.

  18. Protein-protein interaction networks identify targets which rescue the MPP+ cellular model of Parkinson’s disease

    PubMed Central

    Keane, Harriet; Ryan, Brent J.; Jackson, Brendan; Whitmore, Alan; Wade-Martins, Richard

    2015-01-01

    Neurodegenerative diseases are complex multifactorial disorders characterised by the interplay of many dysregulated physiological processes. As an exemplar, Parkinson’s disease (PD) involves multiple perturbed cellular functions, including mitochondrial dysfunction and autophagic dysregulation in preferentially-sensitive dopamine neurons, a selective pathophysiology recapitulated in vitro using the neurotoxin MPP+. Here we explore a network science approach for the selection of therapeutic protein targets in the cellular MPP+ model. We hypothesised that analysis of protein-protein interaction networks modelling MPP+ toxicity could identify proteins critical for mediating MPP+ toxicity. Analysis of protein-protein interaction networks constructed to model the interplay of mitochondrial dysfunction and autophagic dysregulation (key aspects of MPP+ toxicity) enabled us to identify four proteins predicted to be key for MPP+ toxicity (P62, GABARAP, GBRL1 and GBRL2). Combined, but not individual, knockdown of these proteins increased cellular susceptibility to MPP+ toxicity. Conversely, combined, but not individual, over-expression of the network targets provided rescue of MPP+ toxicity associated with the formation of autophagosome-like structures. We also found that modulation of two distinct proteins in the protein-protein interaction network was necessary and sufficient to mitigate neurotoxicity. Together, these findings validate our network science approach to multi-target identification in complex neurological diseases. PMID:26608097

  19. Cellular heterogeneity in cultured human chondrocytes identified by antibodies specific for alpha 2(XI) collagen chains

    PubMed Central

    1989-01-01

    Collagen type XI is a component of hyaline cartilage consisting of alpha 1(XI), alpha 2(XI), and alpha 3(XI) chains; with 5-10% of the total collagen content, it is a minor but significant component next to type II collagen, but its function and precise localization in cartilaginous tissues is still unclear. Owing to the homology of the alpha 3(XI) and alpha 1(II) collagen chains, attempts to prepare specific antibodies to native type XI collagen have been unsuccessful in the past. In this study, we report on the preparation and use for immunohistochemistry of a polyclonal antibody specific for alpha 2(XI) denatured collagen chains. The antibody was prepared by immunization with the isolated alpha 2(XI) chain and reacts neither with native type XI collagen nor type I, II, V, or IX by ELISA or immunoblotting, nor with alpha 1(XI) or alpha 3(XI), but with alpha 2(XI) chains. Using this antibody, it was possible to specifically localize alpha 2(XI) in cartilage by pretreating tissue sections with 6 M urea. In double immunofluorescence staining experiments, the distribution of alpha 2(XI) as indicative for type XI collagen in fetal bovine and human cartilage was compared with that of type II collagen, using a monoclonal antibody to alpha 1(II). Type XI collagen was found throughout the matrix of hyaline cartilage. However, owing to cross- reactivity of the monoclonal anti-alpha 1(II) with alpha 3(XI), both antibodies produced the same staining pattern. Cellular heterogeneity was, however, detected in monolayer cultures of human chondrocytes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2670958

  20. Chemical Genomics Identifies the PERK-Mediated Unfolded Protein Stress Response as a Cellular Target for Influenza Virus Inhibition.

    PubMed

    Landeras-Bueno, Sara; Fernández, Yolanda; Falcón, Ana; Oliveros, Juan Carlos; Ortín, Juan

    2016-04-19

    Influenza A viruses generate annual epidemics and occasional pandemics of respiratory disease with important consequences for human health and the economy. Therefore, a large effort has been devoted to the development of new anti-influenza virus drugs directed to viral targets, as well as to the identification of cellular targets amenable to anti-influenza virus therapy. Here we have addressed the identification of such potential cellular targets by screening collections of drugs approved for human use. We reasoned that screening with a green fluorescent protein-based recombinant replicon system would identify cellular targets involved in virus transcription/replication and/or gene expression and hence address an early stage of virus infection. By using such a strategy, we identified Montelukast (MK) as an inhibitor of virus multiplication. MK inhibited virus gene expression but did not alter viral RNA synthesis in vitro or viral RNA accumulation in vivo The low selectivity index of MK prevented its use as an antiviral, but it was sufficient to identify a new cellular pathway suitable for anti-influenza virus intervention. By deep sequencing of RNA isolated from mock- and virus-infected human cells, treated with MK or left untreated, we showed that it stimulates the PERK-mediated unfolded protein stress response. The phosphorylation of PERK was partly inhibited in virus-infected cells but stimulated in MK-treated cells. Accordingly, pharmacological inhibition of PERK phosphorylation led to increased viral gene expression, while inhibition of PERK phosphatase reduced viral protein synthesis. These results suggest the PERK-mediated unfolded protein response as a potential cellular target to modulate influenza virus infection. Influenza A viruses are responsible for annual epidemics and occasional pandemics with important consequences for human health and the economy. The unfolded protein response is a defense mechanism fired by cells when the demand of protein

  1. Cofactor engineering for advancing chemical biotechnology.

    PubMed

    Wang, Yipeng; San, Ka-Yiu; Bennett, George N

    2013-12-01

    Cofactors provide redox carriers for biosynthetic reactions, catabolic reactions and act as important agents in transfer of energy for the cell. Recent advances in manipulating cofactors include culture conditions or additive alterations, genetic modification of host pathways for increased availability of desired cofactor, changes in enzyme cofactor specificity, and introduction of novel redox partners to form effective circuits for biochemical processes and biocatalysts. Genetic strategies to employ ferredoxin, NADH and NADPH most effectively in natural or novel pathways have improved yield and efficiency of large-scale processes for fuels and chemicals and have been demonstrated with a variety of microbial organisms.

  2. Nuclear Morphometry Identifies a Distinct Aggressive Cellular Phenotype in Cutaneous Squamous Cell Carcinoma

    PubMed Central

    Glazer, Evan S.; Bartels, Peter H.; Prasad, Anil R.; Yozwiak, Michael L.; Bartels, Hubert G.; Einspahr, Janine G.; Alberts, David S.; Krouse, Robert S.

    2011-01-01

    By identifying aggressive cutaneous squamous cell carcinoma (cSCC) in patients who are at high risk for recurrences or second primaries after resection, intensive surveillance and therapy may decrease morbidity and mortality. We investigated the role of nuclear morphometry (karyometry) in differentiating between aggressive and nonaggressive cSCC. We retrospectively analyzed cSCC lesions from 40 male patients. 22 patients had evidence of aggressive cSCC (local/regional recurrence or a second primary cSCC), and 18 patients were identified with similar ages and sites of disease as control patients with nonaggressive cSCC (no evidence of recurrence, metastasis, or second primary). We performed karyometric analysis to identify nuclear features that discriminate between aggressive and nonaggressive cSCC nuclei. We used statistically significant differences (Kruskal-Wallis test P < 0.0001) to compose a quantitative aggressive classification score (proportion of aggressive nuclei from 0% to 100%). For comparisons, we used Fisher’s exact test or Student t test. The mean age was 79 ± 7 years for aggressive cSCC and 80 ± 9 years for nonaggressive cSCC (P = 0.66). We analyzed a mean of 96 nuclei in each group. The mean classification score for aggressive cSCC was significantly higher (69% ± 6%) than for nonaggressive cSCC (28% ± 5%, P = 0.00002). Overall, the classification score accurately categorized 80% of our patients (P = 0.0004). In most patients, karyometry differentiated between aggressive and nonaggressive cSCC. We found that classification scores, which provide information on individual lesions, could be used for risk stratification. PMID:21636541

  3. Nuclear morphometry identifies a distinct aggressive cellular phenotype in cutaneous squamous cell carcinoma.

    PubMed

    Glazer, Evan S; Bartels, Peter H; Prasad, Anil R; Yozwiak, Michael L; Bartels, Hubert G; Einspahr, Janine G; Alberts, David S; Krouse, Robert S

    2011-11-01

    By identifying aggressive cutaneous squamous cell carcinoma (cSCC) in patients who are at high risk for recurrences or second primaries after resection, intensive surveillance and therapy may decrease morbidity and mortality. We investigated the role of nuclear morphometry (karyometry) in differentiating between aggressive and nonaggressive cSCC. We retrospectively analyzed cSCC lesions from 40 male patients. Twenty-two patients had evidence of aggressive cSCC (local/regional recurrence or a second primary cSCC), and 18 patients were identified with similar ages and sites of disease as control patients with nonaggressive cSCC (no evidence of recurrence, metastasis, or second primary). We carried out karyometric analysis to identify nuclear features that discriminate between aggressive and nonaggressive cSCC nuclei. We used statistically significant differences (Kruskal-Wallis test, P < 0.0001) to compose a quantitative aggressive classification score (proportion of aggressive nuclei from 0% to 100%). For comparisons, we used Fisher's exact test or Student's t test. The mean age was 79 ± 7 years for aggressive cSCC and 80 ± 9 years for nonaggressive cSCC (P = 0.66). We analyzed a mean of 96 nuclei in each group. The mean classification score for aggressive cSCC was significantly higher (69% ± 6%) than for nonaggressive cSCC (28% ± 5%, P = 0.00002). Overall, the classification score accurately categorized 80% of our patients (P = 0.0004). In most patients, karyometry differentiated between aggressive and nonaggressive cSCC. We found that classification scores, which provide information on individual lesions, could be used for risk stratification.

  4. Analysis of individual cells identifies cell-to-cell variability following induction of cellular senescence.

    PubMed

    Wiley, Christopher D; Flynn, James M; Morrissey, Christapher; Lebofsky, Ronald; Shuga, Joe; Dong, Xiao; Unger, Marc A; Vijg, Jan; Melov, Simon; Campisi, Judith

    2017-10-01

    Senescent cells play important roles in both physiological and pathological processes, including cancer and aging. In all cases, however, senescent cells comprise only a small fraction of tissues. Senescent phenotypes have been studied largely in relatively homogeneous populations of cultured cells. In vivo, senescent cells are generally identified by a small number of markers, but whether and how these markers vary among individual cells is unknown. We therefore utilized a combination of single-cell isolation and a nanofluidic PCR platform to determine the contributions of individual cells to the overall gene expression profile of senescent human fibroblast populations. Individual senescent cells were surprisingly heterogeneous in their gene expression signatures. This cell-to-cell variability resulted in a loss of correlation among the expression of several senescence-associated genes. Many genes encoding senescence-associated secretory phenotype (SASP) factors, a major contributor to the effects of senescent cells in vivo, showed marked variability with a subset of highly induced genes accounting for the increases observed at the population level. Inflammatory genes in clustered genomic loci showed a greater correlation with senescence compared to nonclustered loci, suggesting that these genes are coregulated by genomic location. Together, these data offer new insights into how genes are regulated in senescent cells and suggest that single markers are inadequate to identify senescent cells in vivo. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  5. Molybdenum cofactor and human disease.

    PubMed

    Schwarz, Guenter

    2016-04-01

    Four molybdenum-dependent enzymes are known in humans, each harboring a pterin-based molybdenum cofactor (Moco) in the active site. They catalyze redox reactions using water as oxygen acceptor or donator. Moco is synthesized by a conserved biosynthetic pathway. Moco deficiency results in a severe inborn error of metabolism causing often early childhood death. Disease-causing symptoms mainly go back to the lack of sulfite oxidase (SO) activity, an enzyme in cysteine catabolism. Besides their name-giving functions, Mo-enzymes have been recognized to catalyze novel reactions, including the reduction of nitrite to nitric oxide. In this review we cover the biosynthesis of Moco, key features of Moco-enzymes and focus on their deficiency. Underlying disease mechanisms as well as treatment options will be discussed. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Chemical proteomics approaches for identifying the cellular targets of natural products.

    PubMed

    Wright, M H; Sieber, S A

    2016-05-04

    Covering: 2010 up to 2016Deconvoluting the mode of action of natural products and drugs remains one of the biggest challenges in chemistry and biology today. Chemical proteomics is a growing area of chemical biology that seeks to design small molecule probes to understand protein function. In the context of natural products, chemical proteomics can be used to identify the protein binding partners or targets of small molecules in live cells. Here, we highlight recent examples of chemical probes based on natural products and their application for target identification. The review focuses on probes that can be covalently linked to their target proteins (either via intrinsic chemical reactivity or via the introduction of photocrosslinkers), and can be applied "in situ" - in living systems rather than cell lysates. We also focus here on strategies that employ a click reaction, the copper-catalysed azide-alkyne cycloaddition reaction (CuAAC), to allow minimal functionalisation of natural product scaffolds with an alkyne or azide tag. We also discuss 'competitive mode' approaches that screen for natural products that compete with a well-characterised chemical probe for binding to a particular set of protein targets. Fuelled by advances in mass spectrometry instrumentation and bioinformatics, many modern strategies are now embracing quantitative proteomics to help define the true interacting partners of probes, and we highlight the opportunities this rapidly evolving technology provides in chemical proteomics. Finally, some of the limitations and challenges of chemical proteomics approaches are discussed.

  7. Mitochondrial Membrane Potential Identifies Cells with Enhanced Stemness for Cellular Therapy.

    PubMed

    Sukumar, Madhusudhanan; Liu, Jie; Mehta, Gautam U; Patel, Shashank J; Roychoudhuri, Rahul; Crompton, Joseph G; Klebanoff, Christopher A; Ji, Yun; Li, Peng; Yu, Zhiya; Whitehill, Greg D; Clever, David; Eil, Robert L; Palmer, Douglas C; Mitra, Suman; Rao, Mahadev; Keyvanfar, Keyvan; Schrump, David S; Wang, Ena; Marincola, Francesco M; Gattinoni, Luca; Leonard, Warren J; Muranski, Pawel; Finkel, Toren; Restifo, Nicholas P

    2016-01-12

    Long-term survival and antitumor immunity of adoptively transferred CD8(+) T cells is dependent on their metabolic fitness, but approaches to isolate therapeutic T cells based on metabolic features are not well established. Here we utilized a lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM) to identify and isolate metabolically robust T cells based on their mitochondrial membrane potential (ΔΨm). Comprehensive metabolomic and gene expression profiling demonstrated global features of improved metabolic fitness in low-ΔΨm-sorted CD8(+) T cells. Transfer of these low-ΔΨm T cells was associated with superior long-term in vivo persistence and an enhanced capacity to eradicate established tumors compared with high-ΔΨm cells. Use of ΔΨm-based sorting to enrich for cells with superior metabolic features was observed in CD8(+), CD4(+) T cell subsets, and long-term hematopoietic stem cells. This metabolism-based approach to cell selection may be broadly applicable to therapies involving the transfer of HSC or lymphocytes for the treatment of viral-associated illnesses and cancer.

  8. Identified Cellular Correlates of Neocortical Ripple and High-Gamma Oscillations during Spindles of Natural Sleep.

    PubMed

    Averkin, Robert G; Szemenyei, Viktor; Bordé, Sándor; Tamás, Gábor

    2016-11-23

    Ultra-high-frequency network events in the hippocampus are instrumental in a dialogue with the neocortex during memory formation, but the existence of transient ∼200 Hz network events in the neocortex is not clear. Our recordings from neocortical layer II/III of freely behaving rats revealed field potential events at ripple and high-gamma frequencies repeatedly occurring at troughs of spindle oscillations during sleep. Juxtacellular recordings identified subpopulations of fast-spiking, parvalbumin-containing basket cells with epochs of firing at ripple (∼200 Hz) and high-gamma (∼120 Hz) frequencies detected during spindles and centered with millisecond precision at the trough of spindle waves in phase with field potential events but phase shifted relative to pyramidal cell firing. The results suggest that basket cell subpopulations are involved in spindle-nested, high-frequency network events that hypothetically provide repeatedly occurring neocortical temporal reference states potentially involved in mnemonic processes.

  9. Chemical proteomics approaches for identifying the cellular targets of natural products

    PubMed Central

    Sieber, S. A.

    2016-01-01

    Covering: 2010 up to 2016 Deconvoluting the mode of action of natural products and drugs remains one of the biggest challenges in chemistry and biology today. Chemical proteomics is a growing area of chemical biology that seeks to design small molecule probes to understand protein function. In the context of natural products, chemical proteomics can be used to identify the protein binding partners or targets of small molecules in live cells. Here, we highlight recent examples of chemical probes based on natural products and their application for target identification. The review focuses on probes that can be covalently linked to their target proteins (either via intrinsic chemical reactivity or via the introduction of photocrosslinkers), and can be applied “in situ” – in living systems rather than cell lysates. We also focus here on strategies that employ a click reaction, the copper-catalysed azide–alkyne cycloaddition reaction (CuAAC), to allow minimal functionalisation of natural product scaffolds with an alkyne or azide tag. We also discuss ‘competitive mode’ approaches that screen for natural products that compete with a well-characterised chemical probe for binding to a particular set of protein targets. Fuelled by advances in mass spectrometry instrumentation and bioinformatics, many modern strategies are now embracing quantitative proteomics to help define the true interacting partners of probes, and we highlight the opportunities this rapidly evolving technology provides in chemical proteomics. Finally, some of the limitations and challenges of chemical proteomics approaches are discussed. PMID:27098809

  10. Sub-cellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas

    PubMed Central

    Hong-Hermesdorf, Anne; Miethke, Marcus; Gallaher, Sean D; Kropat, Janette; Dodani, Sheel C; Chan, Jefferson; Barupala, Dulmini; Domaille, Dylan W; Shirasaki, Dyna I; Loo, Joseph A; Weber, Peter K; Pett-Ridge, Jennifer; Stemmler, Timothy L; Chang, Christopher J; Merchant, Sabeeha S

    2014-01-01

    We identified a Cu accumulating structure with a dynamic role in intracellular Cu homeostasis. During Zn limitation, Chlamydomonas reinhardtii hyperaccumulated Cu, dependent on the nutritional Cu sensor CRR1, but was functionally Cu-deficient. Visualization of intracellular Cu revealed major Cu accumulation sites coincident with electron-dense structures that stained positive for low pH and polyphosphate, suggesting that they are lysosome-related organelles. NanoSIMS showed colocalization of Ca and Cu, and X-ray absorption spectroscopy (XAS) was consistent with Cu+ accumulation in an ordered structure. Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures. Cu isotope labeling demonstrated that sequestered Cu+ became bio-available for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1. Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mis-metallation during Zn deficiency and enabling efficient cuproprotein (re)-metallation upon Zn resupply. PMID:25344811

  11. Chemical Screens Identify Drugs that Enhance or Mitigate Cellular Responses to Antibody-Toxin Fusion Proteins

    PubMed Central

    Guha, Rajarshi; Simon, Nathan; Pasetto, Matteo; Keller, Jonathan; Huang, Manjie; Angelus, Evan; Pastan, Ira; Ferrer, Marc; FitzGerald, David J.; Thomas, Craig J.

    2016-01-01

    The intersection of small molecular weight drugs and antibody-based therapeutics is rarely studied in large scale. Both types of agents are currently part of the cancer armamentarium. However, very little is known about how to combine them in optimal ways. Immunotoxins are antibody-toxin gene fusion proteins engineered to target cancer cells via antibody binding to surface antigens. For fusion proteins derived from Pseudomonas exotoxin (PE), potency relies on the enzymatic domain of the toxin which catalyzes the ADP-ribosylation of EF2 causing inhibition of protein synthesis leading to cell death. Candidate immunotoxins have demonstrated clear value in clinical trials but generally have not been curative as single agents. Therefore we undertook three screens to discover effective combinations that could act synergistically. From the MIPE-3 library of compounds we identified various enhancers of immunotoxin action and at least one major class of inhibitor. Follow-up experiments confirmed the screening data and suggested that immunotoxins when administered with everolimus or nilotinib exhibit favorable combinatory activity and would be candidates for preclinical development. Mechanistic studies revealed that everolimus-immunotoxin combinations acted synergistically on elements of the protein synthetic machinery, including S61 kinase and 4E-BP1 of the mTORC1 pathway. Conversely, PARP inhibitors antagonized immunotoxins and also blocked the toxicity due to native ADP-ribosylating toxins. Thus, our goal of investigating a chemical library was justified based on the identification of several approved compounds that could be developed preclinically as ‘enhancers’ and at least one class of mitigator to be avoided. PMID:27556570

  12. Identifying apoptosis-evasion proteins/pathways in human hepatoma cells via induction of cellular hormesis by UV irradiation.

    PubMed

    Hsieh, Sen-Yung; Hsu, Chih-Yun; He, Jung-Ru; Liu, Chiung-Liang; Lo, Shao-Jung; Chen, Ying-Ching; Huang, Hui-Yu

    2009-08-01

    Evading apoptosis is pivotal in both of carcinogenesis and resistance to anticancer therapy. We investigated the molecules and pathways of apoptosis evasion in human hepatoma cells by irradiating hepatoma cells with optimized UV (so-called "hormetic responses"). Proteins and pathways related to hormetic responses were identified via proteomic approaches followed by reconstruction of function-networks. Of the 2326 defined protein spots, 42 distinct proteins significantly changed their expression. Eleven hormetic response proteins (HINT1, PHB, CTSD, ANXA1, LGASL1, TPT1, NPM, PRDX2, UCHL1, CERK, and C1QBP) were involved in 5 death-regulatory pathways, including the p53-dependent apoptotic pathway, protein ubiquinization, cellular redox, calcium-mediated signaling pathway, and sphingomyelin-metabolism pathway. Knockdown of HINT1 expression via RNA interference increased tumor cell resistance to apoptosis induction, while silencing NPM, UCHL1, or CERK greatly sensitized tumor cells to apoptosis induction. In conclusion, NPM, UCHL1, and CERK act as apoptosis-evasion proteins that may serve as therapeutic targets for hepatoma. Silencing their expression would increase therapeutic efficacy, thereby reducing the corresponding doses and side-effects of anticancer therapy. This model of induction of cellular hormetic responses to identify apoptosis-evasion molecules/pathways via proteomic approaches can be applied to other modalities of anticancer therapy.

  13. Engineering redox balance through cofactor systems.

    PubMed

    Chen, Xiulai; Li, Shubo; Liu, Liming

    2014-06-01

    Redox balance plays an important role in the production of enzymes, pharmaceuticals, and chemicals. To meet the demands of industrial production, it is desirable that microbes maintain a maximal carbon flux towards target metabolites with no fluctuations in redox. This requires functional cofactor systems that support dynamic homeostasis between different redox states or functional stability in a given redox state. Redox balance can be achieved by improving the self-balance of a cofactor system, regulating the substrate balance of a cofactor system, and engineering the synthetic balance of a cofactor system. This review summarizes how cofactor systems can be manipulated to improve redox balance in microbes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. The human immunodeficiency virus (HIV) Rev-binding protein (HRB) is a co-factor for HIV-1 Nef-mediated CD4 downregulation.

    PubMed

    Landi, Alessia; Timermans, Cristina Garcia; Naessens, Evelien; Vanderstraeten, Hanne; Stove, Veronique; Verhasselt, Bruno

    2016-03-01

    Human immunodeficiency virus type 1 (HIV-1)-mediated CD4 downregulation is an important determinant of viral replication in vivo. Research on cellular co-factors involved in this process could lead to the identification of potential therapeutic targets. We found that CD4 surface levels were significantly higher in HIV-1-infected cells knocked-down for the HIV Rev-binding protein (HRB) compared with control cells. HRB knock-down affected CD4 downregulation induced by Nef but not by HIV-1 Vpu. Interestingly, the knock-down of the related protein HRBL (HRB-like), but not of the HRB interaction partner EPS15 (epidermal growth factor receptor pathway substrate 15), increased CD4 levels in Vpu-expressing cells significantly. Both of these proteins are known to be involved in HIV-1-mediated CD4 downregulation as co-factors of HIV-1 Nef. These results identify HRB as a previously unknown co-factor for HIV-1 Nef-mediated CD4 downregulation and highlight differences with the related protein HRBL, which affects the CD4 downregulation in a dual role as co-factor of both HIV-1 Nef and Vpu.

  15. Multicolor flow cytometry-based cellular phenotyping identifies osteoprogenitors and inflammatory cells in the osteoarthritic subchondral bone marrow compartment.

    PubMed

    Pippenger, B E; Duhr, R; Muraro, M G; Pagenstert, G I; Hügle, T; Geurts, J

    2015-11-01

    The cellular component of subchondral bone is thought to be responsible for aberrant bone remodeling in osteoarthritis (OA). Direct phenotypical analysis of the cellular compartment is critical to better understand the OA disease process. This study provides proof-of-principle for flow cytometry-based phenotyping of isolated subchondral trabecular bone (STB) marrow cells without prior use of cell culture techniques. Tibial plateaus were obtained from OA patients undergoing total knee arthroplasty. Subchondral bone chips were digested with collagenase IA and single cell suspensions were directly phenotyped using flow cytometry. Cells were analyzed for the expression of alkaline phosphatase (ALP) as osteoblast/osteoprogenitor marker and monocyte/macrophage markers (CD14, CD68, HLA-DR, CD115). MTT staining revealed abundant viable cells in the bone marrow compartment of STB prior to digestion, which were efficiently released by collagenase. Within the CD45-negative cell fraction, approximately 20% of the cells were positive for the early osteoblast/osteoprogenitor marker ALP. Within the CD45+ hematopoietic cell fraction, the majority of cells were of monocytic origin (>80%) displaying strong surface expression of CD14. Discreet macrophage populations (CD14+/HLA-DR+/CD68+) and putative osteoclast progenitors (CD45+/HLA-DR-/CD115+) were unequivocally identified. Osteoblast, macrophage and osteoclast progenitor presence in the subchondral bone unit (SBU) was confirmed by (immuno)histochemical staining for osteocalcin, CD68 and tartrate-resistant acid phosphatase, respectively. Flow cytometric analysis is a valuable methodology to study the cellular compartment of STB marrow. This method provides a proof of principle that the whole resident cell population can be directly phenotypically characterized without the prior use of cell culture techniques. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  16. General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH

    PubMed Central

    Brinkmann-Chen, Sabine; Flock, Tilman; Cahn, Jackson K. B.; Snow, Christopher D.; Brustad, Eric M.; McIntosh, John A.; Meinhold, Peter; Zhang, Liang; Arnold, Frances H.

    2013-01-01

    To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymes having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. High-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch. PMID:23776225

  17. General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH.

    PubMed

    Brinkmann-Chen, Sabine; Flock, Tilman; Cahn, Jackson K B; Snow, Christopher D; Brustad, Eric M; McIntosh, John A; Meinhold, Peter; Zhang, Liang; Arnold, Frances H

    2013-07-02

    To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymes having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. High-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.

  18. General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH

    SciTech Connect

    Brinkmann-Chen, Sabine; Flock, Tilman; Cahn, Jackson K. B.; Snow, Christopher D.; Brustad, Eric M.; McIntosh, John A.; Meinhold, Peter; Zhang, Liang; Arnold, Frances H.

    2013-06-17

    To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymes having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. As a result, high-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.

  19. General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH

    DOE PAGES

    Brinkmann-Chen, Sabine; Flock, Tilman; Cahn, Jackson K. B.; ...

    2013-06-17

    To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymesmore » having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. As a result, high-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.« less

  20. Inhibition of chymotrypsin by heparin cofactor II.

    PubMed Central

    Church, F C; Noyes, C M; Griffith, M J

    1985-01-01

    Human heparin cofactor II is a plasma protein that is known to inhibit thrombin. The rate of thrombin inhibition by heparin cofactor II is accelerated (greater than or equal to 1000-fold) in the presence of the glycosaminoglycans, heparin and dermatan sulfate. We have found that chymotrypsin A alpha is also inhibited by heparin cofactor II with a second-order rate constant value of 1.8 X 10(6) M-1 X min-1 at pH 8.0 and 25 degrees C. However, there was no measurable effect of heparin or dermatan sulfate on the rate of chymotrypsin inhibition. Arginine-modified heparin cofactor II showed a comparable percentage loss of both antichymotrypsin and antithrombin activities. Heparin cofactor II and chymotrypsin formed a stable complex with a Mr value near 90,000 when analyzed by NaDodSO4/polyacrylamide gel electrophoresis; this suggests a 1:1 reaction stoichiometry. The chymotrypsin cleavage site in heparin cofactor II was the same as that for thrombin, and primary structure analysis of the inhibitor showed a P'1-P'8 sequence of Ser-Thr-Gln-Val-Arg-Phe-Thr-Val ... . The results indicate that, in contrast to alpha 1-antichymotrypsin, which does not inhibit trypsin-like enzymes, including thrombin, heparin cofactor II can effectively inhibit both thrombin and chymotrypsin. PMID:3863104

  1. EPR monitored redox titration of the cofactors of Saccharomyces cerevisiae Nar1.

    PubMed

    Hagedoorn, Peter-Leon; van der Weel, Laura; Hagen, Wilfred R

    2014-11-26

    Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state. The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor. A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 µM. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.

  2. EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

    PubMed Central

    Hagedoorn, Peter-Leon; van der Weel, Laura; Hagen, Wilfred R.

    2014-01-01

    Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state. The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor. A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 µM. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor. PMID:25490157

  3. The AAA+ ATPase p97, a cellular multitool.

    PubMed

    Stach, Lasse; Freemont, Paul S

    2017-08-17

    The AAA+ (ATPases associated with diverse cellular activities) ATPase p97 is essential to a wide range of cellular functions, including endoplasmic reticulum-associated degradation, membrane fusion, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and chromatin-associated processes, which are regulated by ubiquitination. p97 acts downstream from ubiquitin signaling events and utilizes the energy from ATP hydrolysis to extract its substrate proteins from cellular structures or multiprotein complexes. A multitude of p97 cofactors have evolved which are essential to p97 function. Ubiquitin-interacting domains and p97-binding domains combine to form bi-functional cofactors, whose complexes with p97 enable the enzyme to interact with a wide range of ubiquitinated substrates. A set of mutations in p97 have been shown to cause the multisystem proteinopathy inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia. In addition, p97 inhibition has been identified as a promising approach to provoke proteotoxic stress in tumors. In this review, we will describe the cellular processes governed by p97, how the cofactors interact with both p97 and its ubiquitinated substrates, p97 enzymology and the current status in developing p97 inhibitors for cancer therapy. © 2017 The Author(s).

  4. Genome-wide siRNA screen identifies the retromer as a cellular entry factor for human papillomavirus

    PubMed Central

    Lipovsky, Alex; Popa, Andreea; Pimienta, Genaro; Wyler, Michael; Bhan, Ashima; Kuruvilla, Leena; Guie, Marie-Aude; Poffenberger, Adrian C.; Nelson, Christian D. S.; Atwood, Walter J.; DiMaio, Daniel

    2013-01-01

    Despite major advances in our understanding of many aspects of human papillomavirus (HPV) biology, HPV entry is poorly understood. To identify cellular genes required for HPV entry, we conducted a genome-wide screen for siRNAs that inhibited infection of HeLa cells by HPV16 pseudovirus. Many retrograde transport factors were required for efficient infection, including multiple subunits of the retromer, which initiates retrograde transport from the endosome to the trans-Golgi network (TGN). The retromer has not been previously implicated in virus entry. Furthermore, HPV16 capsid proteins arrive in the TGN/Golgi in a retromer-dependent fashion during entry, and incoming HPV proteins form a stable complex with retromer subunits. We propose that HPV16 directly engages the retromer at the early or late endosome and traffics to the TGN/Golgi via the retrograde pathway during cell entry. These results provide important insights into HPV entry, identify numerous potential antiviral targets, and suggest that the role of the retromer in infection by other viruses should be assessed. PMID:23569269

  5. Genome-wide siRNA screen identifies the retromer as a cellular entry factor for human papillomavirus.

    PubMed

    Lipovsky, Alex; Popa, Andreea; Pimienta, Genaro; Wyler, Michael; Bhan, Ashima; Kuruvilla, Leena; Guie, Marie-Aude; Poffenberger, Adrian C; Nelson, Christian D S; Atwood, Walter J; DiMaio, Daniel

    2013-04-30

    Despite major advances in our understanding of many aspects of human papillomavirus (HPV) biology, HPV entry is poorly understood. To identify cellular genes required for HPV entry, we conducted a genome-wide screen for siRNAs that inhibited infection of HeLa cells by HPV16 pseudovirus. Many retrograde transport factors were required for efficient infection, including multiple subunits of the retromer, which initiates retrograde transport from the endosome to the trans-Golgi network (TGN). The retromer has not been previously implicated in virus entry. Furthermore, HPV16 capsid proteins arrive in the TGN/Golgi in a retromer-dependent fashion during entry, and incoming HPV proteins form a stable complex with retromer subunits. We propose that HPV16 directly engages the retromer at the early or late endosome and traffics to the TGN/Golgi via the retrograde pathway during cell entry. These results provide important insights into HPV entry, identify numerous potential antiviral targets, and suggest that the role of the retromer in infection by other viruses should be assessed.

  6. Synthesis, Delivery and Regulation of Eukaryotic Heme and Fe-S Cluster Cofactors

    PubMed Central

    Barupala, Dulmini P.; Dzul, Stephen P.; Riggs-Gelasco, Pamela Jo; Stemmler, Timothy L.

    2016-01-01

    In humans, the bulk of iron in the body (over 75%) is directed towards heme- or Fe-S cluster cofactor synthesis, and the complex, highly regulated pathways in place to accomplish biosynthesis have evolved to safely assemble and load these cofactors into apoprotein partners. In eukaryotes, heme biosynthesis is both initiated and finalized within the mitochondria, while cellular Fe-S cluster assembly is controlled by correlated pathways both within the mitochondria and within the cytosol. Iron plays a vital role in a wide array of metabolic processes and defects in iron cofactor assembly leads to human diseases. This review describes progress towards our molecular-level understanding of cellular heme and Fe-S cluster biosynthesis, focusing on the regulation and mechanistic details that are essential for understanding human disorders related to the breakdown in these essential pathways. PMID:26785297

  7. Myocardial Gene Expression Profiling to Predict and Identify Cardiac Allograft Acute Cellular Rejection: The GET-Study

    PubMed Central

    Bodez, Diane; Hocini, Hakim; Tchitchek, Nicolas; Tisserand, Pascaline; Benhaiem, Nicole; Barau, Caroline; Kharoubi, Mounira; Guellich, Aziz; Guendouz, Soulef; Radu, Costin; Couetil, Jean-Paul; Ghaleh, Bijan; Dubois-Randé, Jean-Luc; Teiger, Emmanuel; Hittinger, Luc

    2016-01-01

    Aims Serial invasive endomyocardial biopsies (EMB) remain the gold standard for acute cellular rejection (ACR) diagnosis. However histological grading has several limitations. We aimed to explore the value of myocardial Gene Expression Profiling (GEP) for diagnosing and identifying predictive biomarkers of ACR. Methods A case-control study nested within a retrospective heart transplant patients cohort included 126 patients with median (IQR) age 50 (41–57) years and 111 (88%) males. Among 1157 EMB performed, 467 were eligible (i.e, corresponding to either ISHLT grade 0 or ≥3A), among which 36 were selected for GEP according to the grading: 0 (CISHLT, n = 13); rejection ≥3A (RISHLT, n = 13); 0 one month before ACR (BRISHLT, n = 10). Results We found 294 genes differentially expressed between CISHLT and RISHLT, mainly involved in immune activation, and inflammation. Hierarchical clustering showed a clear segregation of CISHLT and RISHLT groups and heterogeneity of GEP within RISHLT. All EMB presented immune activation, but some RISHLT EMB were strongly subject to inflammation, whereas others, closer to CISHLT, were characterized by structural modifications with lower inflammation level. We identified 15 probes significantly different between BRISHLT and CISHLT, including the gene of the muscular protein TTN. This result suggests that structural alterations precede inflammation in ACR. Linear Discriminant Analysis based on these 15 probes was able to identify the histological status of every 36 samples. Conclusion Myocardial GEP is a helpful method to accurately diagnose ACR, and predicts rejection one month before its histological occurrence. These results should be considered in cardiac allograft recipients’ care. PMID:27898719

  8. The biosynthesis of the molybdenum cofactors.

    PubMed

    Mendel, Ralf R; Leimkühler, Silke

    2015-03-01

    The biosynthesis of the molybdenum cofactors (Moco) is an ancient, ubiquitous, and highly conserved pathway leading to the biochemical activation of molybdenum. Moco is the essential component of a group of redox enzymes, which are diverse in terms of their phylogenetic distribution and their architectures, both at the overall level and in their catalytic geometry. A wide variety of transformations are catalyzed by these enzymes at carbon, sulfur and nitrogen atoms, which include the transfer of an oxo group or two electrons to or from the substrate. More than 50 molybdoenzymes were identified to date. In all molybdoenzymes except nitrogenase, molybdenum is coordinated to a dithiolene group on the 6-alkyl side chain of a pterin called molybdopterin (MPT). The biosynthesis of Moco can be divided into three general steps, with a fourth one present only in bacteria and archaea: (1) formation of the cyclic pyranopterin monophosphate, (2) formation of MPT, (3) insertion of molybdenum into molybdopterin to form Moco, and (4) additional modification of Moco in bacteria with the attachment of a nucleotide to the phosphate group of MPT, forming the dinucleotide variant of Moco. This review will focus on the biosynthesis of Moco in bacteria, humans and plants.

  9. Interactions of the Mcm1 MADS Box Protein with Cofactors That Regulate Mating in Yeast

    PubMed Central

    Mead, Janet; Bruning, Adrian R.; Gill, Michael K.; Steiner, Andrew M.; Acton, Thomas B.; Vershon, Andrew K.

    2002-01-01

    The yeast Mcm1 protein is a member of the MADS box family of transcriptional regulatory factors, a class of DNA-binding proteins that control numerous cellular and developmental processes in yeast, Drosophila melanogaster, plants, and mammals. Although these proteins bind DNA on their own, they often combine with different cofactors to bind with increased affinity and specificity to their target sites. To understand how this class of proteins functions, we have made a series of alanine substitutions in the MADS box domain of Mcm1 and examined the effects of these mutations in combination with its cofactors that regulate mating in yeast. Our results indicate which residues of Mcm1 are essential for viability and transcriptional regulation with its cofactors in vivo. Most of the mutations in Mcm1 that are lethal affect DNA-binding affinity. Interestingly, the lethality of many of these mutations can be suppressed if the MCM1 gene is expressed from a high-copy-number plasmid. Although many of the alanine substitutions affect the ability of Mcm1 to activate transcription alone or in combination with the α1 and Ste12 cofactors, most mutations have little or no effect on Mcm1-mediated repression in combination with the α2 cofactor. Even nonconservative amino acid substitutions of residues in Mcm1 that directly contact α2 do not significantly affect repression. These results suggest that within the same region of the Mcm1 MADS box domain, there are different requirements for interaction with α2 than for interaction with either α1 or Ste12. Our results suggest how a small domain, the MADS box, interacts with multiple cofactors to achieve specificity in transcriptional regulation and how subtle differences in the sequences of different MADS box proteins can influence the interactions with specific cofactors while not affecting the interactions with common cofactors. PMID:12052870

  10. An in vivo RNAi assay identifies major genetic and cellular requirements for primary piRNA biogenesis in Drosophila

    PubMed Central

    Olivieri, Daniel; Sykora, Martina M; Sachidanandam, Ravi; Mechtler, Karl; Brennecke, Julius

    2010-01-01

    In Drosophila, PIWI proteins and bound PIWI-interacting RNAs (piRNAs) form the core of a small RNA-mediated defense system against selfish genetic elements. Within germline cells, piRNAs are processed from piRNA clusters and transposons to be loaded into Piwi/Aubergine/AGO3 and a subset of piRNAs undergoes target-dependent amplification. In contrast, gonadal somatic support cells express only Piwi, lack signs of piRNA amplification and exhibit primary piRNA biogenesis from piRNA clusters. Neither piRNA processing/loading nor Piwi-mediated target silencing is understood at the genetic, cellular or molecular level. We developed an in vivo RNAi assay for the somatic piRNA pathway and identified the RNA helicase Armitage, the Tudor domain containing RNA helicase Yb and the putative nuclease Zucchini as essential factors for primary piRNA biogenesis. Lack of any of these proteins leads to transposon de-silencing, to a collapse in piRNA levels and to a failure in Piwi-nuclear accumulation. We show that Armitage and Yb interact physically and co-localize in cytoplasmic Yb bodies, which flank P bodies. Loss of Zucchini leads to an accumulation of Piwi and Armitage in Yb bodies, indicating that Yb bodies are sites of primary piRNA biogenesis. PMID:20818334

  11. Redox cofactors insertion in prokaryotic molybdoenzymes occurs via a conserved folding mechanism

    PubMed Central

    Arias-Cartin, Rodrigo; Ceccaldi, Pierre; Schoepp-Cothenet, Barbara; Frick, Klaudia; Blanc, Jean-Michel; Guigliarelli, Bruno; Walburger, Anne; Grimaldi, Stéphane; Friedrich, Thorsten; Receveur-Brechot, Véronique; Magalon, Axel

    2016-01-01

    A major gap of knowledge in metalloproteins is the identity of the prefolded state of the protein before cofactor insertion. This holds for molybdoenzymes serving multiple purposes for life, especially in energy harvesting. This large group of prokaryotic enzymes allows for coordination of molybdenum or tungsten cofactors (Mo/W-bisPGD) and Fe/S clusters. Here we report the structural data on a cofactor-less enzyme, the nitrate reductase respiratory complex and characterize the conformational changes accompanying Mo/W-bisPGD and Fe/S cofactors insertion. Identified conformational changes are shown to be essential for recognition of the dedicated chaperone involved in cofactors insertion. A solvent-exposed salt bridge is shown to play a key role in enzyme folding after cofactors insertion. Furthermore, this salt bridge is shown to be strictly conserved within this prokaryotic molybdoenzyme family as deduced from a phylogenetic analysis issued from 3D structure-guided multiple sequence alignment. A biochemical analysis with a distantly-related member of the family, respiratory complex I, confirmed the critical importance of the salt bridge for folding. Overall, our results point to a conserved cofactors insertion mechanism within the Mo/W-bisPGD family. PMID:27886223

  12. brp and blh are required for synthesis of the retinal cofactor of bacteriorhodopsin in Halobacterium salinarum.

    PubMed

    Peck, R F; Echavarri-Erasun, C; Johnson, E A; Ng, W V; Kennedy, S P; Hood, L; DasSarma, S; Krebs, M P

    2001-02-23

    Bacteriorhodopsin, the light-driven proton pump of Halobacterium salinarum, consists of the membrane apoprotein bacterioopsin and a covalently bound retinal cofactor. The mechanism by which retinal is synthesized and bound to bacterioopsin in vivo is unknown. As a step toward identifying cellular factors involved in this process, we constructed an in-frame deletion of brp, a gene implicated in bacteriorhodopsin biogenesis. In the Deltabrp strain, bacteriorhodopsin levels are decreased approximately 4.0-fold compared with wild type, whereas bacterioopsin levels are normal. The probable precursor of retinal, beta-carotene, is increased approximately 3.8-fold, whereas retinal is decreased by approximately 3.7-fold. These results suggest that brp is involved in retinal synthesis. Additional cellular factors may substitute for brp function in the Deltabrp strain because retinal production is not abolished. The in-frame deletion of blh, a brp paralog identified by analysis of the Halobacterium sp. NRC-1 genome, reduced bacteriorhodopsin accumulation on solid medium but not in liquid. However, deletion of both brp and blh abolished bacteriorhodopsin and retinal production in liquid medium, again without affecting bacterioopsin accumulation. The level of beta-carotene increased approximately 5.3-fold. The simplest interpretation of these results is that brp and blh encode similar proteins that catalyze or regulate the conversion of beta-carotene to retinal.

  13. Broadening the cofactor specificity of a thermostable alcohol dehydrogenase using rational protein design introduces novel kinetic transient behavior.

    PubMed

    Campbell, Elliot; Wheeldon, Ian R; Banta, Scott

    2010-12-01

    Cofactor specificity in the aldo-keto reductase (AKR) superfamily has been well studied, and several groups have reported the rational alteration of cofactor specificity in these enzymes. Although most efforts have focused on mesostable AKRs, several putative AKRs have recently been identified from hyperthermophiles. The few that have been characterized exhibit a strong preference for NAD(H) as a cofactor, in contrast to the NADP(H) preference of the mesophilic AKRs. Using the design rules elucidated from mesostable AKRs, we introduced two site-directed mutations in the cofactor binding pocket to investigate cofactor specificity in a thermostable AKR, AdhD, which is an alcohol dehydrogenase from Pyrococcus furiosus. The resulting double mutant exhibited significantly improved activity and broadened cofactor specificity as compared to the wild-type. Results of previous pre-steady-state kinetic experiments suggest that the high affinity of the mesostable AKRs for NADP(H) stems from a conformational change upon cofactor binding which is mediated by interactions between a canonical arginine and the 2'-phosphate of the cofactor. Pre-steady-state kinetics with AdhD and the new mutants show a rich conformational behavior that is independent of the canonical arginine or the 2'-phosphate. Additionally, experiments with the highly active double mutant using NADPH as a cofactor demonstrate an unprecedented transient behavior where the binding mechanism appears to be dependent on cofactor concentration. These results suggest that the structural features involved in cofactor specificity in the AKRs are conserved within the superfamily, but the dynamic interactions of the enzyme with cofactors are unexpectedly complex.

  14. Organic cofactors in the metabolism of Dehalococcoides mccartyi strains

    PubMed Central

    Schipp, Christian J.; Marco-Urrea, Ernest; Kublik, Anja; Seifert, Jana; Adrian, Lorenz

    2013-01-01

    Dehalococcoides mccartyi strains are strictly anaerobic organisms specialized to grow with halogenated compounds as electron acceptor via a respiratory process. Their genomes are among the smallest known for free-living organisms, and the embedded gene set reflects their strong specialization. Here, we briefly review main characteristics of published Dehalococcoides genomes and show how genome information together with cultivation and biochemical experiments have contributed to our understanding of Dehalococcoides physiology and biochemistry. We extend this approach by the detailed analysis of cofactor metabolism in Dehalococcoides strain CBDB1. Dehalococcoides genomes were screened for encoded proteins annotated to contain or interact with organic cofactors, and the expression of these proteins was analysed by shotgun proteomics to shed light on cofactor requirements. In parallel, cultivation experiments testing for vitamin requirements showed that cyanocobalamin (vitamin B12), thiamine and biotin were essential supplements and that cyanocobalamin could be substituted by dicyanocobinamide and dimethylbenzimidazole. Dehalococcoides genome analysis, detection of single enzymes by shotgun proteomics and inhibition studies confirmed the expression of the biosynthetic pathways for pyridoxal-5-phosphate, flavin nucleotides, folate, S-adenosylmethionine, pantothenate and nicotinic acids in strain CBDB1. Haem/cytochromes, quinones and lipoic acids were not necessary for cultivation or dechlorination activity and no biosynthetic pathways were identified in the genomes. PMID:23479751

  15. Resolution of the cellular proteome of the nucleocapsid protein from a highly pathogenic isolate of porcine reproductive and respiratory syndrome virus identifies PARP-1 as a cellular target whose interaction is critical for virus biology.

    PubMed

    Liu, Long; Lear, Zoe; Hughes, David J; Wu, Weining; Zhou, En-min; Whitehouse, Adrian; Chen, Hongying; Hiscox, Julian A

    2015-03-23

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to the swine industry and food security worldwide. The nucleocapsid (N) protein is a major structural protein of PRRSV. The primary function of this protein is to encapsidate the viral RNA genome, and it is also thought to participate in the modulation of host cell biology and recruitment of cellular factors to facilitate virus infection. In order to the better understand these latter roles the cellular interactome of PRRSV N protein was defined using label free quantitative proteomics. This identified several cellular factors that could interact with the N protein including poly [ADP-ribose] polymerase 1 (PARP-1), a cellular protein, which can add adenosine diphosphate ribose to a protein. Use of the PARP-1 small molecule inhibitor, 3-AB, in PRRSV infected cells demonstrated that PARP-1 was required and acted as an enhancer factor for virus biology. Serial growth of PRRSV in different concentrations of 3-AB did not yield viruses that were able to grow with wild type kinetics, suggesting that by targeting a cellular protein crucial for virus biology, resistant phenotypes did not emerge. This study provides further evidence that cellular proteins, which are critical for virus biology, can also be targeted to ablate virus growth and provide a high barrier for the emergence of drug resistance.

  16. A Genome-Wide Screen in Yeast Identifies Specific Oxidative Stress Genes Required for the Maintenance of Sub-Cellular Redox Homeostasis

    PubMed Central

    Ayer, Anita; Fellermeier, Sina; Fife, Christopher; Li, Simone S.; Smits, Gertien; Meyer, Andreas J.; Dawes, Ian W.; Perrone, Gabriel G.

    2012-01-01

    Maintenance of an optimal redox environment is critical for appropriate functioning of cellular processes and cell survival. Despite the importance of maintaining redox homeostasis, it is not clear how the optimal redox potential is sensed and set, and the processes that impact redox on a cellular/organellar level are poorly understood. The genetic bases of cellular redox homeostasis were investigated using a green fluorescent protein (GFP) based redox probe, roGFP2 and a pH sensitive GFP-based probe, pHluorin. The use of roGFP2, in conjunction with pHluorin, enabled determination of pH-adjusted sub-cellular redox potential in a non-invasive and real-time manner. A genome-wide screen using both the non-essential and essential gene collections was carried out in Saccharomyces cerevisiae using cytosolic-roGFP2 to identify factors essential for maintenance of cytosolic redox state under steady-state conditions. 102 genes of diverse function were identified that are required for maintenance of cytosolic redox state. Mutations in these genes led to shifts in the half-cell glutathione redox potential by 75-10 mV. Interestingly, some specific oxidative stress-response processes were identified as over-represented in the data set. Further investigation of the role of oxidative stress-responsive systems in sub-cellular redox homeostasis was conducted using roGFP2 constructs targeted to the mitochondrial matrix and peroxisome and EGSH was measured in cells in exponential and stationary phase. Analyses allowed for the identification of key redox systems on a sub-cellular level and the identification of novel genes involved in the regulation of cellular redox homeostasis. PMID:22970195

  17. A genome-wide screen in yeast identifies specific oxidative stress genes required for the maintenance of sub-cellular redox homeostasis.

    PubMed

    Ayer, Anita; Fellermeier, Sina; Fife, Christopher; Li, Simone S; Smits, Gertien; Meyer, Andreas J; Dawes, Ian W; Perrone, Gabriel G

    2012-01-01

    Maintenance of an optimal redox environment is critical for appropriate functioning of cellular processes and cell survival. Despite the importance of maintaining redox homeostasis, it is not clear how the optimal redox potential is sensed and set, and the processes that impact redox on a cellular/organellar level are poorly understood. The genetic bases of cellular redox homeostasis were investigated using a green fluorescent protein (GFP) based redox probe, roGFP2 and a pH sensitive GFP-based probe, pHluorin. The use of roGFP2, in conjunction with pHluorin, enabled determination of pH-adjusted sub-cellular redox potential in a non-invasive and real-time manner. A genome-wide screen using both the non-essential and essential gene collections was carried out in Saccharomyces cerevisiae using cytosolic-roGFP2 to identify factors essential for maintenance of cytosolic redox state under steady-state conditions. 102 genes of diverse function were identified that are required for maintenance of cytosolic redox state. Mutations in these genes led to shifts in the half-cell glutathione redox potential by 75-10 mV. Interestingly, some specific oxidative stress-response processes were identified as over-represented in the data set. Further investigation of the role of oxidative stress-responsive systems in sub-cellular redox homeostasis was conducted using roGFP2 constructs targeted to the mitochondrial matrix and peroxisome and E(GSH) was measured in cells in exponential and stationary phase. Analyses allowed for the identification of key redox systems on a sub-cellular level and the identification of novel genes involved in the regulation of cellular redox homeostasis.

  18. The Tight Junction Proteins Claudin-1, -6, and -9 Are Entry Cofactors for Hepatitis C Virus▿

    PubMed Central

    Meertens, Laurent; Bertaux, Claire; Cukierman, Lisa; Cormier, Emmanuel; Lavillette, Dimitri; Cosset, François-Loïc; Dragic, Tatjana

    2008-01-01

    Hepatitis C virus (HCV) is a major cause of liver disease in humans. The CD81 tetraspanin is necessary but not sufficient for HCV penetration into hepatocytes, and it was recently reported that the tight junction protein claudin-1 is a critical HCV entry cofactor. Here, we confirm the role of claudin-1 in HCV entry. In addition, we show that claudin-6 and claudin-9 expressed in CD81+ cells also enable the entry of HCV pseudoparticles derived from six of the major genotypes. Whereas claudin-1, -6, and -9 function equally well as entry cofactors in endothelial cells, claudin-1 is more efficient in hepatoma cells. This suggests that additional cellular factors modulate the ability of claudins to function as HCV entry cofactors. Our work has generated novel and essential means to investigate the mechanism of HCV penetration into hepatocytes and the role of the claudin protein family in HCV dissemination, replication, and pathogenesis. PMID:18234789

  19. Enzymatic regeneration of adenosine triphosphate cofactor

    NASA Technical Reports Server (NTRS)

    Marshall, D. L.

    1974-01-01

    Regenerating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by enzymatic process which utilizes carbamyl phosphate as phosphoryl donor is technique used to regenerate expensive cofactors. Process allows complex enzymatic reactions to be considered as candidates for large-scale continuous processes.

  20. Molybdenum Enzymes, Cofactors, and Model Systems.

    ERIC Educational Resources Information Center

    Burgmayer, S. J. N; Stiefel, E. I.

    1985-01-01

    Discusses: (l) molybdoenzymes (examining their distribution and metabolic role, composition and redox strategy, cofactors, substrate reactions, and mechanistic possibilities); (2) structural information on molybdenum (Mo) centers; (3) modeling studies (Mo-co models, nitrogenase models, and the MO-S duo); and (4) the copper-molybdenum antagonism.…

  1. Molybdenum Enzymes, Cofactors, and Model Systems.

    ERIC Educational Resources Information Center

    Burgmayer, S. J. N; Stiefel, E. I.

    1985-01-01

    Discusses: (l) molybdoenzymes (examining their distribution and metabolic role, composition and redox strategy, cofactors, substrate reactions, and mechanistic possibilities); (2) structural information on molybdenum (Mo) centers; (3) modeling studies (Mo-co models, nitrogenase models, and the MO-S duo); and (4) the copper-molybdenum antagonism.…

  2. Exogenous cofactors for the improvement of bioremoval and biotransformation of sulfamethoxazole by Alcaligenes faecalis.

    PubMed

    Zhang, Yi-Bi; Zhou, Jiao; Xu, Qiu-Man; Cheng, Jing-Sheng; Luo, Yu-Lu; Yuan, Ying-Jin

    2016-09-15

    Sulfamethoxazole (SMX), an extensively prescribed or administered antibiotic pharmaceutical product, is usually detected in aquatic environments, because of its incomplete metabolism and elimination. This study investigated the effects of exogenous cofactors on the bioremoval and biotransformation of SMX by Alcaligenes faecalis. High concentration (100mg·L(-1)) of exogenous vitamin C (VC), vitamin B6 (VB6) and oxidized glutathione (GSSG) enhanced SMX bioremoval, while the additions of vitamin B2 (VB2) and vitamin B12 (VB12) did not significantly alter the SMX removal efficiency. Globally, cellular growth of A. faecalis and SMX removal both initially increased and then gradually decreased, indicating that SMX bioremoval is likely dependent on the primary biomass activity of A. faecalis. The decreases in the SMX removal efficiency indicated that some metabolites of SMX might be transformed into parent compound at the last stage of incubation. Two transformation products of SMX, N-hydroxy sulfamethoxazole (HO-SMX) and N4-acetyl sulfamethoxazole (Ac-SMX), were identified by a high-performance liquid chromatograph coupled with mass spectrometer. High concentrations of VC, nicotinamide adenine dinucleotide hydrogen (NADH, 7.1mg·L(-1)), and nicotinamide adenine dinucleotide (NAD(+), 6.6mg·L(-1)), and low concentrations of reduced glutathione (GSH, 0.1 and 10mg·L(-1)) and VB2 (1mg·L(-1)) remarkably increased the formation of HO-SMX, while VB12 showed opposite effects on HO-SMX formation. In addition, low concentrations of GSH and NADH enhanced Ac-SMX formation by the addition of A. faecalis, whereas cofactors (VC, VB2, VB12, NAD(+), and GSSG) had no obvious impact on the formation of Ac-SMX compared with the controls. The levels of Ac-SMX were stable when biomass of A. faecalis gradually decreased, indicating the direct effect of biomass on the formation of Ac-SMX by A. faecalis. In sum, these results help us understand the roles played by exogenous cofactors in

  3. Multibody cofactor and substrate molecular recognition in the myo-inositol monophosphatase enzyme

    PubMed Central

    Ferruz, Noelia; Tresadern, Gary; Pineda-Lucena, Antonio; De Fabritiis, Gianni

    2016-01-01

    Molecular recognition is rarely a two-body protein-ligand problem, as it often involves the dynamic interplay of multiple molecules that together control the binding process. Myo-inositol monophosphatase (IMPase), a drug target for bipolar disorder, depends on 3 Mg2+ ions as cofactor for its catalytic activity. Although the crystallographic pose of the pre-catalytic complex is well characterized, the binding process by which substrate, cofactor and protein cooperate is essentially unknown. Here, we have characterized cofactor and substrate cooperative binding by means of large-scale molecular dynamics. Our study showed the first and second Mg2+ ions identify the binding pocket with fast kinetics whereas the third ion presents a much higher energy barrier. Substrate binding can occur in cooperation with cofactor, or alone to a binary or ternary cofactor-IMPase complex, although the last scenario occurs several orders of magnitude faster. Our atomic description of the three-body mechanism offers a particularly challenging example of pathway reconstruction, and may prove particularly useful in realistic contexts where water, ions, cofactors or other entities cooperate and modulate the binding process. PMID:27440438

  4. A survey of synthetic nicotinamide cofactors in enzymatic processes.

    PubMed

    Paul, Caroline E; Hollmann, Frank

    2016-06-01

    Synthetic nicotinamide cofactors are analogues of the natural cofactors used by oxidoreductases as redox intermediates. Their ability to be fine-tuned makes these biomimetics an attractive alternative to the natural cofactors in terms of stability, reactivity, and cost. The following mini-review focuses on the current state of the art of those biomimetics in enzymatic processes.

  5. Comparative analysis of Salmonella susceptibility and tolerance to the biocide chlorhexidine identifies a complex cellular defense network

    PubMed Central

    Condell, Orla; Power, Karen A.; Händler, Kristian; Finn, Sarah; Sheridan, Aine; Sergeant, Kjell; Renaut, Jenny; Burgess, Catherine M.; Hinton, Jay C. D.; Nally, Jarlath E.; Fanning, Séamus

    2014-01-01

    Chlorhexidine is one of the most widely used biocides in health and agricultural settings as well as in the modern food industry. It is a cationic biocide of the biguanide class. Details of its mechanism of action are largely unknown. The frequent use of chlorhexidine has been questioned recently, amidst concerns that an overuse of this compound may select for bacteria displaying an altered susceptibility to antimicrobials, including clinically important anti-bacterial agents. We generated a Salmonella enterica serovar Typhimurium isolate (ST24CHX) that exhibited a high-level tolerant phenotype to chlorhexidine, following several rounds of in vitro selection, using sub-lethal concentrations of the biocide. This mutant showed altered suceptibility to a panel of clinically important antimicrobial compounds. Here we describe a genomic, transcriptomic, proteomic, and phenotypic analysis of the chlorhexidine tolerant S. Typhimurium compared with its isogenic sensitive progenitor. Results from this study describe a chlorhexidine defense network that functions in both the reference chlorhexidine sensitive isolate and the tolerant mutant. The defense network involved multiple cell targets including those associated with the synthesis and modification of the cell wall, the SOS response, virulence, and a shift in cellular metabolism toward anoxic pathways, some of which were regulated by CreB and Fur. In addition, results indicated that chlorhexidine tolerance was associated with more extensive modifications of the same cellular processes involved in this proposed network, as well as a divergent defense response involving the up-regulation of additional targets such as the flagellar apparatus and an altered cellular phosphate metabolism. These data show that sub-lethal concentrations of chlorhexidine induce distinct changes in exposed Salmonella, and our findings provide insights into the mechanisms of action and tolerance to this biocidal agent. PMID:25136333

  6. Biosynthesis and Insertion of the Molybdenum Cofactor.

    PubMed

    Magalon, Axel; Mendel, Ralf R

    2015-01-01

    The transition element molybdenum (Mo) is of primordial importance for biological systems, because it is required by enzymes catalyzing key reactions in the global carbon, sulfur, and nitrogen metabolism. To gain biological activity, Mo has to be complexed by a special cofactor. With the exception of bacterial nitrogenase, all Mo-dependent enzymes contain a unique pyranopterin-based cofactor coordinating a Mo atom at their catalytic site. Various types of reactions are catalyzed by Mo-enzymes in prokaryotes including oxygen atom transfer, sulfur or proton transfer, hydroxylation, or even nonredox reactions. Mo-enzymes are widespread in prokaryotes and many of them were likely present in the Last Universal Common Ancestor. To date, more than 50--mostly bacterial--Mo-enzymes are described in nature. In a few eubacteria and in many archaea, Mo is replaced by tungsten bound to the same unique pyranopterin. How Mo-cofactor is synthesized in bacteria is reviewed as well as the way until its insertion into apo-Mo-enzymes.

  7. Protocols for cofactor isolation of nitrogenase.

    PubMed

    Fay, Aaron W; Lee, Chi-Chung; Wiig, Jared A; Hu, Yilin; Ribbe, Markus W

    2011-01-01

    The iron-molybdenum cofactor (FeMoco) of the nitrogenase MoFe protein has remained a focal point in the field of bioinorganic chemistry for decades. This unique metal cluster has long been regarded as the actual site of dinitrogen reduction, and it is structurally complex and chemically unprecedented. A detailed characterization of the isolated FeMoco is crucial for elucidating the physiochemical properties of this biologically important cofactor. Such a study requires an effective technique to extract FeMoco intact, and in high yield, from the MoFe protein. A method involving the acid treatment of the MoFe protein and the subsequent extraction of FeMoco into an organic solvent was developed over 30 years ago and has been improved upon ever since. FeMoco isolated by this strategy is catalytically active and spectrally interesting, which provides a useful platform for future structure-function analyses of this unique cofactor. A general working protocol for FeMoco isolation is described in this chapter, along with some of the major modifications reported in the past years.

  8. The phylogenomic roots of modern biochemistry: origins of proteins, cofactors and protein biosynthesis.

    PubMed

    Caetano-Anollés, Gustavo; Kim, Kyung Mo; Caetano-Anollés, Derek

    2012-02-01

    The complexity of modern biochemistry developed gradually on early Earth as new molecules and structures populated the emerging cellular systems. Here, we generate a historical account of the gradual discovery of primordial proteins, cofactors, and molecular functions using phylogenomic information in the sequence of 420 genomes. We focus on structural and functional annotations of the 54 most ancient protein domains. We show how primordial functions are linked to folded structures and how their interaction with cofactors expanded the functional repertoire. We also reveal protocell membranes played a crucial role in early protein evolution and show translation started with RNA and thioester cofactor-mediated aminoacylation. Our findings allow elaboration of an evolutionary model of early biochemistry that is firmly grounded in phylogenomic information and biochemical, biophysical, and structural knowledge. The model describes how primordial α-helical bundles stabilized membranes, how these were decorated by layered arrangements of β-sheets and α-helices, and how these arrangements became globular. Ancient forms of aminoacyl-tRNA synthetase (aaRS) catalytic domains and ancient non-ribosomal protein synthetase (NRPS) modules gave rise to primordial protein synthesis and the ability to generate a code for specificity in their active sites. These structures diversified producing cofactor-binding molecular switches and barrel structures. Accretion of domains and molecules gave rise to modern aaRSs, NRPS, and ribosomal ensembles, first organized around novel emerging cofactors (tRNA and carrier proteins) and then more complex cofactor structures (rRNA). The model explains how the generation of protein structures acted as scaffold for nucleic acids and resulted in crystallization of modern translation.

  9. Recent trends and novel concepts in cofactor-dependent biotransformations.

    PubMed

    Kara, Selin; Schrittwieser, Joerg H; Hollmann, Frank; Ansorge-Schumacher, Marion B

    2014-02-01

    Cofactor-dependent enzymes catalyze a broad range of synthetically useful transformations. However, the cofactor requirement also poses economic and practical challenges for the application of these biocatalysts. For three decades, considerable research effort has been devoted to the development of reliable in situ regeneration methods for the most commonly employed cofactors, particularly NADH and NADPH. Today, researchers can choose from a plethora of options, and oxidoreductases are routinely employed even on industrial scale. Nevertheless, more efficient cofactor regeneration methods are still being developed, with the aim of achieving better atom economy, simpler reaction setups, and higher productivities. Besides, cofactor dependence has been recognized as an opportunity to confer novel reactivity upon enzymes by engineering their cofactors, and to couple (redox) biotransformations in multi-enzyme cascade systems. These novel concepts will help to further establish cofactor-dependent biotransformations as an attractive option for the synthesis of biologically active compounds, chiral building blocks, and bio-based platform molecules.

  10. An integrated analysis of enzyme activities, cofactor pools and metabolic fluxes in baculovirus-infected Spodoptera frugiperda Sf9 cells.

    PubMed

    Bernal, Vicente; Monteiro, Francisca; Carinhas, Nuno; Ambrósio, Raquel; Alves, Paula M

    2010-11-01

    The scarcity of fundamental knowledge on the baculovirus-host cell interaction is a major drawback for the improvement of bioprocesses through Metabolic Engineering. After the first hours post-infection, the virus takes over the control of cellular machinery, leading to the repression of host gene expression and imposing a high metabolic burden to insect cells. Nevertheless, there is a lack of detailed data on the metabolic responses to infection, which are ultimately responsible for system productivity performance. In this work, a further insight into the central metabolism of Sf9 cells is achieved by a combined analysis of enzyme activities, cellular cofactors (ATP and NAD(P)(+)/NAD(P)H) and metabolic fluxes. Hexokinase and isocitrate dehydrogenase were identified as feasible limiting steps of metabolism; carbon and nitrogen metabolism enzymes were differentially regulated during batch cultures. Moreover, alterations occurring after infection demonstrated the importance of maintaining the energetic state of the cells for baculovirus replication, since ATP accumulated in a MOI-dependent way, and the glutamate dehydrogenase anaplerotic pathway was greatly activated. Altogether, cellular de-energization and stress responses are relevant factors in the metabolic burden imposed by infection. The implications for the improvement of bioprocess performance are discussed. Copyright © 2010 Elsevier B.V. All rights reserved.

  11. DEAH-RHA helicase•Znf cofactor systems in kinetoplastid RNA editing and evolutionarily distant RNA processes

    PubMed Central

    Cruz-Reyes, Jorge; Mooers, Blaine H.M.; Abu-Adas, Zakaria; Kumar, Vikas; Gulati, Shelly

    2016-01-01

    Multi-zinc finger proteins are an emerging class of cofactors in DEAH-RHA RNA helicases across highly divergent eukaryotic lineages. DEAH-RHA helicase•zinc finger cofactor partnerships predate the split of kinetoplastid protozoa, which include several human pathogens, from other eukaryotic lineages 100–400 Ma. Despite a long evolutionary history, the prototypical DEAH-RHA domains remain highly conserved. This short review focuses on a recently identified DEAH-RHA helicase•zinc finger cofactor system in kinetoplastid RNA editing, and its potential functional parallels with analogous systems in embryogenesis control in nematodes and antivirus protection in humans. PMID:27540585

  12. A phylogenomic census of molecular functions identifies modern thermophilic archaea as the most ancient form of cellular life.

    PubMed

    Nasir, Arshan; Kim, Kyung Mo; Caetano-Anollés, Gustavo

    2014-01-01

    The origins of diversified life remain mysterious despite considerable efforts devoted to untangling the roots of the universal tree of life. Here we reconstructed phylogenies that described the evolution of molecular functions and the evolution of species directly from a genomic census of gene ontology (GO) definitions. We sampled 249 free-living genomes spanning organisms in the three superkingdoms of life, Archaea, Bacteria, and Eukarya, and used the abundance of GO terms as molecular characters to produce rooted phylogenetic trees. Results revealed an early thermophilic origin of Archaea that was followed by genome reduction events in microbial superkingdoms. Eukaryal genomes displayed extraordinary functional diversity and were enriched with hundreds of novel molecular activities not detected in the akaryotic microbial cells. Remarkably, the majority of these novel functions appeared quite late in evolution, synchronized with the diversification of the eukaryal superkingdom. The distribution of GO terms in superkingdoms confirms that Archaea appears to be the simplest and most ancient form of cellular life, while Eukarya is the most diverse and recent.

  13. Characterization of transcriptional regulatory domains of ankyrin repeat cofactor-1

    SciTech Connect

    Zhang, Aihua; Li, Chia-Wei; Chen, J. Don . E-mail: chenjd@umdnj.edu

    2007-07-13

    The ankyrin repeats cofactor-1 (ANCO-1) was recently identified as a p160 coactivator-interacting protein that may inhibit transcriptional activity of nuclear receptors. Here, we have characterized the transcriptional regulatory domains of ANCO-1. Two intrinsic repression domains (RD) were identified: an N-terminal RD1 at residues 318-611 and a C-terminal RD2 at 2369-2663. ANCO-1 also contains an activation domain (AD) capable of stimulating transcription in both mammalian and yeast cells. The minimal AD was delimited to a 70-amino acid region at residues 2076-2145. Overall, full-length ANCO-1 exhibited transcriptional repressor activity, suggesting that RD domains may suppress the AD activity. We further demonstrated that ANCO-1 silencing by siRNA enhanced progesterone receptor-mediated transcription. Together, these results indicate that the transcriptional potential of ANCO-1 may be modulated by a combination of repression and activation signals.

  14. Regulation of Estrogen-Dependent Transcription by the LIM Cofactors CLIM and RLIM in Breast Cancer

    PubMed Central

    Johnsen, Steven A.; Güngör, Cenap; Prenzel, Tanja; Riethdorf, Sabine; Riethdorf, Lutz; Taniguchi-Ishigaki, Naoko; Rau, Thomas; Tursun, Baris; Furlow, J. David; Sauter, Guido; Scheffner, Martin; Pantel, Klaus; Gannon, Frank; Bach, Ingolf

    2009-01-01

    Mammary oncogenesis is profoundly influenced by signaling pathways controlled by Estrogen Receptor-alpha (ERα). Although it is known that ERα exerts its oncogenic effect by stimulating the proliferation of many human breast cancers through the activation of target genes, our knowledge of the underlying transcriptional mechanisms remains limited. Our published work has shown that the in vivo activity of LIM homeodomain transcription factors (LIM-HDs) is critically regulated by Cofactors of LIM-HD proteins (CLIM) and the ubiquitin ligase RING finger LIM domain interacting protein (RLIM). Here, we identify CLIM and RLIM as novel ERα cofactors that co-localize and interact with ERα in primary human breast tumors. We show that both cofactors associate with estrogen responsive promoters and regulate the expression of endogenous ERα target genes in breast cancer cells. Surprisingly, our results indicate opposing functions of LIM cofactors for ERα and LIM-HDs: whereas CLIM enhances transcriptional activity of LIM-HDs, it inhibits transcriptional activation mediated by ERα on most target genes in vivo. In turn, the ubiquitin ligase RLIM inhibits transcriptional activity of LIM-HDs, but enhances transcriptional activation of endogenous ERα target genes. Results from a human breast cancer tissue microarray (TMA) of 1,335 patients revealed a highly significant correlation of elevated CLIM levels to ER/PR positivity and poor differentiation of tumors. Combined, these results indicate that LIM cofactors CLIM and RLIM regulate the biological activity of ERα during the development of human breast cancer. PMID:19117995

  15. The nuclear cofactor DOR regulates autophagy in mammalian and Drosophila cells

    PubMed Central

    Mauvezin, Caroline; Orpinell, Meritxell; Francis, Víctor A; Mansilla, Francisco; Duran, Jordi; Ribas, Vicent; Palacín, Manuel; Boya, Patricia; Teleman, Aurelio A; Zorzano, Antonio

    2010-01-01

    The regulation of autophagy in metazoans is only partly understood, and there is a need to identify the proteins that control this process. The diabetes- and obesity-regulated gene (DOR), a recently reported nuclear cofactor of thyroid hormone receptors, is expressed abundantly in metabolically active tissues such as muscle. Here, we show that DOR shuttles between the nucleus and the cytoplasm, depending on cellular stress conditions, and re-localizes to autophagosomes on autophagy activation. We demonstrate that DOR interacts physically with autophagic proteins Golgi-associated ATPase enhancer of 16 kDa (GATE16) and microtubule-associated protein 1A/1B-light chain 3. Gain-of-function and loss-of-function studies indicate that DOR stimulates autophagosome formation and accelerates the degradation of stable proteins. CG11347, the DOR Drosophila homologue, has been predicted to interact with the Drosophila Atg8 homologues, which suggests functional conservation in autophagy. Flies lacking CG11347 show reduced autophagy in the fat body during pupal development. All together, our data indicate that DOR regulates autophagosome formation and protein degradation in mammalian and Drosophila cells. PMID:20010805

  16. Structure determination of the UDP-disaccharide fragment of cytoplasmic cofactor isolated from Methanobacterium thermoautotrophicum.

    PubMed

    Marsden, B J; Sauer, F D; Blackwell, B A; Kramer, J K

    1989-03-31

    The methylcoenzyme M methylreductase reaction has an absolute requirement for 7-mercaptoheptanoylthreonine phosphate or component B, which is the active component of the intact molecule previously referred to as cytoplasmic cofactor. A hydrolytic fragment of cytoplasmic cofactor has been purified and identified as uridine 5'-(O-2-acetamido-2-deoxy-beta-manno-pyranuronosyl acid (1----4)-2-acetamido-2-deoxy-alpha-glucopyranosyl diphosphate) by high resolution NMR and fast atom bombardment mass spectro-metry. It is postulated that UDP-disaccharide may function to anchor 7-mercaptoheptanoyl threonine phosphate at the active site of the methyl-reductase enzyme complex.

  17. A short hairpin RNA screen of interferon-stimulated genes identifies a novel negative regulator of the cellular antiviral response.

    PubMed

    Li, Jianqing; Ding, Steve C; Cho, Hyelim; Chung, Brian C; Gale, Michael; Chanda, Sumit K; Diamond, Michael S

    2013-06-18

    The type I interferon (IFN) signaling pathway restricts infection of many divergent families of RNA and DNA viruses by inducing hundreds of IFN-stimulated genes (ISGs), some of which have direct antiviral activity. We screened 813 short hairpin RNA (shRNA) constructs targeting 245 human ISGs using a flow cytometry approach to identify genes that modulated infection of West Nile virus (WNV) in IFN-β-treated human cells. Thirty ISGs with inhibitory effects against WNV were identified, including several novel genes that had antiviral activity against related and unrelated positive-strand RNA viruses. We also defined one ISG, activating signal cointegrator complex 3 (ASCC3), which functioned as a negative regulator of the host defense response. Silencing of ASCC3 resulted in upregulation of multiple antiviral ISGs, which correlated with inhibition of infection of several positive-strand RNA viruses. Reciprocally, ectopic expression of human ASCC3 or mouse Ascc3 resulted in downregulation of ISGs and increased viral infection. Mechanism-of-action and RNA sequencing studies revealed that ASCC3 functions to modulate ISG expression in an IRF-3- and IRF-7-dependent manner. Compared to prior ectopic ISG expression studies, our shRNA screen identified novel ISGs that restrict infection of WNV and other viruses and defined a new counterregulatory ISG, ASCC3, which tempers cell-intrinsic immunity.

  18. An Image-Based Genetic Assay Identifies Genes in T1D Susceptibility Loci Controlling Cellular Antiviral Immunity in Mouse

    PubMed Central

    Liao, Juan; Jijon, Humberto B.; Kim, Ira R.; Goel, Gautam; Doan, Aivi; Sokol, Harry; Bauer, Hermann; Herrmann, Bernhard G.; Lassen, Kara G.; Xavier, Ramnik J.

    2014-01-01

    The pathogenesis of complex diseases, such as type 1 diabetes (T1D), derives from interactions between host genetics and environmental factors. Previous studies have suggested that viral infection plays a significant role in initiation of T1D in genetically predisposed individuals. T1D susceptibility loci may therefore be enriched in previously uncharacterized genes functioning in antiviral defense pathways. To identify genes involved in antiviral immunity, we performed an image-based high-throughput genetic screen using short hairpin RNAs (shRNAs) against 161 genes within T1D susceptibility loci. RAW 264.7 cells transduced with shRNAs were infected with GFP-expressing herpes simplex virus type 1 (HSV-1) and fluorescent microscopy was performed to assess the viral infectivity by fluorescence reporter activity. Of the 14 candidates identified with high confidence, two candidates were selected for further investigation, Il27 and Tagap. Administration of recombinant IL-27 during viral infection was found to act synergistically with interferon gamma (IFN-γ) to activate expression of type I IFNs and proinflammatory cytokines, and to enhance the activities of interferon regulatory factor 3 (IRF3). Consistent with a role in antiviral immunity, Tagap-deficient macrophages demonstrated increased viral replication, reduced expression of proinflammatory chemokines and cytokines, and decreased production of IFN-β. Taken together, our unbiased loss-of-function genetic screen identifies genes that play a role in host antiviral immunity and delineates roles for IL-27 and Tagap in the production of antiviral cytokines. PMID:25268627

  19. Monitoring oxidative and nitrative modification of cellular proteins; a paradigm for identifying key disease related markers of oxidative stress.

    PubMed

    Murray, James; Oquendo, C Elisa; Willis, John H; Marusich, Michael F; Capaldi, Roderick A

    2008-01-01

    High levels of free radicals produced by the mitochondrial respiratory chain, with subsequent damage to mitochondria have been implicated in a large and growing number of diseases. The underlying pathology of these diseases is oxidative damage to mitochondrial DNA, lipids and proteins which accumulate over time to produce a metabolic deficiency. We are developing an antibody based immunocapture array for many important mitochondrial proteins involved in free radical production, detoxification and mitochondrial energy production. Our array is capable of a multi-parameter measurement including enzyme activity, quantity, and oxidative protein modifications. Here we demonstrate the use of this array by analyzing the proteomic differences in OXPHOS (oxidative phosphorylation) enzymes between human heart and liver tissues, cells grown in media promoting aerobic versus anaerobic metabolism, and the catalytic/proteomic effects of mitochondria exposed to oxidative stress. Protein oxidation is identified as carbonyl formation arising from reactive oxygen species and 3-nitrotyrosine as a marker of reactive nitrogen species. Several identified modifications are confirmed by electrophoresis and mass spectrometry of immunocaptured material. We continue to expand this array as antibodies for enzyme isolation and detection become available.

  20. Caenorhabditis elegans UBX cofactors for CDC-48/p97 control spermatogenesis.

    PubMed

    Sasagawa, Yohei; Yamanaka, Kunitoshi; Saito-Sasagawa, Yuko; Ogura, Teru

    2010-12-01

    UBX (ubiquitin regulatory X) domain-containing proteins act as cofactors for CDC-48/p97. CDC-48/p97 is essential for various cellular processes including retro-translocation in endoplasmic reticulum-associated degradation, homotypic membrane fusion, nuclear envelope assembly, degradation of ubiquitylated proteins, and cell cycle progression. CDC-48/p97-dependent processes are determined by differential binding of cofactors including UBX proteins, but the cellular functions of UBX proteins have not yet been elucidated, especially in multicellular organisms. Therefore, we investigated the functions of UBX family members using Caenorhabditis elegans, which expresses six UBX proteins, UBXN-1 to UBXN-6. All six UBXN proteins directly interacted with CDC-48.1 and CDC-48.2, and simultaneous knockdown of the expression of three genes, ubxn-1, ubxn-2 and ubxn-3, induced embryonic lethal and sterile phenotypes, but knockdown of either one or two did not. The sterile worms had a feminized germ-line phenotype, producing oocytes but no sperm. UBXN-1, UBXN-2 and UBXN-3 colocalized with CDC-48 in spermatocytes but not mature sperm. TRA-1A, which is a key factor in the sex determination pathway and inhibits spermatogenesis, accumulated in worms in which UBXN-1, UBXN-2 and UBXN-3 had been simultaneously knocked down. Taken together, these results suggest that UBXN-1, UBXN-2 and UBXN-3 are redundant cofactors for CDC-48/p97 and control spermatogenesis via the degradation of TRA-1A.

  1. iEzy-drug: a web server for identifying the interaction between enzymes and drugs in cellular networking.

    PubMed

    Min, Jian-Liang; Xiao, Xuan; Chou, Kuo-Chen

    2013-01-01

    With the features of extremely high selectivity and efficiency in catalyzing almost all the chemical reactions in cells, enzymes play vitally important roles for the life of an organism and hence have become frequent targets for drug design. An essential step in developing drugs by targeting enzymes is to identify drug-enzyme interactions in cells. It is both time-consuming and costly to do this purely by means of experimental techniques alone. Although some computational methods were developed in this regard based on the knowledge of the three-dimensional structure of enzyme, unfortunately their usage is quite limited because three-dimensional structures of many enzymes are still unknown. Here, we reported a sequence-based predictor, called "iEzy-Drug," in which each drug compound was formulated by a molecular fingerprint with 258 feature components, each enzyme by the Chou's pseudo amino acid composition generated via incorporating sequential evolution information and physicochemical features derived from its sequence, and the prediction engine was operated by the fuzzy K-nearest neighbor algorithm. The overall success rate achieved by iEzy-Drug via rigorous cross-validations was about 91%. Moreover, to maximize the convenience for the majority of experimental scientists, a user-friendly web server was established, by which users can easily obtain their desired results.

  2. hiPSC-derived cardiomyocytes from Brugada Syndrome patients without identified mutations do not exhibit clear cellular electrophysiological abnormalities

    PubMed Central

    Veerman, Christiaan C.; Mengarelli, Isabella; Guan, Kaomei; Stauske, Michael; Barc, Julien; Tan, Hanno L.; Wilde, Arthur A. M.; Verkerk, Arie O.; Bezzina, Connie R.

    2016-01-01

    Brugada syndrome (BrS) is a rare cardiac rhythm disorder associated with sudden cardiac death. Mutations in the sodium channel gene SCN5A are found in ~20% of cases while mutations in other genes collectively account for <5%. In the remaining patients the genetic defect and the underlying pathogenic mechanism remain obscure. To provide insight into the mechanism of BrS in individuals without identified mutations, we here studied electrophysiological properties of cardiomyocytes (CMs) generated from human induced pluripotent stem cells (hiPSCs) from 3 BrS patients who tested negative for mutations in the known BrS-associated genes. Patch clamp studies revealed no differences in sodium current (INa) in hiPSC-CMs from the 3 BrS patients compared to 2 unrelated controls. Moreover, action potential upstroke velocity (Vmax), reflecting INa, was not different between hiPSC-CMs from the BrS patients and the controls. hiPSC-CMs harboring the BrS-associated SCN5A-1795insD mutation exhibited a reduction in both INa and Vmax, demonstrating our ability to detect reduced sodium channel function. hiPSC-CMs from one of the BrS lines demonstrated a mildly reduced action potential duration, however, the transient outward potassium current (Ito) and the L-type calcium current (ICa,L), both implicated in BrS, were not different compared to the controls. Our findings indicate that ion channel dysfunction, in particular in the cardiac sodium channel, may not be a prerequisite for BrS. PMID:27485484

  3. Catalysis in Enzymatic Decarboxylations: Comparison of Selected Cofactor-dependent and Cofactor-independent Examples

    PubMed Central

    Jordan, Frank; Patel, Hetalben

    2013-01-01

    This review is focused on three types of enzymes decarboxylating very different substrates: (1) Thiamin diphosphate (ThDP)-dependent enzymes reacting with 2-oxo acids; (2) Pyridoxal phosphate (PLP)-dependent enzymes reacting with α-amino acids; and (3) An enzyme with no known co-factors, orotidine 5'-monophosphate decarboxylase (OMPDC). While the first two classes have been much studied for many years, during the past decade studies of both classes have revealed novel mechanistic insight challenging accepted understanding. The enzyme OMPDC has posed a challenge to the enzymologist attempting to explain a 1017-fold rate acceleration in the absence of cofactors or even metal ions. A comparison of the available evidence on the three types of decarboxylases underlines some common features and more differences. The field of decarboxylases remains an interesting and challenging one for the mechanistic enzymologist notwithstanding the large amount of information already available. PMID:23914308

  4. Genetic characterization of the Neurospora crassa molybdenum cofactor biosynthesis.

    PubMed

    Probst, Corinna; Ringel, Phillip; Boysen, Verena; Wirsing, Lisette; Alexander, Mariko Matsuda; Mendel, Ralf R; Kruse, Tobias

    2014-05-01

    Molybdenum (Mo) is a trace element that is essential for important cellular processes. To gain biological activity, Mo must be complexed in the molybdenum cofactor (Moco), a pterin derivative of low molecular weight. Moco synthesis is a multi-step pathway that involves a variable number of genes in eukaryotes, which are assigned to four steps of eukaryotic Moco biosynthesis. Moco biosynthesis mutants lack any Moco-dependent enzymatic activities, including assimilation of nitrate (plants and fungi), detoxification of sulfite (humans and plants) and utilization of hypoxanthine as sole N-source (fungi). We report the first comprehensive genetic characterization of the Neurospora crassa (N. crassa) Moco biosynthesis pathway, annotating five genes which encode all pathway enzymes, and compare it with the characterized Aspergillus nidulans pathway. Biochemical characterization of the corresponding knock-out mutants confirms our annotation model, documenting the N. crassa/A. nidulans (fungal) Moco biosynthesis as unique, combining the organizational structure of both plant and human Moco biosynthesis genes. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Combination of Biological Screening in a Cellular Model of Viral Latency and Virtual Screening Identifies Novel Compounds That Reactivate HIV-1

    PubMed Central

    Gallastegui, Edurne; Marshall, Brett; Vidal, David; Sanchez-Duffhues, Gonzalo; Collado, Juan A.; Alvarez-Fernández, Carmen; Luque, Neus; Terme, Jean-Michel; Gatell, Josep M.; Sánchez-Palomino, Sonsoles; Muñoz, Eduardo; Mestres, Jordi; Verdin, Eric

    2012-01-01

    Although highly active antiretroviral therapy (HAART) has converted HIV into a chronic disease, a reservoir of HIV latently infected resting T cells prevents the eradication of the virus from patients. To achieve eradication, HAART must be combined with drugs that reactivate the dormant viruses. We examined this problem in an established model of HIV postintegration latency by screening a library of small molecules. Initially, we identified eight molecules that reactivated latent HIV. Using them as templates, additional hits were identified by means of similarity-based virtual screening. One of those hits, 8-methoxy-6-methylquinolin-4-ol (MMQO), proved to be useful to reactivate HIV-1 in different cellular models, especially in combination with other known reactivating agents, without causing T-cell activation and with lower toxicity than that of the initial hits. Interestingly, we have established that MMQO produces Jun N-terminal protein kinase (JNK) activation and enhances the T-cell receptor (TCR)/CD3 stimulation of HIV-1 reactivation from latency but inhibits CD3-induced interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-α) gene transcription. Moreover, MMQO prevents TCR-induced cell cycle progression and proliferation in primary T cells. The present study documents that the combination of biological screening in a cellular model of viral latency with virtual screening is useful for the identification of novel agents able to reactivate HIV-1. Moreover, we set the bases for a hypothetical therapy to reactivate latent HIV by combining MMQO with physiological or pharmacological TCR/CD3 stimulation. PMID:22258251

  6. Cofactor dependent conformational switching of GTPases.

    PubMed

    Hauryliuk, Vasili; Hansson, Sebastian; Ehrenberg, Måns

    2008-08-01

    This theoretical work covers structural and biochemical aspects of nucleotide binding and GDP/GTP exchange of GTP hydrolases belonging to the family of small GTPases. Current models of GDP/GTP exchange regulation are often based on two specific assumptions. The first is that the conformation of a GTPase is switched by the exchange of the bound nucleotide from GDP to GTP or vice versa. The second is that GDP/GTP exchange is regulated by a guanine nucleotide exchange factor, which stabilizes a GTPase conformation with low nucleotide affinity. Since, however, recent biochemical and structural data seem to contradict this view, we present a generalized scheme for GTPase action. This novel ansatz accounts for those important cases when conformational switching in addition to guanine nucleotide exchange requires the presence of cofactors, and gives a more nuanced picture of how the nucleotide exchange is regulated. The scheme is also used to discuss some problems of interpretation that may arise when guanine nucleotide exchange mechanisms are inferred from experiments with analogs of GTP, like GDPNP, GDPCP, and GDP gamma S.

  7. Cofactor Dependent Conformational Switching of GTPases

    PubMed Central

    Hauryliuk, Vasili; Hansson, Sebastian; Ehrenberg, Måns

    2008-01-01

    This theoretical work covers structural and biochemical aspects of nucleotide binding and GDP/GTP exchange of GTP hydrolases belonging to the family of small GTPases. Current models of GDP/GTP exchange regulation are often based on two specific assumptions. The first is that the conformation of a GTPase is switched by the exchange of the bound nucleotide from GDP to GTP or vice versa. The second is that GDP/GTP exchange is regulated by a guanine nucleotide exchange factor, which stabilizes a GTPase conformation with low nucleotide affinity. Since, however, recent biochemical and structural data seem to contradict this view, we present a generalized scheme for GTPase action. This novel ansatz accounts for those important cases when conformational switching in addition to guanine nucleotide exchange requires the presence of cofactors, and gives a more nuanced picture of how the nucleotide exchange is regulated. The scheme is also used to discuss some problems of interpretation that may arise when guanine nucleotide exchange mechanisms are inferred from experiments with analogs of GTP, like GDPNP, GDPCP, and GDP \\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\usepackage[Euler]{upgreek} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}{\\gamma}\\end{equation*}\\end{document} S. PMID:18502805

  8. Engineering the Assembly of Heme Cofactors in Man-Made Proteins

    PubMed Central

    2015-01-01

    Timely ligation of one or more chemical cofactors at preselected locations in proteins is a critical preamble for catalysis in many natural enzymes, including the oxidoreductases and allied transport and signaling proteins. Likewise, ligation strategies must be directly addressed when designing oxidoreductase and molecular transport functions in man-made, first-principle protein constructs intended to operate in vitro or in vivo. As one of the most common catalytic cofactors in biology, we have chosen heme B, along with its chemical analogues, to determine the kinetics and barriers to cofactor incorporation and bishistidine ligation in a range of 4-α-helix proteins. We compare five elementary synthetic designs (maquettes) and the natural cytochrome b562 that differ in oligomeric forms, apo- and holo-tertiary structural stability; qualities that we show can either assist or hinder assembly. The cofactor itself also imposes an assembly barrier if amphiphilicity ranges toward too hydrophobic or hydrophilic. With progressive removal of identified barriers, we achieve maquette assembly rates as fast as native cytochrome b562, paving the way to in vivo assembly of man-made hemoprotein maquettes and integration of artificial proteins into enzymatic pathways. PMID:24495285

  9. Cofactor mobility determines reaction outcome in the IMPDH and GMPR (β-α)8 barrel enzymes.

    PubMed

    Patton, Gregory C; Stenmark, Pål; Gollapalli, Deviprasad R; Sevastik, Robin; Kursula, Petri; Flodin, Susanne; Schuler, Herwig; Swales, Colin T; Eklund, Hans; Himo, Fahmi; Nordlund, Pär; Hedstrom, Lizbeth

    2011-10-30

    Inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate reductase (GMPR) belong to the same structural family, share a common set of catalytic residues and bind the same ligands. The structural and mechanistic features that determine reaction outcome in the IMPDH and GMPR family have not been identified. Here we show that the GMPR reaction uses the same intermediate E-XMP* as IMPDH, but in this reaction the intermediate reacts with ammonia instead of water. A single crystal structure of human GMPR type 2 with IMP and NADPH fortuitously captures three different states, each of which mimics a distinct step in the catalytic cycle of GMPR. The cofactor is found in two conformations: an 'in' conformation poised for hydride transfer and an 'out' conformation in which the cofactor is 6 Å from IMP. Mutagenesis along with substrate and cofactor analog experiments demonstrate that the out conformation is required for the deamination of GMP. Remarkably, the cofactor is part of the catalytic machinery that activates ammonia.

  10. A NADH-accepting imine reductase variant: Immobilization and cofactor regeneration by oxidative deamination.

    PubMed

    Gand, Martin; Thöle, Christian; Müller, Hubertus; Brundiek, Henrike; Bashiri, Ghader; Höhne, Matthias

    2016-07-20

    Engineering cofactor specificity of enzymes is a promising approach that can expand the application of enzymes for biocatalytic production of industrially relevant chemicals. Until now, only NADPH-dependent imine reductases (IREDs) are known. This limits their applications to reactions employing whole cells as a cost-efficient cofactor regeneration system. For applications of IREDs as cell-free catalysts, (i) we created an IRED variant showing an improved activity for NADH. With rational design we were able to identify four residues in the (R)-selective IRED from Streptomyces GF3587 (IR-Sgf3587), which coordinate the 2'-phosphate moiety of the NADPH cofactor. From a set of 15 variants, the highest NADH activity was caused by the single amino acid exchange K40A resulting in a 3-fold increased acceptance of NADH. (ii) We showed its applicability using an immobilisate obtained either from purified enzyme or from lysate using the EziG(™) carriers. Applying the variant and NADH, we reached 88% conversion in a preparative scale biotransformation when employing 4% (w/v) 2-methylpyrroline. (iii) We demonstrated a one-enzyme cofactor regeneration approach using the achiral amine N-methyl-3-aminopentanone as a hydrogen donor co-substrate.

  11. Engineering the assembly of heme cofactors in man-made proteins.

    PubMed

    Solomon, Lee A; Kodali, Goutham; Moser, Christopher C; Dutton, P Leslie

    2014-02-26

    Timely ligation of one or more chemical cofactors at preselected locations in proteins is a critical preamble for catalysis in many natural enzymes, including the oxidoreductases and allied transport and signaling proteins. Likewise, ligation strategies must be directly addressed when designing oxidoreductase and molecular transport functions in man-made, first-principle protein constructs intended to operate in vitro or in vivo. As one of the most common catalytic cofactors in biology, we have chosen heme B, along with its chemical analogues, to determine the kinetics and barriers to cofactor incorporation and bishistidine ligation in a range of 4-α-helix proteins. We compare five elementary synthetic designs (maquettes) and the natural cytochrome b562 that differ in oligomeric forms, apo- and holo-tertiary structural stability; qualities that we show can either assist or hinder assembly. The cofactor itself also imposes an assembly barrier if amphiphilicity ranges toward too hydrophobic or hydrophilic. With progressive removal of identified barriers, we achieve maquette assembly rates as fast as native cytochrome b562, paving the way to in vivo assembly of man-made hemoprotein maquettes and integration of artificial proteins into enzymatic pathways.

  12. Molecular Determinants of the Cofactor Specificity of Ribitol Dehydrogenase, a Short-Chain Dehydrogenase/Reductase

    PubMed Central

    Moon, Hee-Jung; Tiwari, Manish Kumar; Singh, Ranjitha

    2012-01-01

    Ribitol dehydrogenase from Zymomonas mobilis (ZmRDH) catalyzes the conversion of ribitol to d-ribulose and concomitantly reduces NAD(P)+ to NAD(P)H. A systematic approach involving an initial sequence alignment-based residue screening, followed by a homology model-based screening and site-directed mutagenesis of the screened residues, was used to study the molecular determinants of the cofactor specificity of ZmRDH. A homologous conserved amino acid, Ser156, in the substrate-binding pocket of the wild-type ZmRDH was identified as an important residue affecting the cofactor specificity of ZmRDH. Further insights into the function of the Ser156 residue were obtained by substituting it with other hydrophobic nonpolar or polar amino acids. Substituting Ser156 with the negatively charged amino acids (Asp and Glu) altered the cofactor specificity of ZmRDH toward NAD+ (S156D, [kcat/Km,NAD]/[kcat/Km,NADP] = 10.9, where Km,NAD is the Km for NAD+ and Km,NADP is the Km for NADP+). In contrast, the mutants containing positively charged amino acids (His, Lys, or Arg) at position 156 showed a higher efficiency with NADP+ as the cofactor (S156H, [kcat/Km,NAD]/[kcat/Km,NADP] = 0.11). These data, in addition to those of molecular dynamics and isothermal titration calorimetry studies, suggest that the cofactor specificity of ZmRDH can be modulated by manipulating the amino acid residue at position 156. PMID:22344653

  13. Molecular determinants of the cofactor specificity of ribitol dehydrogenase, a short-chain dehydrogenase/reductase.

    PubMed

    Moon, Hee-Jung; Tiwari, Manish Kumar; Singh, Ranjitha; Kang, Yun Chan; Lee, Jung-Kul

    2012-05-01

    Ribitol dehydrogenase from Zymomonas mobilis (ZmRDH) catalyzes the conversion of ribitol to d-ribulose and concomitantly reduces NAD(P)(+) to NAD(P)H. A systematic approach involving an initial sequence alignment-based residue screening, followed by a homology model-based screening and site-directed mutagenesis of the screened residues, was used to study the molecular determinants of the cofactor specificity of ZmRDH. A homologous conserved amino acid, Ser156, in the substrate-binding pocket of the wild-type ZmRDH was identified as an important residue affecting the cofactor specificity of ZmRDH. Further insights into the function of the Ser156 residue were obtained by substituting it with other hydrophobic nonpolar or polar amino acids. Substituting Ser156 with the negatively charged amino acids (Asp and Glu) altered the cofactor specificity of ZmRDH toward NAD(+) (S156D, [k(cat)/K(m)(,NAD)]/[k(cat)/K(m)(,NADP)] = 10.9, where K(m)(,NAD) is the K(m) for NAD(+) and K(m)(,NADP) is the K(m) for NADP(+)). In contrast, the mutants containing positively charged amino acids (His, Lys, or Arg) at position 156 showed a higher efficiency with NADP(+) as the cofactor (S156H, [k(cat)/K(m)(,NAD)]/[k(cat)/K(m)(,NADP)] = 0.11). These data, in addition to those of molecular dynamics and isothermal titration calorimetry studies, suggest that the cofactor specificity of ZmRDH can be modulated by manipulating the amino acid residue at position 156.

  14. The methanogenic redox cofactor F420 is widely synthesized by aerobic soil bacteria.

    PubMed

    Ney, Blair; Ahmed, F Hafna; Carere, Carlo R; Biswas, Ambarish; Warden, Andrew C; Morales, Sergio E; Pandey, Gunjan; Watt, Stephen J; Oakeshott, John G; Taylor, Matthew C; Stott, Matthew B; Jackson, Colin J; Greening, Chris

    2017-01-01

    F420 is a low-potential redox cofactor that mediates the transformations of a wide range of complex organic compounds. Considered one of the rarest cofactors in biology, F420 is best known for its role in methanogenesis and has only been chemically identified in two phyla to date, the Euryarchaeota and Actinobacteria. In this work, we show that this cofactor is more widely distributed than previously reported. We detected the genes encoding all five known F420 biosynthesis enzymes (cofC, cofD, cofE, cofG and cofH) in at least 653 bacterial and 173 archaeal species, including members of the dominant soil phyla Proteobacteria, Chloroflexi and Firmicutes. Metagenome datamining validated that these genes were disproportionately abundant in aerated soils compared with other ecosystems. We confirmed through high-performance liquid chromatography analysis that aerobically grown stationary-phase cultures of three bacterial species, Paracoccus denitrificans, Oligotropha carboxidovorans and Thermomicrobium roseum, synthesized F420, with oligoglutamate sidechains of different lengths. To understand the evolution of F420 biosynthesis, we also analyzed the distribution, phylogeny and genetic organization of the cof genes. Our data suggest that although the Fo precursor to F420 originated in methanogens, F420 itself was first synthesized in an ancestral actinobacterium. F420 biosynthesis genes were then disseminated horizontally to archaea and other bacteria. Together, our findings suggest that the cofactor is more significant in aerobic bacterial metabolism and soil ecosystem composition than previously thought. The cofactor may confer several competitive advantages for aerobic soil bacteria by mediating their central metabolic processes and broadening the range of organic compounds they can synthesize, detoxify and mineralize.

  15. Identification and Biochemical Characterization of Molybdenum Cofactor-binding Proteins from Arabidopsis thaliana*

    PubMed Central

    Kruse, Tobias; Gehl, Christian; Geisler, Mirco; Lehrke, Markus; Ringel, Phillip; Hallier, Stephan; Hänsch, Robert; Mendel, Ralf R.

    2010-01-01

    The molybdenum cofactor (Moco) forms part of the catalytic center in all eukaryotic molybdenum enzymes and is synthesized in a highly conserved pathway. Among eukaryotes, very little is known about the processes taking place subsequent to Moco biosynthesis, i.e. Moco transfer, allocation, and insertion into molybdenum enzymes. In the model plant Arabidopsis thaliana, we identified a novel protein family consisting of nine members that after recombinant expression are able to bind Moco with KD values in the low micromolar range and are therefore named Moco-binding proteins (MoBP). For two of the nine proteins atomic structures are available in the Protein Data Bank. Surprisingly, both crystal structures lack electron density for the C terminus, which may indicate a high flexibility of this part of the protein. C-terminal truncated MoBPs showed significantly decreased Moco binding stoichiometries. Experiments where the MoBP C termini were exchanged among MoBPs converted a weak Moco-binding MoBP into a strong binding MoBP, thus indicating that the MoBP C terminus, which is encoded by a separate exon, is involved in Moco binding. MoBPs were able to enhance Moco transfer to apo-nitrate reductase in the Moco-free Neurospora crassa mutant nit-1. Furthermore, we show that the MoBPs are localized in the cytosol and undergo protein-protein contact with both the Moco donor protein Cnx1 and the Moco acceptor protein nitrate reductase under in vivo conditions, thus indicating for the MoBPs a function in Arabidopsis cellular Moco distribution. PMID:20040598

  16. Dietary restriction: critical co-factors to separate health span from life span benefits.

    PubMed

    Mendelsohn, Andrew R; Larrick, James W

    2012-10-01

    Dietary restriction (DR), typically a 20%-40% reduction in ad libitum or "normal" nutritional energy intake, has been reported to extend life span in diverse organisms, including yeast, nematodes, spiders, fruit flies, mice, rats, and rhesus monkeys. The magnitude of the life span enhancement appears to diminish with increasing organismal complexity. However, the extent of life span extension has been notoriously inconsistent, especially in mammals. Recently, Mattison et al. reported that DR does not extend life span in rhesus monkeys in contrast to earlier work of Colman et al. Examination of these papers identifies multiple potential confounding factors. Among these are the varied genetic backgrounds and composition of the "normal" and DR diets. In monkeys, the correlation of DR with increased health span is stronger than that seen with life span and indeed may be separable. Recent mechanistic studies in Drosophila implicate non-genetic co-factors such as level of physical activity and muscular fatty acid metabolism in the benefits of DR. These results should be followed up in mammals. Perhaps levels of physical activity among the cohorts of rhesus monkeys contribute to inconsistent DR effects. To understand the maximum potential benefits from DR requires differentiating fundamental effects on aging at the cellular and molecular levels from suppression of age-associated diseases, such as cancer. To that end, it is important that investigators carefully evaluate the effects of DR on biomarkers of molecular aging, such as mutation rate and epigenomic alterations. Several short-term studies show that humans may benefit from DR in as little as 6 months, by achieving lowered fasting insulin levels and improved cardiovascular health. Optimized health span engineering will require a much deeper understanding of DR.

  17. Discovery of cofactor-specific, bactericidal Mycobacterium tuberculosis InhA inhibitors using DNA-encoded library technology

    PubMed Central

    Soutter, Holly H.; Centrella, Paolo; Clark, Matthew A.; Cuozzo, John W.; Dumelin, Christoph E.; Guie, Marie-Aude; Habeshian, Sevan; Keefe, Anthony D.; Kennedy, Kaitlyn M.; Sigel, Eric A.; Troast, Dawn M.; Zhang, Ying; Ferguson, Andrew D.; Davies, Gareth; Stead, Eleanor R.; Breed, Jason; Madhavapeddi, Prashanti; Read, Jon A.

    2016-01-01

    Millions of individuals are infected with and die from tuberculosis (TB) each year, and multidrug-resistant (MDR) strains of TB are increasingly prevalent. As such, there is an urgent need to identify novel drugs to treat TB infections. Current frontline therapies include the drug isoniazid, which inhibits the essential NADH-dependent enoyl–acyl-carrier protein (ACP) reductase, InhA. To inhibit InhA, isoniazid must be activated by the catalase-peroxidase KatG. Isoniazid resistance is linked primarily to mutations in the katG gene. Discovery of InhA inhibitors that do not require KatG activation is crucial to combat MDR TB. Multiple discovery efforts have been made against InhA in recent years. Until recently, despite achieving high potency against the enzyme, these efforts have been thwarted by lack of cellular activity. We describe here the use of DNA-encoded X-Chem (DEX) screening, combined with selection of appropriate physical properties, to identify multiple classes of InhA inhibitors with cell-based activity. The utilization of DEX screening allowed the interrogation of very large compound libraries (1011 unique small molecules) against multiple forms of the InhA enzyme in a multiplexed format. Comparison of the enriched library members across various screening conditions allowed the identification of cofactor-specific inhibitors of InhA that do not require activation by KatG, many of which had bactericidal activity in cell-based assays. PMID:27864515

  18. Discovery of cofactor-specific, bactericidal Mycobacterium tuberculosis InhA inhibitors using DNA-encoded library technology.

    PubMed

    Soutter, Holly H; Centrella, Paolo; Clark, Matthew A; Cuozzo, John W; Dumelin, Christoph E; Guie, Marie-Aude; Habeshian, Sevan; Keefe, Anthony D; Kennedy, Kaitlyn M; Sigel, Eric A; Troast, Dawn M; Zhang, Ying; Ferguson, Andrew D; Davies, Gareth; Stead, Eleanor R; Breed, Jason; Madhavapeddi, Prashanti; Read, Jon A

    2016-12-06

    Millions of individuals are infected with and die from tuberculosis (TB) each year, and multidrug-resistant (MDR) strains of TB are increasingly prevalent. As such, there is an urgent need to identify novel drugs to treat TB infections. Current frontline therapies include the drug isoniazid, which inhibits the essential NADH-dependent enoyl-acyl-carrier protein (ACP) reductase, InhA. To inhibit InhA, isoniazid must be activated by the catalase-peroxidase KatG. Isoniazid resistance is linked primarily to mutations in the katG gene. Discovery of InhA inhibitors that do not require KatG activation is crucial to combat MDR TB. Multiple discovery efforts have been made against InhA in recent years. Until recently, despite achieving high potency against the enzyme, these efforts have been thwarted by lack of cellular activity. We describe here the use of DNA-encoded X-Chem (DEX) screening, combined with selection of appropriate physical properties, to identify multiple classes of InhA inhibitors with cell-based activity. The utilization of DEX screening allowed the interrogation of very large compound libraries (10(11) unique small molecules) against multiple forms of the InhA enzyme in a multiplexed format. Comparison of the enriched library members across various screening conditions allowed the identification of cofactor-specific inhibitors of InhA that do not require activation by KatG, many of which had bactericidal activity in cell-based assays.

  19. In vivo generation of flavoproteins with modified cofactors.

    PubMed

    Mathes, Tilo; Vogl, Christian; Stolz, Jürgen; Hegemann, Peter

    2009-02-06

    To understand flavoprotein mechanisms and reactivity, biochemical and biophysical methods are usually employed, and differences between wild-type and mutated proteins with altered primary structures are placed under specific consideration. Alternatively, the cofactor can be modified, and modified flavoproteins can be studied accordingly. Here we present an efficient and general method for modifying the cofactor of flavoproteins in vivo. The modified cofactor is incorporated into apoprotein during protein biosynthesis in a riboflavin-auxotrophic Escherichia coli strain, which expresses a bacterial riboflavin transporter to import flavins from the medium. This system was used to introduce roseoflavin into the riboflavin-binding protein dodecin and into microbial blue-light photoreceptors of the BLUF (blue-light sensors using FAD) and LOV (light oxygen voltage) families. The modified photoreceptors showed absorption and fluorescence different from those of proteins carrying their natural cofactor or chromophores in solution, but did not show any photochemical reaction as implied by former physiological studies.

  20. Host co-factors of the retrovirus-like transposon Ty1

    PubMed Central

    2012-01-01

    Background Long-terminal repeat (LTR) retrotransposons have complex modes of mobility involving reverse transcription of their RNA genomes in cytoplasmic virus-like particles (VLPs) and integration of the cDNA copies into the host genome. The limited coding capacity of retrotransposons necessitates an extensive reliance on host co-factors; however, it has been challenging to identify co-factors that are required for endogenous retrotransposon mobility because retrotransposition is such a rare event. Results To circumvent the low frequency of Ty1 LTR-retrotransposon mobility in Saccharomyces cerevisiae, we used iterative synthetic genetic array (SGA) analysis to isolate host mutations that reduce retrotransposition. Query strains that harbor a chromosomal Ty1his3AI reporter element and either the rtt101Δ or med1Δ mutation, both of which confer a hypertransposition phenotype, were mated to 4,847 haploid ORF deletion strains. Retrotransposition was measured in the double mutant progeny, and a set of 275 ORF deletions that suppress the hypertransposition phenotypes of both rtt101Δ and med1Δ were identified. The corresponding set of 275 retrotransposition host factors (RHFs) includes 45 previously identified Ty1 or Ty3 co-factors. More than half of the RHF genes have statistically robust human homologs (E < 1 x 10-10). The level of unintegrated Ty1 cDNA in 181 rhfΔ single mutants was altered <2-fold, suggesting that the corresponding co-factors stimulate retrotransposition at a step after cDNA synthesis. However, deletion of 43 RHF genes, including specific ribosomal protein and ribosome biogenesis genes and RNA degradation, modification and transport genes resulted in low Ty1 cDNA levels. The level of Ty1 Gag but not RNA was reduced in ribosome biogenesis mutants bud21Δ, hcr1Δ, loc1Δ, and puf6Δ. Conclusion Ty1 retrotransposition is dependent on multiple co-factors acting at different steps in the replication cycle. Human orthologs of these RHFs are

  1. An array of Escherichia coli clones over-expressing essential proteins: A new strategy of identifying cellular targets of potent antibacterial compounds

    SciTech Connect

    Xu, H. Howard . E-mail: hxu3@calstatela.edu; Real, Lilian; Bailey, Melissa Wu

    2006-11-03

    With the advancement of high throughput screening, it has become easier and faster to discover hit compounds that inhibit proliferation of bacterial cells. However, development in technologies used to identify cellular targets of potent antibacterial inhibitors has lagged behind. Here, we describe a novel strategy of target identification for antibacterial inhibitors using an array of Escherichia coli clones each over-expressing one essential protein. In a proof-of-concept study, eight essential genes were cloned into pLex5BA vector under the control of an inducible promoter. Over-expression of target proteins was confirmed. For two clones, one over-expressing FabI and the other over-expressing MurA enzymes, the host cells became 17- and 139-fold more resistant to the specific inhibitors triclosan and phosphomycin, respectively, while the susceptibility of other clones towards these inhibitors remained unchanged after induction of gene expression. Target identification via target protein over-expression was demonstrated using both mixed clone and individual clone assay formats.

  2. A proteomic screen with Drosophila Opa1-like identifies Hsc70-5/Mortalin as a regulator of mitochondrial morphology and cellular homeostasis.

    PubMed

    Banerjee, Shamik; Chinthapalli, Balaji

    2014-09-01

    Mitochondrial morphology is regulated by conserved proteins involved in fusion and fission processes. The mammalian Optic atrophy 1 (OPA1) that functions in mitochondrial fusion is associated with Optic Atrophy and has been implicated in inner membrane cristae remodeling during cell death. Here, we show Drosophila Optic atrophy 1-like (Opa1-like) influences mitochondrial morphology through interaction with 'mitochondria-shaping' proteins like Mitochondrial assembly regulatory factor (Marf) and Drosophila Mitofilin (dMitofilin). To gain an insight into Opa1-like's network, we delineated bonafide interactors like dMitofilin, Marf, Serine protease High temperature requirement protein A2 (HTRA2), Rhomboid-7 (Rho-7) along with novel interactors such as Mortalin ortholog (Hsc70-5) from Drosophila mitochondrial extract. Interestingly, RNAi mediated down-regulation of hsc70-5 in Drosophila wing imaginal disc's peripodial cells resulted in fragmented mitochondria with reduced membrane potential leading to proteolysis of Opa1-like. Increased ecdysone activity induced dysfunctional fragmented mitochondria for clearance through lysosomes, an effect enhanced in hsc70-5 RNAi leading to increased cell death. Over-expression of Opa1-like rescues mitochondrial morphology and cell death in prepupal tissues expressing hsc70-5 RNAi. Taken together, we have identified a novel interaction between Hsc70-5/Mortalin and Opa1-like that influences cellular homeostasis through mitochondrial fusion.

  3. Hyperoxia-Induced Protein Alterations in Renal Rat Tissue: A Quantitative Proteomic Approach to Identify Hyperoxia-Induced Effects in Cellular Signaling Pathways

    PubMed Central

    Hinkelbein, Jochen; Böhm, Lennert; Spelten, Oliver; Sander, David; Soltész, Stefan; Braunecker, Stefan

    2015-01-01

    Introduction. In renal tissue as well as in other organs, supranormal oxygen pressure may lead to deleterious consequences on a cellular level. Additionally, hyperoxia-induced effect in cells and related free radicals may potentially contribute to renal failure. The aim of this study was to analyze time-dependent alterations of rat kidney protein expression after short-term normobaric hyperoxia using proteomics and bioinformatic approaches. Material and Methods. N = 36 Wistar rats were randomized into six different groups: three groups with normobaric hyperoxia (exposure to 100% oxygen for 3 h) and three groups with normobaric normoxia (NN; room air). After hyperoxia exposure, kidneys were removed immediately, after 3 days and after 7 days. Kidney lysates were analyzed by two-dimensional gel electrophoresis followed by peptide mass fingerprinting using tandem mass spectrometry. Statistical analysis was performed with DeCyder 2D software (p < 0.01). Biological functions of differential regulated proteins were studied using functional network analysis (Ingenuity Pathways Analysis and PathwayStudio). Results. Expression of 14 proteins was significantly altered (p < 0.01): eight proteins (MEP1A_RAT, RSSA_RAT, F16P1_RAT, STML2_RAT, BPNT1_RAT, LGMN_RAT, ATPA_RAT, and VDAC1_RAT) were downregulated and six proteins (MTUS1_RAT, F16P1_RAT, ACTG_RAT, ACTB_RAT, 2ABA_RAT, and RAB1A_RAT) were upregulated. Bioinformatic analyses revealed an association of regulated proteins with inflammation. Conclusions. Significant alterations in renal protein expression could be demonstrated for up to 7 days even after short-term hyperoxia. The identified proteins indicate an association with inflammation signaling cascades. MEP1A and VDAC1 could be promising candidates to identify hyperoxic injury in kidney cells. PMID:26106253

  4. The modulation of WTI transcription function by cofactors.

    PubMed

    Roberts, Stefan G E

    2006-01-01

    Wilms' tumour is a paediatric malignancy of the kidneys that affects one in every 10,000 live births, making it the most common solid tumour in the young. This cancer arises due to a failure of the metanephric mesenchyme to differentiate and form the kidney filtration units and tubules, which instead undergo uncontrolled proliferation. WT1 (Wilms' tumour 1) was identified as a factor that is frequently mutated in Wilms' tumours. WT1 plays a central role in the development of the genito-urinary organs and also other regions of the embryo. A major function of WT1 is to act as a regulator of transcription, controlling the expression of genes that are involved in proliferation and differentiation. WT1 can either activate or repress transcription of its target genes. Thus the transcription function of WT1 is highly context-specific, and can be modulated by a number of cofactors. Here, the known interaction partners of WT1 and the mechanisms by which they modulate WT1 transcription function will be discussed.

  5. Identifying Candidate Genes that Underlie Cellular pH Sensitivity in Serotonin Neurons Using Transcriptomics: A Potential Role for Kir5.1 Channels

    PubMed Central

    Puissant, Madeleine M.; Mouradian, Gary C.; Liu, Pengyuan; Hodges, Matthew R.

    2017-01-01

    Ventilation is continuously adjusted by a neural network to maintain blood gases and pH. Acute CO2 and/or pH regulation requires neural feedback from brainstem cells that encode CO2/pH to modulate ventilation, including but not limited to brainstem serotonin (5-HT) neurons. Brainstem 5-HT neurons modulate ventilation and are stimulated by hypercapnic acidosis, the sensitivity of which increases with increasing postnatal age. The proper function of brainstem 5-HT neurons, particularly during post-natal development is critical given that multiple abnormalities in the 5-HT system have been identified in victims of Sudden Infant Death Syndrome. Here, we tested the hypothesis that there are age-dependent increases in expression of pH-sensitive ion channels in brainstem 5-HT neurons, which may underlie their cellular CO2/pH sensitivity. Midline raphe neurons were acutely dissociated from neonatal and mature transgenic SSePet-eGFP rats [which have enhanced green fluorescent protein (eGFP) expression in all 5-HT neurons] and sorted with fluorescence-activated cell sorting (FACS) into 5-HT-enriched and non-5-HT cell pools for subsequent RNA extraction, cDNA library preparation and RNA sequencing. Overlapping differential expression analyses pointed to age-dependent shifts in multiple ion channels, including but not limited to the pH-sensitive potassium ion (K+) channel genes kcnj10 (Kir4.1), kcnj16 (Kir5.1), kcnk1 (TWIK-1), kcnk3 (TASK-1) and kcnk9 (TASK-3). Intracellular contents isolated from single adult eGFP+ 5-HT neurons confirmed gene expression of Kir4.1, Kir5.1 and other K+ channels, but also showed heterogeneity in the expression of multiple genes. 5-HT neuron-enriched cell pools from selected post-natal ages showed increases in Kir4.1, Kir5.1, and TWIK-1, fitting with age-dependent increases in Kir4.1 and Kir5.1 protein expression in raphe tissue samples. Immunofluorescence imaging confirmed Kir5.1 protein was co-localized to brainstem neurons and glia including 5

  6. Variable motif utilization in homeotic selector (Hox)-cofactor complex formation controls specificity.

    PubMed

    Lelli, Katherine M; Noro, Barbara; Mann, Richard S

    2011-12-27

    Homeotic selector (Hox) proteins often bind DNA cooperatively with cofactors such as Extradenticle (Exd) and Homothorax (Hth) to achieve functional specificity in vivo. Previous studies identified the Hox YPWM motif as an important Exd interaction motif. Using a comparative approach, we characterize the contribution of this and additional conserved sequence motifs to the regulation of specific target genes for three Drosophila Hox proteins. We find that Sex combs reduced (Scr) uses a simple interaction mechanism, where a single tryptophan-containing motif is necessary for Exd-dependent DNA-binding and in vivo functions. Abdominal-A (AbdA) is more complex, using multiple conserved motifs in a context-dependent manner. Lastly, Ultrabithorax (Ubx) is the most flexible, in that it uses multiple conserved motifs that function in parallel to regulate target genes in vivo. We propose that using different binding mechanisms with the same cofactor may be one strategy to achieve functional specificity in vivo.

  7. Transcriptional co-factor Transducin beta-like (TBL) 1 acts as a checkpoint in pancreatic cancer malignancy

    PubMed Central

    Stoy, Christian; Sundaram, Aishwarya; Rios Garcia, Marcos; Wang, Xiaoyue; Seibert, Oksana; Zota, Annika; Wendler, Susann; Männle, David; Hinz, Ulf; Sticht, Carsten; Muciek, Maria; Gretz, Norbert; Rose, Adam J; Greiner, Vera; Hofmann, Thomas G; Bauer, Andrea; Hoheisel, Jörg; Berriel Diaz, Mauricio; Gaida, Matthias M; Werner, Jens; Schafmeier, Tobias; Strobel, Oliver; Herzig, Stephan

    2015-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer fatalities in Western societies, characterized by high metastatic potential and resistance to chemotherapy. Critical molecular mechanisms of these phenotypical features still remain unknown, thus hampering the development of effective prognostic and therapeutic measures in PDAC. Here, we show that transcriptional co-factor Transducin beta-like (TBL) 1 was over-expressed in both human and murine PDAC. Inactivation of TBL1 in human and mouse pancreatic cancer cells reduced cellular proliferation and invasiveness, correlating with diminished glucose uptake, glycolytic flux, and oncogenic PI3 kinase signaling which in turn could rescue TBL1 deficiency-dependent phenotypes. TBL1 deficiency both prevented and reversed pancreatic tumor growth, mediated transcriptional PI3 kinase inhibition, and increased chemosensitivity of PDAC cells in vivo. As TBL1 mRNA levels were also found to correlate with PI3 kinase levels and overall survival in a cohort of human PDAC patients, TBL1 was identified as a checkpoint in the malignant behavior of pancreatic cancer and its expression may serve as a novel molecular target in the treatment of human PDAC. PMID:26070712

  8. Interaction between the AAA+ ATPase p97 and its cofactor ataxin3 in health and disease: Nucleotide-induced conformational changes regulate cofactor binding.

    PubMed

    Rao, Maya V; Williams, Dewight R; Cocklin, Simon; Loll, Patrick J

    2017-09-22

    p97 is an essential ATPase associated with various cellular activities (AAA+) that functions as a segregase in diverse cellular processes, including the maintenance of proteostasis. p97 interacts with different cofactors that target it to distinct pathways; an important example is the deubiquitinase ataxin3, which collaborates with p97 in endoplasmic reticulum associated degradation (ERAD). However, the molecular details of this interaction have been unclear. Here, we characterized the binding of ataxin3 to p97, showing that ataxin3 binds with low-micromolar affinity to both wild-type p97 and mutants linked to degenerative disorders known as multisystem proteinopathy 1 (MSP1); we further showed that the stoichiometry of binding is one ataxin3 molecule per p97 hexamer. We mapped the binding determinants on each protein, demonstrating that ataxin3's p97/VCP-binding motif (VBM) interacts with the inter-lobe cleft in the N-domain of p97. We also probed the nucleotide dependence of this interaction, confirming that ataxin3 and p97 associate in the presence of ATP and in the absence of nucleotide, but not in the presence of ADP. Our experiments suggest that an ADP-driven downward movement of the p97 N-terminal domain dislodges ataxin3 by inducing a steric clash between the D1-domain and ataxin3's C-terminus. In contrast, MSP1 mutants of p97 bind ataxin3 irrespective of their nucleotide state, indicating a failure by these mutants to translate ADP binding into a movement of the N-terminal domain. Our model provides a mechanistic explanation for how nucleotides regulate the p97-ataxin3 interaction and why atypical cofactor binding is observed with MSP1 mutants. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  9. Coordinated remodeling of cellular metabolism during iron deficiency through targeted mRNA degradation.

    PubMed

    Puig, Sergi; Askeland, Eric; Thiele, Dennis J

    2005-01-14

    Iron (Fe) is an essential micronutrient for virtually all organisms and serves as a cofactor for a wide variety of vital cellular processes. Although Fe deficiency is the primary nutritional disorder in the world, cellular responses to Fe deprivation are poorly understood. We have discovered a posttranscriptional regulatory process controlled by Fe deficiency, which coordinately drives widespread metabolic reprogramming. We demonstrate that, in response to Fe deficiency, the Saccharomyces cerevisiae Cth2 protein specifically downregulates mRNAs encoding proteins that participate in many Fe-dependent processes. mRNA turnover requires the binding of Cth2, an RNA binding protein conserved in plants and mammals, to specific AU-rich elements in the 3' untranslated region of mRNAs targeted for degradation. These studies elucidate coordinated global metabolic reprogramming in response to Fe deficiency and identify a mechanism for achieving this by targeting specific mRNA molecules for degradation, thereby facilitating the utilization of limited cellular Fe levels.

  10. Cofactor Engineering for Enhancing the Flux of Metabolic Pathways

    PubMed Central

    Akhtar, M. Kalim; Jones, Patrik R.

    2014-01-01

    The manufacture of a diverse array of chemicals is now possible with biologically engineered strains, an approach that is greatly facilitated by the emergence of synthetic biology. This is principally achieved through pathway engineering in which enzyme activities are coordinated within a genetically amenable host to generate the product of interest. A great deal of attention is typically given to the quantitative levels of the enzymes with little regard to their overall qualitative states. This highly constrained approach fails to consider other factors that may be necessary for enzyme functionality. In particular, enzymes with physically bound cofactors, otherwise known as holoenzymes, require careful evaluation. Herein, we discuss the importance of cofactors for biocatalytic processes and show with empirical examples why the synthesis and integration of cofactors for the formation of holoenzymes warrant a great deal of attention within the context of pathway engineering. PMID:25221776

  11. Genetics Home Reference: molybdenum cofactor deficiency

    MedlinePlus

    ... affected individuals have high levels of chemicals called sulfite, S-sulfocysteine, xanthine, and hypoxanthine in the urine and ... activity leads to buildup of certain chemicals, including sulfite, S-sulfocysteine, xanthine, and hypoxanthine (which can be identified ...

  12. Developmental expression patterns of candidate co-factors for vertebrate Six family transcription factors

    PubMed Central

    Neilson, Karen M.; Pignoni, Francesca; Yan, Bo; Moody, Sally A.

    2010-01-01

    Six family transcription factors play important roles in craniofacial development. Their transcriptional activity can be modified by co-factor proteins. Two Six genes and one co-factor gene (Eya1) are involved in the human Branchio-otic (BO) and Branchio-otic-renal (BOR) syndromes. However, mutations in Six and Eya genes only account for about half of these patients. To discover potential new causative genes, we searched the Xenopus genome for orthologues of Drosophila co-factor proteins that interact with the fly Six-related factor, SO. We identified 33 Xenopus genes with high sequence identity to 20 of the 25 fly SO-interacting proteins. We provide the developmental expression patterns of the Xenopus orthologues for 11 of the fly genes, and demonstrate that all are expressed in developing craniofacial tissues with at least partial overlap with Six1/Six2. We speculate that these genes may function as Six-interacting partners with important roles in vertebrate craniofacial development and perhaps congenital syndromes. PMID:21089078

  13. The role of FeS clusters for molybdenum cofactor biosynthesis and molybdoenzymes in bacteria.

    PubMed

    Yokoyama, Kenichi; Leimkühler, Silke

    2015-06-01

    The biosynthesis of the molybdenum cofactor (Moco) has been intensively studied, in addition to its insertion into molybdoenzymes. In particular, a link between the assembly of molybdoenzymes and the biosynthesis of FeS clusters has been identified in the recent years: 1) the synthesis of the first intermediate in Moco biosynthesis requires an FeS-cluster containing protein, 2) the sulfurtransferase for the dithiolene group in Moco is also involved in the synthesis of FeS clusters, thiamin and thiolated tRNAs, 3) the addition of a sulfido-ligand to the molybdenum atom in the active site additionally involves a sulfurtransferase, and 4) most molybdoenzymes in bacteria require FeS clusters as redox active cofactors. In this review we will focus on the biosynthesis of the molybdenum cofactor in bacteria, its modification and insertion into molybdoenzymes, with an emphasis to its link to FeS cluster biosynthesis and sulfur transfer. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. The interplay between genotype, metabolic state and cofactor treatment governs phenylalanine hydroxylase function and drug response.

    PubMed

    Staudigl, Michael; Gersting, Søren W; Danecka, Marta K; Messing, Dunja D; Woidy, Mathias; Pinkas, Daniel; Kemter, Kristina F; Blau, Nenad; Muntau, Ania C

    2011-07-01

    The discovery of a pharmacological treatment for phenylketonuria (PKU) raised new questions about function and dysfunction of phenylalanine hydroxylase (PAH), the enzyme deficient in this disease. To investigate the interdependence of the genotype, the metabolic state (phenylalanine substrate) and treatment (BH(4) cofactor) in the context of enzyme function in vitro and in vivo, we (i) used a fluorescence-based method for fast enzyme kinetic analyses at an expanded range of phenylalanine and BH(4) concentrations, (ii) depicted PAH function as activity landscapes, (iii) retraced the analyses in eukaryotic cells, and (iv) translated this into the human system by analyzing the outcome of oral BH(4) loading tests. PAH activity landscapes uncovered the optimal working range of recombinant wild-type PAH and provided new insights into PAH kinetics. They demonstrated how mutations might alter enzyme function in the space of varying substrate and cofactor concentrations. Experiments in eukaryotic cells revealed that the availability of the active PAH enzyme depends on the phenylalanine-to-BH(4) ratio. Finally, evaluation of data from BH(4) loading tests indicated that the patient's genotype influences the impact of the metabolic state on drug response. The results allowed for visualization and a better understanding of PAH function in the physiological and pathological state as well as in the therapeutic context of cofactor treatment. Moreover, our data underscore the need for more personalized procedures to safely identify and treat patients with BH(4)-responsive PAH deficiency.

  15. The role of FeS clusters for molybdenum cofactor biosynthesis and molybdoenzymes in bacteria

    PubMed Central

    Yokoyama, Kenichi; Leimkühler, Silke

    2016-01-01

    Molybdenum is the only second row transition metal essential for biological systems, which is biologically available as molybdate ion. In eukarya, bacteria and archaea, molybdenum is bound to either to a tricyclic pyranopterin, thereby forming the molybdenum cofactor (Moco), or in some bacteria to the FeS cluster based iron-molybdenum cofactor (FeMoco), which forms the active site of nitrogenase. To date more than 50 Moco-containing enzymes have been purified and biochemically or structurally characterized. The physiological role of molybdenum in these enzymes is fundamental to organisms, since the reactions include the catalysis of key steps in carbon, nitrogen and sulfur metabolism. The catalyzed reactions are in most cases oxo-transfer reactions or the hydroxylation of carbon centers. The biosynthesis of Moco has been intensively studied, in addition to its insertion into molybdoenzymes. In particular, a link between the biosynthesis and maturation of molybdoenzymes and the biosynthesis and distribution of FeS clusters has been identified in the last years: 1) The synthesis of the first intermediate in Moco biosynthesis requires an FeS-cluster containing protein, 2) The sulfurtransferase for the dithiolene group in Moco is common also for the synthesis of FeS clusters, thiamin and thiolated tRNAs, 3) the modification of the active site with a sulfur atom additionally involves a sulfurtransferase, 4) most molybdoenzymes in bacteria require FeS clusters as additional redox active cofactors. In this review we will focus on the biosynthesis of the molybdenum cofactor in bacteria, its modification and insertion into molybdoenzymes, with an emphasis to its link to FeS cluster biosynthesis and sulfur transfer. PMID:25268953

  16. Identification of common and cell type specific LXXLL motif EcR cofactors using a bioinformatics refined candidate RNAi screen in Drosophila melanogaster cell lines

    PubMed Central

    2011-01-01

    Background During Drosophila development, titers of the steroid ecdysone trigger and maintain temporal and tissue specific biological transitions. Decades of evidence reveal that the ecdysone response is both unique to specific tissues and distinct among developmental timepoints. To achieve this diversity in response, the several isoforms of the Ecdysone Receptor, which transduce the hormone signal to the genome level, are believed to interact with tissue specific cofactors. To date, little is known about the identity of these cofactor interactions; therefore, we conducted a bioinformatics informed, RNAi luciferase reporter screen against a subset of putative candidate cofactors identified through an in silico proteome screen. Candidates were chosen based on criteria obtained from bioinformatic consensus of known nuclear receptor cofactors and homologs, including amino acid sequence motif content and context. Results The bioinformatics pre-screen of the Drosophila melanogaster proteome was successful in identifying an enriched putative candidate gene cohort. Over 80% of the genes tested yielded a positive hit in our reporter screen. We have identified both cell type specific and common cofactors which appear to be necessary for proper ecdysone induced gene regulation. We have determined that certain cofactors act as co-repressors to reduce target gene expression, while others act as co-activators to increase target gene expression. Interestingly, we find that a few of the cofactors shared among cell types have a reversible roles to function as co-repressors in certain cell types while in other cell types they serve as co-activators. Lastly, these proteins are highly conserved, with higher order organism homologs also harboring the LXXLL steroid receptor interaction domains, suggesting a highly conserved mode of steroid cell target specificity. Conclusions In conclusion, we submit these cofactors as novel components of the ecdysone signaling pathway in order to

  17. Pterin chemistry and its relationship to the molybdenum cofactor

    PubMed Central

    Basu, Partha; Burgmayer, Sharon J.N.

    2011-01-01

    The molybdenum cofactor is composed of a molybdenum coordinated by one or two rather complicated ligands known as either molybdopterin or pyranopterin. Pterin is one of a large family of bicyclic N-heterocycles called pteridines. Such molecules are widely found in Nature, having various forms to perform a variety of biological functions. This article describes the basic nomenclature of pterin, their biological roles, structure, chemical synthesis and redox reactivity. In addition, the biosynthesis of pterins and current models of the molybdenum cofactor are discussed. PMID:21607119

  18. Generation of protein-derived redox cofactors by posttranslational modification.

    PubMed

    Davidson, Victor L

    2011-01-01

    Redox enzymes which catalyze the oxidation and reduction of substrates are ubiquitous in nature. These enzymes typically possess exogenous cofactors to allow them to perform catalytic functions which cannot be accomplished using only amino acid residues. It is now evident that nature also employs an alternative strategy of generating catalytic and redox-active sites in proteins by posttranslational modification of amino acid residues. This review describes the structures and functions of several of these protein-derived cofactors and the diverse mechanisms of posttranslational modification through which they are generated.

  19. Dendrite arborization requires the dynein cofactor NudE.

    PubMed

    Arthur, Ashley L; Yang, Sihui Z; Abellaneda, Allison M; Wildonger, Jill

    2015-06-01

    The microtubule-based molecular motor dynein is essential for proper neuronal morphogenesis. Dynein activity is regulated by cofactors, and the role(s) of these cofactors in shaping neuronal structure are still being elucidated. Using Drosophila melanogaster, we reveal that the loss of the dynein cofactor NudE results in abnormal dendrite arborization. Our data show that NudE associates with Golgi outposts, which mediate dendrite branching, suggesting that NudE normally influences dendrite patterning by regulating Golgi outpost transport. Neurons lacking NudE also have increased microtubule dynamics, reflecting a change in microtubule stability that is likely to also contribute to abnormal dendrite growth and branching. These defects in dendritogenesis are rescued by elevating levels of Lis1, another dynein cofactor that interacts with NudE as part of a tripartite complex. Our data further show that the NudE C-terminus is dispensable for dendrite morphogenesis and is likely to modulate NudE activity. We propose that a key function of NudE is to enhance an interaction between Lis1 and dynein that is crucial for motor activity and dendrite architecture.

  20. Chemistry and bioactivity of an artificial adenosylpeptide B(12) cofactor.

    PubMed

    Zhou, Kai; Oetterli, René M; Brandl, Helmut; Lyatuu, Fredrick E; Buckel, Wolfgang; Zelder, Felix

    2012-09-24

    Artificial influence: We describe a semi-artificial adenosylpeptide B(12) that behaves as a cofactor in B(12)-dependent enzymatic reactions and demonstrate that the peptide backbone influences its chemical properties and modulates its bioactivity in vitro and in vivo. Inhibition of the growth of L. delbrueckii is demonstrated, thus providing a potentially powerful approach for the development of antibacterial and antiproliferative compounds.

  1. Iron-molybdenum cofactor synthesis in Azotobacter vinelandii Nif- mutants.

    PubMed Central

    Imperial, J; Shah, V K; Ugalde, R A; Ludden, P W; Brill, W J

    1987-01-01

    Nif- mutants of Azotobacter vinelandii defective in dinitrogenase activity synthesized iron-molybdenum cofactor (FeMo-co) and accumulated it in two protein-bound forms: inactive dinitrogenase and a possible intermediate involved in the FeMo-co biosynthetic pathway. FeMo-co from both these proteins could activate apo-dinitrogenase from FeMo-co-deficient mutants. PMID:3470286

  2. Cofactor Trapping, a New Method To Produce Flavin Mononucleotide ▿

    PubMed Central

    Krauss, Ulrich; Svensson, Vera; Wirtz, Astrid; Knieps-Grünhagen, Esther; Jaeger, Karl-Erich

    2011-01-01

    We have purified flavin mononucleotide (FMN) from a flavoprotein-overexpressing Escherichia coli strain by cofactor trapping. This approach uses an overexpressed flavoprotein to trap FMN, which is thus removed from the cascade regulating FMN production in E. coli. This, in turn, allows the isolation of highly pure FMN. PMID:21131527

  3. Studies on free radicals, antioxidants, and co-factors

    PubMed Central

    Rahman, Khalid

    2007-01-01

    The interplay between free radicals, antioxidants, and co-factors is important in maintaining health, aging and age-related diseases. Free radicals induce oxidative stress, which is balanced by the body’s endogenous antioxidant systems with an input from co-factors, and by the ingestion of exogenous antioxidants. If the generation of free radicals exceeds the protective effects of antioxidants, and some co-factors, this can cause oxidative damage which accumulates during the life cycle, and has been implicated in aging, and age dependent diseases such as cardiovascular disease, cancer, neurodegenerative disorders, and other chronic conditions. The life expectancy of the world population is increasing, and it is estimated that by 2025, 29% of the world population will be aged ≥60 years, and this will lead to an increase in the number of older people acquiring age-related chronic diseases. This will place greater financial burden on health services and high social cost for individuals and society. In order to acheive healthy aging the older people should be encouraged to acquire healthy life styles which should include diets rich in antioxidants. The aim of this review is to highlight the main themes from studies on free radicals, antioxidants and co-factors, and to propose an evidence-based strategy for healthy aging. PMID:18044138

  4. Recognition of enzymes lacking bound cofactor by protein quality control

    PubMed Central

    Martínez-Limón, Adrián; Alriquet, Marion; Lang, Wei-Han; Calloni, Giulia; Wittig, Ilka; Vabulas, R. Martin

    2016-01-01

    Protein biogenesis is tightly linked to protein quality control (PQC). The role of PQC machinery in recognizing faulty polypeptides is becoming increasingly understood. Molecular chaperones and cytosolic and vacuolar degradation systems collaborate to detect, repair, or hydrolyze mutant, damaged, and mislocalized proteins. On the other hand, the contribution of PQC to cofactor binding-related enzyme maturation remains largely unexplored, although the loading of a cofactor represents an all-or-nothing transition in regard to the enzymatic function and thus must be surveyed carefully. Combining proteomics and biochemical analysis, we demonstrate here that cells are able to detect functionally immature wild-type enzymes. We show that PQC-dedicated ubiquitin ligase C-terminal Hsp70-interacting protein (CHIP) recognizes and marks for degradation not only a mutant protein but also its wild-type variant as long as the latter remains cofactor free. A distinct structural feature, the protruding C-terminal tail, which appears in both the mutant and wild-type polypeptides, contributes to recognition by CHIP. Our data suggest that relative insufficiency of apoprotein degradation caused by cofactor shortage can increase amyloidogenesis and aggravate protein aggregation disorders. PMID:27733512

  5. Structural Basis for Cofactor-Independent Dioxygenation in Vancomycin Biosynthesis

    SciTech Connect

    Widboom,P.; Fielding, E.; Liu, Y.; Bruner, S.

    2007-01-01

    Enzyme-catalyzed oxidations are some of the most common transformations in primary and secondary metabolism. The vancomycin biosynthetic enzyme DpgC belongs to a small class of oxygenation enzymes that are not dependent on an accessory cofactor or metal ion1. The detailed mechanism of cofactor-independent oxygenases has not been established. Here we report the first structure of an enzyme of this oxygenase class in complex with a bound substrate mimic. The use of a designed, synthetic substrate analogue allows unique insights into the chemistry of oxygen activation. The structure confirms the absence of cofactors, and electron density consistent with molecular oxygen is present adjacent to the site of oxidation on the substrate. Molecular oxygen is bound in a small hydrophobic pocket and the substrate provides the reducing power to activate oxygen for downstream chemical steps. Our results resolve the unique and complex chemistry of DpgC, a key enzyme in the biosynthetic pathway of an important class of antibiotics. Furthermore, mechanistic parallels exist between DpgC and cofactor-dependent flavoenzymes, providing information regarding the general mechanism of enzymatic oxygen activation.

  6. Iron as a Cofactor That Limits the Promotion of Cyanobacteria in Lakes Across a Tropic Gradient

    NASA Astrophysics Data System (ADS)

    Sorichetti, R. J.; Creed, I. F.; Trick, C. G.

    2014-12-01

    The frequency and intensity of cyanobacterial blooms (cyanoblooms) is increasing globally. While cyanoblooms in eutrophic (nutrient-rich) freshwater lakes are expected to persist and worsen with climate change projections, many of the "new" cyanobloom reports pertain to oligotrophic (nutrient-poor) freshwater lakes with no prior history of cyanobloom occurrence. Under the pressures of a changing climate, there exists a critical research need to revisit existing conceptual models and identify cyanobloom regulating factors currently unaccounted for. Iron (Fe) is required in nearly all pathways of cyanobacterial macronutrient use, though its precise role in regulating cyanobacterial biomass across the lake trophic gradient is not fully understood. The hypotheses tested were: (1) cyanobacteria will predominate in lakes when bioavailable Fe concentration is low, and (2) cyanobacteria overcome this Fe limitation in all lakes using the siderophore-based Fe acquisition strategy to scavenge Fe providing a competitive advantage over other phytoplankton. These hypotheses were tested using natural lakes across an oligo-meso-eutrophic gradient across Canada. In all lakes sampled, the relative cyanobacterial biomass was highest at low predicted Fe bioavailability (< 1.0 × 10-19 mol L-1). Within this range of low bioavailable Fe, iron-binding organic ligands were measured. Concentrations of ligands with reactive hydroxamate moieties were positively correlated to cyanobacterial biomass in both the oligotrophic (r2 = 0.77, p < 0.001) and eutrophic (r2 = 0.81, p < 0.001) lakes suggesting a possible low-Fe mediated cellular origin, siderophores. Fe-binding ligands with catecholate-type binding sites were detected in all lakes, although lack of a relationship with cyanobacterial biomass and a significant relationship with dissolved organic carbon (DOC) in oligotrophic (r2 = 0.65, p < 0.001) and eutrophic (r2 = 0.65, p < 0.001) lakes may indicate an allochthonous source that is not

  7. Dual Role of the Molybdenum Cofactor Biosynthesis Protein MOCS3 in tRNA Thiolation and Molybdenum Cofactor Biosynthesis in Humans*

    PubMed Central

    Chowdhury, Mita Mullick; Dosche, Carsten; Löhmannsröben, Hans-Gerd; Leimkühler, Silke

    2012-01-01

    We studied two pathways that involve the transfer of persulfide sulfur in humans, molybdenum cofactor biosynthesis and tRNA thiolation. Investigations using human cells showed that the two-domain protein MOCS3 is shared between both pathways. MOCS3 has an N-terminal adenylation domain and a C-terminal rhodanese-like domain. We showed that MOCS3 activates both MOCS2A and URM1 by adenylation and a subsequent sulfur transfer step for the formation of the thiocarboxylate group at the C terminus of each protein. MOCS2A and URM1 are β-grasp fold proteins that contain a highly conserved C-terminal double glycine motif. The role of the terminal glycine of MOCS2A and URM1 was examined for the interaction and the cellular localization with MOCS3. Deletion of the C-terminal glycine of either MOCS2A or URM1 resulted in a loss of interaction with MOCS3. Enhanced cyan fluorescent protein and enhanced yellow fluorescent protein fusions of the proteins were constructed, and the fluorescence resonance energy transfer efficiency was determined by the decrease in the donor lifetime. The cellular localization results showed that extension of the C terminus with an additional glycine of MOCS2A and URM1 altered the localization of MOCS3 from the cytosol to the nucleus. PMID:22453920

  8. The dual role of fission yeast Tbc1/cofactor C orchestrates microtubule homeostasis in tubulin folding and acts as a GAP for GTPase Alp41/Arl2

    PubMed Central

    Mori, Risa; Toda, Takashi

    2013-01-01

    Supplying the appropriate amount of correctly folded α/β-tubulin heterodimers is critical for microtubule dynamics. Formation of assembly-competent heterodimers is remarkably elaborate at the molecular level, in which the α- and β-tubulins are separately processed in a chaperone-dependent manner. This sequential step is performed by the tubulin-folding cofactor pathway, comprising a specific set of regulatory proteins: cofactors A–E. We identified the fission yeast cofactor: the orthologue of cofactor C, Tbc1. In addition to its roles in tubulin folding, Tbc1 acts as a GAP in regulating Alp41/Arl2, a highly conserved small GTPase. Of interest, the expression of GDP- or GTP-bound Alp41 showed the identical microtubule loss phenotype, suggesting that continuous cycling between these forms is important for its functions. In addition, we found that Alp41 interacts with Alp1D, the orthologue of cofactor D, specifically when in the GDP-bound form. Intriguingly, Alp1D colocalizes with microtubules when in excess, eventually leading to depolymerization, which is sequestered by co-overproducing GDP-bound Alp41. We present a model of the final stages of the tubulin cofactor pathway that includes a dual role for both Tbc1 and Alp1D in opposing regulation of the microtubule. PMID:23576550

  9. Effect of organic matter on nitrogenase metal cofactors homeostasis in Azotobacter vinelandii under diazotrophic conditions.

    PubMed

    Noumsi, Christelle Jouogo; Pourhassan, Nina; Darnajoux, Romain; Deicke, Michael; Wichard, Thomas; Burrus, Vincent; Bellenger, Jean-Philippe

    2016-02-01

    Biological nitrogen fixation can be catalysed by three isozymes of nitrogenase: molybdenum (Mo)-nitrogenase, vanadium (V)-nitrogenase and iron-only (Fe)-nitrogenase. The activity of these isozymes strongly depends on their metal cofactors, molybdenum, vanadium and iron, and their bioavailability in ecosystems. Here, we show how metal bioavailability can be affected by the presence of tannic acid (organic matter), and the subsequent consequences on diazotrophic growth of the soil bacterium Azotobacter vinelandii. In the presence of tannic acids, A. vinelandii produces a higher amount of metallophores, which coincides with an active, regulated and concomitant acquisition of molybdenum and vanadium under cellular conditions that are usually considered not molybdenum limiting. The associated nitrogenase genes exhibit decreased nifD expression and increased vnfD expression. Thus, in limiting bioavailable metal conditions, A. vinelandii takes advantage of its nitrogenase diversity to ensure optimal diazotrophic growth. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  10. [Cofactors in the course of HIV infection].

    PubMed

    Meyer, L; Carré, N

    Cohorts of patients infected with the human immunodeficiency virus (HIV), and followed-up since their infection, have identified risk factors of progression to acquired immunodeficiency syndrome (AIDS). The risk of progression increases with the subject's age at contamination by 40% for each decade. Other host factors such as certain HLA subtypes would be related to progression. Virus-related factors have also been described. Sexual or transfusional transmission from a highly immunodepressed subject increases the risk of progression in the infected subject. Progression is more rapid in male homosexuals than in heterosexuals, even after exclusion of Kaposi's syndrome. There has been little success in isolating co-infections which might explain this finding. The more rapid progression in homosexuals could be due to infection with particularly virulent strains or particular subtypes. Finally, progresion is more rapid when signs of primary infection are major or prolonged, an observation which probably results from a complex host-virus interaction. Behavioral factors occurring after contamination (pregnancy, continued intravenous drug abuse, tobacco, alcohol) have not been demonstrated until now to play a role in progression.

  11. An Ancient Fingerprint Indicates the Common Ancestry of Rossmann-Fold Enzymes Utilizing Different Ribose-Based Cofactors

    PubMed Central

    Laurino, Paola; Tóth-Petróczy, Ágnes; Meana-Pañeda, Rubén; Lin, Wei; Truhlar, Donald G.; Tawfik, Dan S.

    2016-01-01

    Nucleoside-based cofactors are presumed to have preceded proteins. The Rossmann fold is one of the most ancient and functionally diverse protein folds, and most Rossmann enzymes utilize nucleoside-based cofactors. We analyzed an omnipresent Rossmann ribose-binding interaction: a carboxylate side chain at the tip of the second β-strand (β2-Asp/Glu). We identified a canonical motif, defined by the β2-topology and unique geometry. The latter relates to the interaction being bidentate (both ribose hydroxyls interacting with the carboxylate oxygens), to the angle between the carboxylate and the ribose, and to the ribose’s ring configuration. We found that this canonical motif exhibits hallmarks of divergence rather than convergence. It is uniquely found in Rossmann enzymes that use different cofactors, primarily SAM (S-adenosyl methionine), NAD (nicotinamide adenine dinucleotide), and FAD (flavin adenine dinucleotide). Ribose-carboxylate bidentate interactions in other folds are not only rare but also have a different topology and geometry. We further show that the canonical geometry is not dictated by a physical constraint—geometries found in noncanonical interactions have similar calculated bond energies. Overall, these data indicate the divergence of several major Rossmann-fold enzyme classes, with different cofactors and catalytic chemistries, from a common pre-LUCA (last universal common ancestor) ancestor that possessed the β2-Asp/Glu motif. PMID:26938925

  12. An Ancient Fingerprint Indicates the Common Ancestry of Rossmann-Fold Enzymes Utilizing Different Ribose-Based Cofactors.

    PubMed

    Laurino, Paola; Tóth-Petróczy, Ágnes; Meana-Pañeda, Rubén; Lin, Wei; Truhlar, Donald G; Tawfik, Dan S

    2016-03-01

    Nucleoside-based cofactors are presumed to have preceded proteins. The Rossmann fold is one of the most ancient and functionally diverse protein folds, and most Rossmann enzymes utilize nucleoside-based cofactors. We analyzed an omnipresent Rossmann ribose-binding interaction: a carboxylate side chain at the tip of the second β-strand (β2-Asp/Glu). We identified a canonical motif, defined by the β2-topology and unique geometry. The latter relates to the interaction being bidentate (both ribose hydroxyls interacting with the carboxylate oxygens), to the angle between the carboxylate and the ribose, and to the ribose's ring configuration. We found that this canonical motif exhibits hallmarks of divergence rather than convergence. It is uniquely found in Rossmann enzymes that use different cofactors, primarily SAM (S-adenosyl methionine), NAD (nicotinamide adenine dinucleotide), and FAD (flavin adenine dinucleotide). Ribose-carboxylate bidentate interactions in other folds are not only rare but also have a different topology and geometry. We further show that the canonical geometry is not dictated by a physical constraint--geometries found in noncanonical interactions have similar calculated bond energies. Overall, these data indicate the divergence of several major Rossmann-fold enzyme classes, with different cofactors and catalytic chemistries, from a common pre-LUCA (last universal common ancestor) ancestor that possessed the β2-Asp/Glu motif.

  13. Structural alteration of cofactor specificity in Corynebacterium 2,5-diketo-D-gluconic acid reductase

    PubMed Central

    Sanli, Gulsah; Banta, Scott; Anderson, Stephen; Blaber, Michael

    2004-01-01

    Corynebacterium 2,5-Diketo-D-gluconic acid reductase (2,5-DKGR) catalyzes the reduction of 2,5-diketo-D-gluconic acid (2,5-DKG) to 2-Keto-L-gulonic acid (2-KLG). 2-KLG is an immediate precursor to L-ascorbic acid (vitamin C), and 2,5-DKGR is, therefore, an important enzyme in a novel industrial method for the production of vitamin C. 2,5-DKGR, as with most other members of the aldo-keto reductase (AKR) superfamily, exhibits a preference for NADPH compared to NADH as a cofactor in the stereo-specific reduction of substrate. The application of 2,5-DKGR in the industrial production of vitamin C would be greatly enhanced if NADH could be efficiently utilized as a cofactor. A mutant form of 2,5-DKGR has previously been identified that exhibits two orders of magnitude higher activity with NADH in comparison to the wild-type enzyme, while retaining a high level of activity with NADPH. We report here an X-ray crystal structure of the holo form of this mutant in complex with NADH cofactor, as well as thermodynamic stability data. By comparing the results to our previously reported X-ray structure of the holo form of wild-type 2,5-DKGR in complex with NADPH, the structural basis of the differential NAD(P)H selectivity of wild-type and mutant 2,5-DKGR enzymes has been identified. PMID:14718658

  14. Structures and Activities of the Elongator Complex and Its Cofactors.

    PubMed

    Kolaj-Robin, Olga; Séraphin, Bertrand

    2017-01-01

    Elongator is a highly conserved eukaryotic protein complex consisting of two sets of six Elp proteins, while homologues of its catalytic subunit Elp3 are found in all the kingdoms of life. Although it was originally described as a transcription elongation factor, cumulating evidence suggests that its primary function is catalyzing tRNA modifications. In humans, defects in Elongator subunits are associated with neurological disorders and cancer. Although further studies are still required, a clearer picture of the molecular mechanism of action of Elongator and its cofactors has started to emerge within recent years that have witnessed significant development in the field. In this review we summarize recent Elongator-related findings provided largely by crystal structures of several subunits of the complex, the electron microscopy structure of the entire yeast holoenzyme, as well as the structure of the Elongator cofactor complex Kti11/Kti13. © 2017 Elsevier Inc. All rights reserved.

  15. Enzymic synthesis and cofactor activity of 3'-pyrophosphocoenzyme A.

    PubMed Central

    Mukai, J I; Sy, J; Lipmann, F

    1983-01-01

    The 3'-pyrophosphate derivative of CoA was synthesized by using the excreted 5'-to-3' pyrophosphoryl-transferring enzyme from Streptomyces adephospholyticus and ATP as donor and dephospho-CoA as acceptor. Cofactor activity of this new coenzyme A derivative was tested with Clostridium kluyveri phosphotransacetylase and hog heart succinic thiokinase. With the phosphotransacetylase, 3'-pyrophospho-CoA was found to be twice as active as CoA whereas dephospho-CoA was inactive. However, succinic thiokinase utilized all three types of CoA equally well. Adenosine 5'-monophosphate 3'-pyrophosphate also was synthesized and used as an analog of adenosine 5'-monophosphate 3'-monophosphate in the dog liver's sulfotransferase-catalyzed sulfate transfer from p-nitrophenyl sulfate to phenol. In contrast to the pyrophospho derivative of coenzyme A, adenosine 5'-monophosphate 3'-pyrophosphate was inactive as a cofactor. PMID:6574458

  16. Copper is a Cofactor of the Formylglycine‐Generating Enzyme

    PubMed Central

    Knop, Matthias; Dang, Thanh Quy; Jeschke, Gunnar

    2016-01-01

    Abstract Formylglycine‐generating enzyme (FGE) is an O2‐utilizing oxidase that converts specific cysteine residues of client proteins to formylglycine. We show that CuI is an integral cofactor of this enzyme and binds with high affinity (K D=of 10−17  m) to a pair of active‐site cysteines. These findings establish FGE as a novel type of copper enzyme. PMID:27862795

  17. Coimmobilization of a Redox Enzyme and a Cofactor Regeneration System

    DTIC Science & Technology

    2006-07-01

    biocatalysis is widely used for redox reactions including asymmetric hydroxylations and epoxidations.2a Unfortunately, whole-cell systems are often...of purified redox enzymes in biocatalysis is limited by the cost of supplying stoichiometric amounts of cofactors for catalysis. An increasing...reduction of nitrobenzene to hydroxylaminobenzene (HAB) and has been successfully employed for whole-cell biocatalysis in o-aminophenol synthesis.4

  18. Nicotinamide Cofactors Suppress Active-Site Labeling of Aldehyde Dehydrogenases.

    PubMed

    Stiti, Naim; Chandrasekar, Balakumaran; Strubl, Laura; Mohammed, Shabaz; Bartels, Dorothea; van der Hoorn, Renier A L

    2016-06-17

    Active site labeling by (re)activity-based probes is a powerful chemical proteomic tool to globally map active sites in native proteomes without using substrates. Active site labeling is usually taken as a readout for the active state of the enzyme because labeling reflects the availability and reactivity of active sites, which are hallmarks for enzyme activities. Here, we show that this relationship holds tightly, but we also reveal an important exception to this rule. Labeling of Arabidopsis ALDH3H1 with a chloroacetamide probe occurs at the catalytic Cys, and labeling is suppressed upon nitrosylation and oxidation, and upon treatment with other Cys modifiers. These experiments display a consistent and strong correlation between active site labeling and enzymatic activity. Surprisingly, however, labeling is suppressed by the cofactor NAD(+), and this property is shared with other members of the ALDH superfamily and also detected for unrelated GAPDH enzymes with an unrelated hydantoin-based probe in crude extracts of plant cell cultures. Suppression requires cofactor binding to its binding pocket. Labeling is also suppressed by ALDH modulators that bind at the substrate entrance tunnel, confirming that labeling occurs through the substrate-binding cavity. Our data indicate that cofactor binding adjusts the catalytic Cys into a conformation that reduces the reactivity toward chloroacetamide probes.

  19. Involvement of the Cys-Tyr cofactor on iron binding in the active site of human cysteine dioxygenase.

    PubMed

    Arjune, Sita; Schwarz, Guenter; Belaidi, Abdel A

    2015-01-01

    Sulfur metabolism has gained increasing medical interest over the last years. In particular, cysteine dioxygenase (CDO) has been recognized as a potential marker in oncology due to its altered gene expression in various cancer types. Human CDO is a non-heme iron-dependent enzyme, which catalyzes the irreversible oxidation of cysteine to cysteine sulfinic acid, which is further metabolized to taurine or pyruvate and sulfate. Several studies have reported a unique post-translational modification of human CDO consisting of a cross-link between cysteine 93 and tyrosine 157 (Cys-Tyr), which increases catalytic efficiency in a substrate-dependent manner. However, the reaction mechanism by which the Cys-Tyr cofactor increases catalytic efficiency remains unclear. In this study, steady-state kinetics were determined for wild type CDO and two different variants being either impaired or saturated with the Cys-Tyr cofactor. Cofactor formation in CDO resulted in an approximately fivefold increase in k cat and tenfold increase in k cat/K m over the cofactor-free CDO variant. Furthermore, iron titration experiments revealed an 18-fold decrease in K d of iron upon cross-link formation. This finding suggests a structural role of the Cys-Tyr cofactor in coordinating the ferrous iron in the active site of CDO in accordance with the previously postulated reaction mechanism of human CDO. Finally, we identified product-based inhibition and α-ketoglutarate and glutarate as CDO inhibitors using a simplified well plate-based activity assay. This assay can be used for high-throughput identification of additional inhibitors, which may contribute to understand the functional importance of CDO in sulfur amino acid metabolism and related diseases.

  20. Identification of 526 conserved metazoan genetic innovations exposes a new role for cofactor E-like in neuronal microtubule homeostasis.

    PubMed

    Frédéric, Melissa Y; Lundin, Victor F; Whiteside, Matthew D; Cueva, Juan G; Tu, Domena K; Kang, S Y Catherine; Singh, Hansmeet; Baillie, David L; Hutter, Harald; Goodman, Miriam B; Brinkman, Fiona S L; Leroux, Michel R

    2013-01-01

    The evolution of metazoans from their choanoflagellate-like unicellular ancestor coincided with the acquisition of novel biological functions to support a multicellular lifestyle, and eventually, the unique cellular and physiological demands of differentiated cell types such as those forming the nervous, muscle and immune systems. In an effort to understand the molecular underpinnings of such metazoan innovations, we carried out a comparative genomics analysis for genes found exclusively in, and widely conserved across, metazoans. Using this approach, we identified a set of 526 core metazoan-specific genes (the 'metazoanome'), approximately 10% of which are largely uncharacterized, 16% of which are associated with known human disease, and 66% of which are conserved in Trichoplax adhaerens, a basal metazoan lacking neurons and other specialized cell types. Global analyses of previously-characterized core metazoan genes suggest a prevalent property, namely that they act as partially redundant modifiers of ancient eukaryotic pathways. Our data also highlights the importance of exaptation of pre-existing genetic tools during metazoan evolution. Expression studies in C. elegans revealed that many metazoan-specific genes, including tubulin folding cofactor E-like (TBCEL/coel-1), are expressed in neurons. We used C. elegans COEL-1 as a representative to experimentally validate the metazoan-specific character of our dataset. We show that coel-1 disruption results in developmental hypersensitivity to the microtubule drug paclitaxel/taxol, and that overexpression of coel-1 has broad effects during embryonic development and perturbs specialized microtubules in the touch receptor neurons (TRNs). In addition, coel-1 influences the migration, neurite outgrowth and mechanosensory function of the TRNs, and functionally interacts with components of the tubulin acetylation/deacetylation pathway. Together, our findings unveil a conserved molecular toolbox fundamental to metazoan

  1. Identification of 526 Conserved Metazoan Genetic Innovations Exposes a New Role for Cofactor E-like in Neuronal Microtubule Homeostasis

    PubMed Central

    Whiteside, Matthew D.; Cueva, Juan G.; Tu, Domena K.; Kang, S. Y. Catherine; Singh, Hansmeet; Baillie, David L.; Hutter, Harald; Goodman, Miriam B.; Brinkman, Fiona S. L.; Leroux, Michel R.

    2013-01-01

    The evolution of metazoans from their choanoflagellate-like unicellular ancestor coincided with the acquisition of novel biological functions to support a multicellular lifestyle, and eventually, the unique cellular and physiological demands of differentiated cell types such as those forming the nervous, muscle and immune systems. In an effort to understand the molecular underpinnings of such metazoan innovations, we carried out a comparative genomics analysis for genes found exclusively in, and widely conserved across, metazoans. Using this approach, we identified a set of 526 core metazoan-specific genes (the ‘metazoanome’), approximately 10% of which are largely uncharacterized, 16% of which are associated with known human disease, and 66% of which are conserved in Trichoplax adhaerens, a basal metazoan lacking neurons and other specialized cell types. Global analyses of previously-characterized core metazoan genes suggest a prevalent property, namely that they act as partially redundant modifiers of ancient eukaryotic pathways. Our data also highlights the importance of exaptation of pre-existing genetic tools during metazoan evolution. Expression studies in C. elegans revealed that many metazoan-specific genes, including tubulin folding cofactor E-like (TBCEL/coel-1), are expressed in neurons. We used C. elegans COEL-1 as a representative to experimentally validate the metazoan-specific character of our dataset. We show that coel-1 disruption results in developmental hypersensitivity to the microtubule drug paclitaxel/taxol, and that overexpression of coel-1 has broad effects during embryonic development and perturbs specialized microtubules in the touch receptor neurons (TRNs). In addition, coel-1 influences the migration, neurite outgrowth and mechanosensory function of the TRNs, and functionally interacts with components of the tubulin acetylation/deacetylation pathway. Together, our findings unveil a conserved molecular toolbox fundamental to metazoan

  2. Constraints on texture zero and cofactor zero models for neutrino mass

    SciTech Connect

    Whisnant, K.; Liao, Jiajun; Marfatia, D.

    2014-06-24

    Imposing a texture or cofactor zero on the neutrino mass matrix reduces the number of independent parameters from nine to seven. Since five parameters have been measured, only two independent parameters would remain in such models. We find the allowed regions for single texture zero and single cofactor zero models. We also find strong similarities between single texture zero models with one mass hierarchy and single cofactor zero models with the opposite mass hierarchy. We show that this correspondence can be generalized to texture-zero and cofactor-zero models with the same homogeneous costraints on the elements and cofactors.

  3. In Vivo Estradiol, Tamoxifen and Raloxifene Modulation of Association/Dissociation Kinetics for Estrogen Receptor, Interacting Co-Factors and DNA Binding Sites

    DTIC Science & Technology

    2002-06-01

    is preferred clinically (3, 5 , 7 , 13). It is our goal to understand the molecular and cellular basis of the tissue-specific actions of these...IFinal (14 May 01 - 23 May 02) 4. TITLE AND SUBTITLE 5 . FUNDING NUMBERS In Vivo Estradiol, Tamoxifen and Raloxifene Modulation of DAMDl7-01-1-0498...Association/Dissociation Kinetics for Estrogen Receptor, Interacting Co-Factors and DNA Binding Sites 6. AUTHOR(S) Fred J. Schaufele, Ph.D. 7

  4. Review of quantitative phase-digital holographic microscopy: promising novel imaging technique to resolve neuronal network activity and identify cellular biomarkers of psychiatric disorders

    PubMed Central

    Marquet, Pierre; Depeursinge, Christian; Magistretti, Pierre J.

    2014-01-01

    Abstract. Quantitative phase microscopy (QPM) has recently emerged as a new powerful quantitative imaging technique well suited to noninvasively explore a transparent specimen with a nanometric axial sensitivity. In this review, we expose the recent developments of quantitative phase-digital holographic microscopy (QP-DHM). Quantitative phase-digital holographic microscopy (QP-DHM) represents an important and efficient quantitative phase method to explore cell structure and dynamics. In a second part, the most relevant QPM applications in the field of cell biology are summarized. A particular emphasis is placed on the original biological information, which can be derived from the quantitative phase signal. In a third part, recent applications obtained, with QP-DHM in the field of cellular neuroscience, namely the possibility to optically resolve neuronal network activity and spine dynamics, are presented. Furthermore, potential applications of QPM related to psychiatry through the identification of new and original cell biomarkers that, when combined with a range of other biomarkers, could significantly contribute to the determination of high risk developmental trajectories for psychiatric disorders, are discussed. PMID:26157976

  5. Cellular Aspects of Prion Replication In Vitro

    PubMed Central

    Grassmann, Andrea; Wolf, Hanna; Hofmann, Julia; Graham, James; Vorberg, Ina

    2013-01-01

    Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders in mammals that are caused by unconventional agents predominantly composed of aggregated misfolded prion protein (PrP). Prions self-propagate by recruitment of host-encoded PrP into highly ordered β-sheet rich aggregates. Prion strains differ in their clinical, pathological and biochemical characteristics and are likely to be the consequence of distinct abnormal prion protein conformers that stably replicate their alternate states in the host cell. Understanding prion cell biology is fundamental for identifying potential drug targets for disease intervention. The development of permissive cell culture models has greatly enhanced our knowledge on entry, propagation and dissemination of TSE agents. However, despite extensive research, the precise mechanism of prion infection and potential strain effects remain enigmatic. This review summarizes our current knowledge of the cell biology and propagation of prions derived from cell culture experiments. We discuss recent findings on the trafficking of cellular and pathologic PrP, the potential sites of abnormal prion protein synthesis and potential co-factors involved in prion entry and propagation. PMID:23340381

  6. Modeling the potential spread of the recently identified non-native panther grouper (Chromileptes altivelis) in the Atlantic using a cellular automaton approach.

    PubMed

    Johnston, Matthew W; Purkis, Sam J

    2013-01-01

    The Indo-pacific panther grouper (Chromileptes altiveli) is a predatory fish species and popular imported aquarium fish in the United States which has been recently documented residing in western Atlantic waters. To date, the most successful marine invasive species in the Atlantic is the lionfish (Pterois volitans/miles), which, as for the panther grouper, is assumed to have been introduced to the wild through aquarium releases. However, unlike lionfish, the panther grouper is not yet thought to have an established breeding population in the Atlantic. Using a proven modeling technique developed to track the lionfish invasion, presented is the first known estimation of the potential spread of panther grouper in the Atlantic. The employed cellular automaton-based computer model examines the life history of the subject species including fecundity, mortality, and reproductive potential and combines this with habitat preferences and physical oceanic parameters to forecast the distribution and periodicity of spread of this potential new invasive species. Simulations were examined for origination points within one degree of capture locations of panther grouper from the United States Geological Survey Nonindigenous Aquatic Species Database to eliminate introduction location bias, and two detailed case studies were scrutinized. The model indicates three primary locations where settlement is likely given the inputs and limits of the model; Jupiter Florida/Vero Beach, the Cape Hatteras Tropical Limit/Myrtle Beach South Carolina, and Florida Keys/Ten Thousand Islands locations. Of these locations, Jupiter Florida/Vero Beach has the highest settlement rate in the model and is indicated as the area in which the panther grouper is most likely to become established. This insight is valuable if attempts are to be made to halt this potential marine invasive species.

  7. Modeling the Potential Spread of the Recently Identified Non-Native Panther Grouper (Chromileptes altivelis) in the Atlantic Using a Cellular Automaton Approach

    PubMed Central

    Johnston, Matthew W.; Purkis, Sam J.

    2013-01-01

    The Indo-pacific panther grouper (Chromileptes altiveli) is a predatory fish species and popular imported aquarium fish in the United States which has been recently documented residing in western Atlantic waters. To date, the most successful marine invasive species in the Atlantic is the lionfish (Pterois volitans/miles), which, as for the panther grouper, is assumed to have been introduced to the wild through aquarium releases. However, unlike lionfish, the panther grouper is not yet thought to have an established breeding population in the Atlantic. Using a proven modeling technique developed to track the lionfish invasion, presented is the first known estimation of the potential spread of panther grouper in the Atlantic. The employed cellular automaton-based computer model examines the life history of the subject species including fecundity, mortality, and reproductive potential and combines this with habitat preferences and physical oceanic parameters to forecast the distribution and periodicity of spread of this potential new invasive species. Simulations were examined for origination points within one degree of capture locations of panther grouper from the United States Geological Survey Nonindigenous Aquatic Species Database to eliminate introduction location bias, and two detailed case studies were scrutinized. The model indicates three primary locations where settlement is likely given the inputs and limits of the model; Jupiter Florida/Vero Beach, the Cape Hatteras Tropical Limit/Myrtle Beach South Carolina, and Florida Keys/Ten Thousand Islands locations. Of these locations, Jupiter Florida/Vero Beach has the highest settlement rate in the model and is indicated as the area in which the panther grouper is most likely to become established. This insight is valuable if attempts are to be made to halt this potential marine invasive species. PMID:24009726

  8. In Vitro Bioconversion of Pyruvate to n-Butanol with Minimized Cofactor Utilization

    PubMed Central

    Reiße, Steven; Haack, Martina; Garbe, Daniel; Sommer, Bettina; Steffler, Fabian; Carsten, Jörg; Bohnen, Frank; Sieber, Volker; Brück, Thomas

    2016-01-01

    Due to enhanced energy content and reduced hygroscopicity compared with ethanol, n-butanol is flagged as the next generation biofuel and platform chemical. In addition to conventional cellular systems, n-butanol bioproduction by enzyme cascades is gaining momentum due to simplified process control. In contrast to other bio-based alcohols like ethanol and isobutanol, cell-free n-butanol biosynthesis from the central metabolic intermediate pyruvate involves cofactors [NAD(P)H, CoA] and acetyl-CoA-dependent intermediates, which complicates redox and energy balancing of the reaction system. We have devised a biochemical process for cell-free n-butanol production that only involves three enzyme activities, thereby eliminating the need for acetyl-CoA. Instead, the process utilizes only NADH as the sole redox mediator. Central to this new process is the amino acid catalyzed enamine–aldol condensation, which transforms acetaldehyde directly into crotonaldehyde. Subsequently, crotonaldehyde is reduced to n-butanol applying a 2-enoate reductase and an alcohol dehydrogenase, respectively. In essence, we achieved conversion of the platform intermediate pyruvate to n-butanol utilizing a biocatalytic cascade comprising only three enzyme activities and NADH as reducing equivalent. With reference to previously reported cell-free n-butanol reaction cascades, we have eliminated five enzyme activities and the requirement of CoA as cofactor. Our proof-of-concept demonstrates that n-butanol was synthesized at neutral pH and 50°C. This integrated reaction concept allowed GC detection of all reaction intermediates and n-butanol production of 148 mg L−1 (2 mM), which compares well with other cell-free n-butanol production processes. PMID:27800475

  9. Design of dinuclear manganese cofactors for bacterial reaction centers.

    PubMed

    Olson, Tien L; Espiritu, Eduardo; Edwardraja, Selvakumar; Simmons, Chad R; Williams, JoAnn C; Ghirlanda, Giovanna; Allen, James P

    2016-05-01

    A compelling target for the design of electron transfer proteins with novel cofactors is to create a model for the oxygen-evolving complex, a Mn4Ca cluster, of photosystem II. A mononuclear Mn cofactor can be added to the bacterial reaction center, but the addition of multiple metal centers is constrained by the native protein architecture. Alternatively, metal centers can be incorporated into artificial proteins. Designs for the addition of dinuclear metal centers to four-helix bundles resulted in three artificial proteins with ligands for one, two, or three dinuclear metal centers able to bind Mn. The three-dimensional structure determined by X-ray crystallography of one of the Mn-proteins confirmed the design features and revealed details concerning coordination of the Mn center. Electron transfer between these artificial Mn-proteins and bacterial reaction centers was investigated using optical spectroscopy. After formation of a light-induced, charge-separated state, the experiments showed that the Mn-proteins can donate an electron to the oxidized bacteriochlorophyll dimer of modified reaction centers, with the Mn-proteins having additional metal centers being more effective at this electron transfer reaction. Modeling of the structure of the Mn-protein docked to the reaction center showed that the artificial protein likely binds on the periplasmic surface similarly to cytochrome c2, the natural secondary donor. Combining reaction centers with exogenous artificial proteins provides the opportunity to create ligands and investigate the influence of inhomogeneous protein environments on multinuclear redox-active metal centers. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

  10. Cofactor-dependent pathways of formaldehyde oxidation in methylotrophic bacteria.

    PubMed

    Vorholt, Julia A

    2002-10-01

    Methylotrophic bacteria can grow on a number of substrates as energy source with only one carbon atom, such as methanol, methane, methylamine, and dichloromethane. These compounds are metabolized via the cytotoxin formaldehyde. The formaldehyde consumption pathways, especially the pathways for the oxidation of formaldehyde to CO(2) for energy metabolism, are a central and critical part of the metabolism of these aerobic bacteria. Principally, two main types of pathways for the conversion of formaldehyde to CO(2) have been described: (1) a cyclic pathway initiated by the condensation of formaldehyde with ribulose monophosphate, and (2) distinct linear pathways that involve a dye-linked formaldehyde dehydrogenase or C(1) unit conversion bound to the cofactors tetrahydrofolate (H(4)F), tetrahydromethanopterin (H(4)MPT), glutathione (GSH), or mycothiol (MySH). The pathways involving the four cofactors have in common the following sequence of events: the spontaneous or enzyme-catalyzed condensation of formaldehyde and the respective C(1) carrier, the oxidation of the cofactor-bound C(1) unit and its conversion to formate, and the oxidation of formate to CO(2). However, the H(4)MPT pathway is more complex and involves intermediates that were previously known solely from the energy metabolism of methanogenic archaea. The occurrence of the different formaldehyde oxidation pathways is not uniform among different methylotrophic bacteria. The pathways are in part also used by other organisms to provide C(1) units for biosynthetic reactions (e.g., H(4)F-dependent enzymes) or detoxification of formaldehyde (e.g., GSH-dependent enzymes).

  11. NADP-dependent enzymes. I: Conserved stereochemistry of cofactor binding.

    PubMed

    Carugo, O; Argos, P

    1997-05-01

    The ubiquitous redox cofactors nicotinamide adenine dinucleotides [NAD and NADP] are very similar molecules, despite their participation in substantially different biochemical processes. NADP differs from NAD in only the presence of an additional phosphate group esterified to the 2'-hydroxyl group of the ribose at the adenine end and yet NADP is confined with few exceptions to the reactions of reductive biosynthesis, whereas NAD is used almost exclusively in oxidative degradations. The discrimination between NAD and NADP is therefore an impressive example of the power of molecular recognition by proteins. The many known tertiary structures of NADP complexes affords the possibility for an analysis of their discrimination. A systematic analysis of several crystal structures of NAD(P)-protein complexes show that: 1) the NADP coenzymes are more flexible in conformation than those of NAD; 2) although the protein-cofactor interactions are largely conserved in the NAD complexes, they are quite variable in those of NADP; and 3) in both cases the pocket around the nicotinamide moiety is substrate dependent. The conserved and variable interactions between protein and cofactors in the respective binding pockets are reported in detail. Discrimination between NAD and NADP is essentially a consequence of the overall pocket and not of a few residues. A clear fingerprint in NAD complexes is a carboxylate side chain that chelates the diol group at the ribose near the adenine, whereas in NADP complexes an arginine side chain faces the adenine plane and interacts with the phosphomonoester. The latter type of interaction might be a general feature of recognition of nucleotides by proteins. Other features such as strand-like hydrogen bonding between the NADP diphosphate moieties and the protein are also significant. The NADP binding pocket properties should prove useful in protein engineering and design.

  12. Role of HOXA9 in leukemia: dysregulation, cofactors and essential targets

    PubMed Central

    Collins, Cailin T.; Hess, Jay L.

    2015-01-01

    HOXA9 is a homeodomain-containing transcription factor that plays an important role in hematopoietic stem cell expansion and is commonly deregulated in acute leukemias. A variety of upstream genetic alterations in acute myeloid leukemia (AML) lead to overexpression of HOXA9, which is a strong predictor of poor prognosis. In many cases, HOXA9 has been shown to be necessary for maintaining leukemic transformation, however the molecular mechanisms through which it promotes leukemogenesis remain elusive. Recent work has established that HOXA9 regulates downstream gene expression through binding at promoter distal enhancers along with a subset of cell-specific cofactor and collaborator proteins. Increasing efforts are being made to identify both the critical cofactors and target genes required for maintaining transformation in HOXA9-overexpressing leukemias. With continued advances in understanding HOXA9-mediated transformation, there is a wealth of opportunity for developing novel therapeutics that would be applicable for the greater than 50% of AML with overexpression of HOXA9. PMID:26028034

  13. Crystal Structures of Cyclohexanone Monooxygenase Reveal Complex Domain Movements and a Sliding Cofactor

    SciTech Connect

    Mirza, I.; Yachnin, B; Wang, S; Grosse, S; Bergeron, H; Imura, A; Iwaki, H; Hasegawa, Y; Lau, P; Berghuis, A

    2009-01-01

    Cyclohexanone monooxygenase (CHMO) is a flavoprotein that carries out the archetypical Baeyer-Villiger oxidation of a variety of cyclic ketones into lactones. Using NADPH and O{sub 2} as cosubstrates, the enzyme inserts one atom of oxygen into the substrate in a complex catalytic mechanism that involves the formation of a flavin-peroxide and Criegee intermediate. We present here the atomic structures of CHMO from an environmental Rhodococcus strain bound with FAD and NADP+ in two distinct states, to resolutions of 2.3 and 2.2 {angstrom}. The two conformations reveal domain shifts around multiple linkers and loop movements, involving conserved arginine 329 and tryptophan 492, which effect a translation of the nicotinamide resulting in a sliding cofactor. Consequently, the cofactor is ideally situated and subsequently repositioned during the catalytic cycle to first reduce the flavin and later stabilize formation of the Criegee intermediate. Concurrent movements of a loop adjacent to the active site demonstrate how this protein can effect large changes in the size and shape of the substrate binding pocket to accommodate a diverse range of substrates. Finally, the previously identified BVMO signature sequence is highlighted for its role in coordinating domain movements. Taken together, these structures provide mechanistic insights into CHMO-catalyzed Baeyer-Villiger oxidation.

  14. Dissecting Torsin/cofactor function at the nuclear envelope: a genetic study

    PubMed Central

    Laudermilch, Ethan; Tsai, Pei-Ling; Graham, Morven; Turner, Elizabeth; Zhao, Chenguang; Schlieker, Christian

    2016-01-01

    The human genome encodes four Torsin ATPases, the functions of which are poorly understood. In this study, we use CRISPR/Cas9 engineering to delete all four Torsin ATPases individually and in combination. Using nuclear envelope (NE) blebbing as a phenotypic measure, we establish a direct correlation between the number of inactivated Torsin alleles and the occurrence of omega-shaped herniations within the lumen of the NE. A similar, although not identical, redundancy is observed for LAP1 and LULL1, which serve as regulatory cofactors for a subset of Torsin ATPases. Unexpectedly, deletion of Tor2A in a TorA/B/3A-deficient background results in a stark increase of bleb formation, even though Tor2A does not respond to LAP1/LULL1 stimulation. The robustness of the observed phenotype in Torsin-deficient cells enables a structural analysis via electron microscopy tomography and a compositional analysis via immunogold labeling. Ubiquitin and nucleoporins were identified as distinctively localizing components of the omega-shaped bleb structure. These findings suggest a functional link between the Torsin/cofactor system and NE/nuclear pore complex biogenesis or homeostasis and establish a Torsin-deficient cell line as a valuable experimental platform with which to decipher Torsin function. PMID:27798237

  15. Crystal structures of cyclohexanone monooxygenase reveal complex domain movements and a sliding cofactor.

    PubMed

    Mirza, I Ahmad; Yachnin, Brahm J; Wang, Shaozhao; Grosse, Stephan; Bergeron, Hélène; Imura, Akihiro; Iwaki, Hiroaki; Hasegawa, Yoshie; Lau, Peter C K; Berghuis, Albert M

    2009-07-01

    Cyclohexanone monooxygenase (CHMO) is a flavoprotein that carries out the archetypical Baeyer-Villiger oxidation of a variety of cyclic ketones into lactones. Using NADPH and O(2) as cosubstrates, the enzyme inserts one atom of oxygen into the substrate in a complex catalytic mechanism that involves the formation of a flavin-peroxide and Criegee intermediate. We present here the atomic structures of CHMO from an environmental Rhodococcus strain bound with FAD and NADP(+) in two distinct states, to resolutions of 2.3 and 2.2 A. The two conformations reveal domain shifts around multiple linkers and loop movements, involving conserved arginine 329 and tryptophan 492, which effect a translation of the nicotinamide resulting in a sliding cofactor. Consequently, the cofactor is ideally situated and subsequently repositioned during the catalytic cycle to first reduce the flavin and later stabilize formation of the Criegee intermediate. Concurrent movements of a loop adjacent to the active site demonstrate how this protein can effect large changes in the size and shape of the substrate binding pocket to accommodate a diverse range of substrates. Finally, the previously identified BVMO signature sequence is highlighted for its role in coordinating domain movements. Taken together, these structures provide mechanistic insights into CHMO-catalyzed Baeyer-Villiger oxidation.

  16. Local synthesis of dynein cofactors matches retrograde transport to acutely changing demands

    PubMed Central

    Villarin, Joseph M.; McCurdy, Ethan P.; Martínez, José C.; Hengst, Ulrich

    2016-01-01

    Cytoplasmic dynein mediates retrograde transport in axons, but it is unknown how its transport characteristics are regulated to meet acutely changing demands. We find that stimulus-induced retrograde transport of different cargos requires the local synthesis of different dynein cofactors. Nerve growth factor (NGF)-induced transport of large vesicles requires local synthesis of Lis1, while smaller signalling endosomes require both Lis1 and p150Glued. Lis1 synthesis is also triggered by NGF withdrawal and required for the transport of a death signal. Association of Lis1 transcripts with the microtubule plus-end tracking protein APC is required for their translation in response to NGF stimulation but not for their axonal recruitment and translation upon NGF withdrawal. These studies reveal a critical role for local synthesis of dynein cofactors for the transport of specific cargos and identify association with RNA-binding proteins as a mechanism to establish functionally distinct pools of a single transcript species in axons. PMID:28000671

  17. Polyphosphate is a cofactor for the activation of factor XI by thrombin

    PubMed Central

    Choi, Sharon H.; Smith, Stephanie A.

    2011-01-01

    Factor XI deficiency is associated with a bleeding diathesis, but factor XII deficiency is not, indicating that, in normal hemostasis, factor XI must be activated in vivo by a protease other than factor XIIa. Several groups have identified thrombin as the most likely activator of factor XI, although this reaction is slow in solution. Although certain nonphysiologic anionic polymers and surfaces have been shown to enhance factor XI activation by thrombin, the physiologic cofactor for this reaction is uncertain. Activated platelets secrete the highly anionic polymer polyphosphate, and our previous studies have shown that polyphosphate has potent procoagulant activity. We now report that polyphosphate potently accelerates factor XI activation by α-thrombin, β-thrombin, and factor XIa and that these reactions are supported by polyphosphate polymers of the size secreted by activated human platelets. We therefore propose that polyphosphate is a natural cofactor for factor XI activation in plasma that may help explain the role of factor XI in hemostasis and thrombosis. PMID:21976677

  18. Structural basis for HIV-1 DNA integration in the human genome, role of the LEDGF/P75 cofactor

    PubMed Central

    Michel, Fabrice; Crucifix, Corinne; Granger, Florence; Eiler, Sylvia; Mouscadet, Jean-François; Korolev, Sergei; Agapkina, Julia; Ziganshin, Rustam; Gottikh, Marina; Nazabal, Alexis; Emiliani, Stéphane; Benarous, Richard; Moras, Dino; Schultz, Patrick; Ruff, Marc

    2009-01-01

    Integration of the human immunodeficiency virus (HIV-1) cDNA into the human genome is catalysed by integrase. Several studies have shown the importance of the interaction of cellular cofactors with integrase for viral integration and infectivity. In this study, we produced a stable and functional complex between the wild-type full-length integrase (IN) and the cellular cofactor LEDGF/p75 that shows enhanced in vitro integration activity compared with the integrase alone. Mass spectrometry analysis and the fitting of known atomic structures in cryo negatively stain electron microscopy (EM) maps revealed that the functional unit comprises two asymmetric integrase dimers and two LEDGF/p75 molecules. In the presence of DNA, EM revealed the DNA-binding sites and indicated that, in each asymmetric dimer, one integrase molecule performs the catalytic reaction, whereas the other one positions the viral DNA in the active site of the opposite dimer. The positions of the target and viral DNAs for the 3′ processing and integration reaction shed light on the integration mechanism, a process with wide implications for the understanding of viral-induced pathologies. PMID:19229293

  19. Rice Bran Metabolome Contains Amino Acids, Vitamins & Cofactors, and Phytochemicals with Medicinal and Nutritional Properties.

    PubMed

    Zarei, Iman; Brown, Dustin G; Nealon, Nora Jean; Ryan, Elizabeth P

    2017-12-01

    Rice bran is a functional food that has shown protection against major chronic diseases (e.g. obesity, diabetes, cardiovascular disease and cancer) in animals and humans, and these health effects have been associated with the presence of bioactive phytochemicals. Food metabolomics uses multiple chromatography and mass spectrometry platforms to detect and identify a diverse range of small molecules with high sensitivity and precision, and has not been completed for rice bran. This study utilized global, non-targeted metabolomics to identify small molecules in rice bran, and conducted a comprehensive search of peer-reviewed literature to determine bioactive compounds. Three U.S. rice varieties (Calrose, Dixiebelle, and Neptune), that have been used for human dietary intervention trials, were assessed herein for bioactive compounds that have disease control and prevention properties. The profiling of rice bran by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) identified 453 distinct phytochemicals, 209 of which were classified as amino acids, cofactors & vitamins, and secondary metabolites, and were further assessed for bioactivity. A scientific literature search revealed 65 compounds with health properties, 16 of which had not been previously identified in rice bran. This suite of amino acids, cofactors & vitamins, and secondary metabolites comprised 46% of the identified rice bran metabolome, which substantially enhanced our knowledge of health-promoting rice bran compounds provided during dietary supplementation. Rice bran metabolite profiling revealed a suite of biochemical molecules that can be further investigated and exploited for multiple nutritional therapies and medical food applications. These bioactive compounds may also be biomarkers of dietary rice bran intake. The medicinal compounds associated with rice bran can function as a network across metabolic pathways and this

  20. Biochemistry: role of PQQ as a mammalian enzyme cofactor?

    PubMed

    Felton, Leigh M; Anthony, Chris

    2005-02-03

    The announcement by Kasahara and Kato of a new redox-cofactor vitamin for mammals, pyrroloquinoline quinone (PQQ), was based on their claim that an enzyme, predicted to be involved in mouse lysine metabolism, is a PQQ-dependent dehydrogenase. However, this claim was dependent on a sequence analysis using databases that inappropriately label beta-propeller sequences as PQQ-binding motifs. What the evidence actually suggests is that the enzyme is an interesting novel protein that has a seven-bladed beta-propeller structure, but there is nothing to indicate that it is a PQQ-dependent dehydrogenase.

  1. Insights into hydrocarbon formation by nitrogenase cofactor homologs.

    PubMed

    Lee, Chi Chung; Hu, Yilin; Ribbe, Markus W

    2015-04-14

    The L-cluster is an all-iron homolog of nitrogenase cofactors. Driven by europium(II) diethylenetriaminepentaacetate [Eu(II)-DTPA], the isolated L-cluster is capable of ATP-independent reduction of CO and CN(-) to C1 to C4 and C1 to C6 hydrocarbons, respectively. Compared to its cofactor homologs, the L-cluster generates considerably more CH4 from the reduction of CO and CN(-), which could be explained by the presence of a "free" Fe atom that is "unmasked" by homocitrate as an additional site for methanation. Moreover, the elevated CH4 formation is accompanied by a decrease in the amount of longer hydrocarbons and/or the lengths of the hydrocarbon products, illustrating a competition between CH4 formation/release and C-C coupling/chain extension. These observations suggest the possibility of designing simpler synthetic clusters for hydrocarbon formation while establishing the L-cluster as a platform for mechanistic investigations of CO and CN(-) reduction without complications originating from the heterometal and homocitrate components. Nitrogenase is a metalloenzyme that is highly complex in structure and uniquely versatile in function. It catalyzes two reactions that parallel two important industrial processes: the reduction of nitrogen to ammonia, which parallels the Haber-Bosch process in ammonia production, and the reduction of carbon monoxide to hydrocarbons, which parallels the Fischer-Tropsch process in fuel production. Thus, the significance of nitrogenase can be appreciated from the perspective of the useful products it generates: (i) ammonia, the "fixed" nitrogen that is essential for the existence of the entire human population; and (ii) hydrocarbons, the "recycled" carbon fuel that could be used to directly address the worldwide energy shortage. This article provides initial insights into the catalytic characteristics of various nitrogenase cofactors in hydrocarbon formation. The reported assay system provides a useful tool for mechanistic

  2. iCDI-PseFpt: identify the channel-drug interaction in cellular networking with PseAAC and molecular fingerprints.

    PubMed

    Xiao, Xuan; Min, Jian-Liang; Wang, Pu; Chou, Kuo-Chen

    2013-11-21

    Many crucial functions in life, such as heartbeat, sensory transduction and central nervous system response, are controlled by cell signalings via various ion channels. Therefore, ion channels have become an excellent drug target, and study of ion channel-drug interaction networks is an important topic for drug development. However, it is both time-consuming and costly to determine whether a drug and a protein ion channel are interacting with each other in a cellular network by means of experimental techniques. Although some computational methods were developed in this regard based on the knowledge of the 3D (three-dimensional) structure of protein, unfortunately their usage is quite limited because the 3D structures for most protein ion channels are still unknown. With the avalanche of protein sequences generated in the post-genomic age, it is highly desirable to develop the sequence-based computational method to address this problem. To take up the challenge, we developed a new predictor called iCDI-PseFpt, in which the protein ion-channel sample is formulated by the PseAAC (pseudo amino acid composition) generated with the gray model theory, the drug compound by the 2D molecular fingerprint, and the operation engine is the fuzzy K-nearest neighbor algorithm. The overall success rate achieved by iCDI-PseFpt via the jackknife cross-validation was 87.27%, which is remarkably higher than that by any of the existing predictors in this area. As a user-friendly web-server, iCDI-PseFpt is freely accessible to the public at the website http://www.jci-bioinfo.cn/iCDI-PseFpt/. Furthermore, for the convenience of most experimental scientists, a step-by-step guide is provided on how to use the web-server to get the desired results without the need to follow the complicated math equations presented in the paper just for its integrity. It has not escaped our notice that the current approach can also be used to study other drug-target interaction networks. © 2013 Elsevier Ltd

  3. Cofactor engineering of ketol-acid reductoisomerase (IlvC) and alcohol dehydrogenase (YqhD) improves the fusel alcohol yield in algal protein anaerobic fermentation

    SciTech Connect

    Wu, Weihua; Tran-Gyamfi, Mary Bao; Jaryenneh, James Dekontee; Davis, Ryan W.

    2016-08-24

    Recently the feasibility of conversion of algal protein to mixed alcohols has been demonstrated with an engineered E.coli strain, enabling comprehensive utilization of the biomass for biofuel applications. However, the yield and titers of mixed alcohol production must be improved for market adoption. A major limiting factor for achieving the necessary yield and titer improvements is cofactor imbalance during the fermentation of algal protein. To resolve this problem, a directed evolution approach was applied to modify the cofactor specificity of two key enzymes (IlvC and YqhD) from NADPH to NADH in the mixed alcohol metabolic pathway. Using high throughput screening, more than 20 YqhD mutants were identified to show activity on NADH as a cofactor. Of these 20 mutants, the top five of YqhD mutants were selected for combination with two IlvC mutants with NADH as a cofactor for the modification of the protein conversion strain. The combination of the IlvC and YqhD mutants yielded a refined E.coli strain, subtype AY3, with increased fusel alcohol yield of ~60% compared to wild type under anaerobic fermentation on amino acid mixtures. When applied to real algal protein hydrolysates, the strain AY3 produced 100% and 38% more total mixed alcohols than the wild type strain on two different algal hydrolysates, respectively. The results indicate that cofactor engineering is a promising approach to improve the feasibility of bioconversion of algal protein into mixed alcohols as advanced biofuels.

  4. Cofactor engineering of ketol-acid reductoisomerase (IlvC) and alcohol dehydrogenase (YqhD) improves the fusel alcohol yield in algal protein anaerobic fermentation

    SciTech Connect

    Wu, Weihua; Tran-Gyamfi, Mary Bao; Jaryenneh, James Dekontee; Davis, Ryan W.

    2016-08-24

    Recently the feasibility of conversion of algal protein to mixed alcohols has been demonstrated with an engineered E.coli strain, enabling comprehensive utilization of the biomass for biofuel applications. However, the yield and titers of mixed alcohol production must be improved for market adoption. A major limiting factor for achieving the necessary yield and titer improvements is cofactor imbalance during the fermentation of algal protein. To resolve this problem, a directed evolution approach was applied to modify the cofactor specificity of two key enzymes (IlvC and YqhD) from NADPH to NADH in the mixed alcohol metabolic pathway. Using high throughput screening, more than 20 YqhD mutants were identified to show activity on NADH as a cofactor. Of these 20 mutants, the top five of YqhD mutants were selected for combination with two IlvC mutants with NADH as a cofactor for the modification of the protein conversion strain. The combination of the IlvC and YqhD mutants yielded a refined E.coli strain, subtype AY3, with increased fusel alcohol yield of ~60% compared to wild type under anaerobic fermentation on amino acid mixtures. When applied to real algal protein hydrolysates, the strain AY3 produced 100% and 38% more total mixed alcohols than the wild type strain on two different algal hydrolysates, respectively. The results indicate that cofactor engineering is a promising approach to improve the feasibility of bioconversion of algal protein into mixed alcohols as advanced biofuels.

  5. Biochemistry of B12-cofactors in human metabolism.

    PubMed

    Kräutler, Bernhard

    2012-01-01

    Vitamin B12, the "antipernicious anaemia factor", is a crystallisable cobalt-complex, which belongs to a group of unique "complete" corrinoids, named cobalamins (Cbl). In humans, instead of the "vitamin", two organometallic B12-forms are coenzymes in two metabolically important enzymes: Methyl-cobalamin, the cofactor of methionine synthase, and coenzyme B12 (adenosyl-cobalamin), the cofactor of methylmalonyl-CoA mutase. The cytoplasmatic methionine synthase catalyzes the transfer of a methyl group from N-methyl-tetrahydrofolate to homocysteine to yield methionine and to liberate tetrahydrofolate. In the mitochondrial methylmalonyl-CoA mutase a radical process transforms methylmalonyl-CoA (a remains e.g. from uneven numbered fatty acids) into succinyl-CoA, for further metabolic use. In addition, in the human mitochondria an adenosyl-transferase incorporates the organometallic group of coenzyme B12. In all these enzymes, the bound B12-derivatives engage (or are formed) in exceptional organometallic enzymatic reactions. This chapter recapitulates the physiological chemistry of vitamin B12, relevant in the context of the metabolic transformation of B12-derivatives into the relevant coenzyme forms and their use in B12-dependent enzymes.

  6. Oxygen diffusion pathways in a cofactor-independent dioxygenase

    PubMed Central

    Di Russo, Natali V.; Condurso, Heather L.; Li, Kunhua; Bruner, Steven D.; Roitberg, Adrian E.

    2015-01-01

    Molecular oxygen plays an important role in a wide variety of enzymatic reactions. Through recent research efforts combining computational and experimental methods a new view of O2 diffusion is emerging, where specific channels guide O2 to the active site. The focus of this work is DpgC, a cofactor-independent oxygenase. Molecular dynamics simulations, together with mutagenesis experiments and xenon-binding data, reveal that O2 reaches the active site of this enzyme using three main pathways and four different access points. These pathways connect a series of dynamic hydrophobic pockets, concentrating O2 at a specific face of the enzyme substrate. Extensive molecular dynamics simulations provide information about which pathways are more frequently used. This data is consistent with the results of kinetic measurements on mutants and is difficult to obtain using computational cavity-location methods. Taken together, our results reveal that although DpgC is rare in its ability of activating O2 in the absence of cofactors or metals, the way O2 reaches the active site is similar to that reported for other O2-using proteins: multiple access channels are available, and the architecture of the pathway network can provide regio- and stereoselectivity. Our results point to the existence of common themes in O2 access that are conserved among very different types of proteins. PMID:26508997

  7. Insights into Hydrocarbon Formation by Nitrogenase Cofactor Homologs

    PubMed Central

    Lee, Chi Chung; Hu, Yilin

    2015-01-01

    ABSTRACT The L-cluster is an all-iron homolog of nitrogenase cofactors. Driven by europium(II) diethylenetriaminepentaacetate [Eu(II)-DTPA], the isolated L-cluster is capable of ATP-independent reduction of CO and CN− to C1 to C4 and C1 to C6 hydrocarbons, respectively. Compared to its cofactor homologs, the L-cluster generates considerably more CH4 from the reduction of CO and CN−, which could be explained by the presence of a “free” Fe atom that is “unmasked” by homocitrate as an additional site for methanation. Moreover, the elevated CH4 formation is accompanied by a decrease in the amount of longer hydrocarbons and/or the lengths of the hydrocarbon products, illustrating a competition between CH4 formation/release and C−C coupling/chain extension. These observations suggest the possibility of designing simpler synthetic clusters for hydrocarbon formation while establishing the L-cluster as a platform for mechanistic investigations of CO and CN− reduction without complications originating from the heterometal and homocitrate components. PMID:25873377

  8. Sulphur shuttling across a chaperone during molybdenum cofactor maturation.

    PubMed

    Arnoux, Pascal; Ruppelt, Christian; Oudouhou, Flore; Lavergne, Jérôme; Siponen, Marina I; Toci, René; Mendel, Ralf R; Bittner, Florian; Pignol, David; Magalon, Axel; Walburger, Anne

    2015-02-04

    Formate dehydrogenases (FDHs) are of interest as they are natural catalysts that sequester atmospheric CO2, generating reduced carbon compounds with possible uses as fuel. FDHs activity in Escherichia coli strictly requires the sulphurtransferase EcFdhD, which likely transfers sulphur from IscS to the molybdenum cofactor (Mo-bisPGD) of FDHs. Here we show that EcFdhD binds Mo-bisPGD in vivo and has submicromolar affinity for GDP-used as a surrogate of the molybdenum cofactor's nucleotide moieties. The crystal structure of EcFdhD in complex with GDP shows two symmetrical binding sites located on the same face of the dimer. These binding sites are connected via a tunnel-like cavity to the opposite face of the dimer where two dynamic loops, each harbouring two functionally important cysteine residues, are present. On the basis of structure-guided mutagenesis, we propose a model for the sulphuration mechanism of Mo-bisPGD where the sulphur atom shuttles across the chaperone dimer.

  9. Identifying quantitative operation principles in metabolic pathways: a systematic method for searching feasible enzyme activity patterns leading to cellular adaptive responses

    PubMed Central

    2009-01-01

    Background Optimization methods allow designing changes in a system so that specific goals are attained. These techniques are fundamental for metabolic engineering. However, they are not directly applicable for investigating the evolution of metabolic adaptation to environmental changes. Although biological systems have evolved by natural selection and result in well-adapted systems, we can hardly expect that actual metabolic processes are at the theoretical optimum that could result from an optimization analysis. More likely, natural systems are to be found in a feasible region compatible with global physiological requirements. Results We first present a new method for globally optimizing nonlinear models of metabolic pathways that are based on the Generalized Mass Action (GMA) representation. The optimization task is posed as a nonconvex nonlinear programming (NLP) problem that is solved by an outer-approximation algorithm. This method relies on solving iteratively reduced NLP slave subproblems and mixed-integer linear programming (MILP) master problems that provide valid upper and lower bounds, respectively, on the global solution to the original NLP. The capabilities of this method are illustrated through its application to the anaerobic fermentation pathway in Saccharomyces cerevisiae. We next introduce a method to identify the feasibility parametric regions that allow a system to meet a set of physiological constraints that can be represented in mathematical terms through algebraic equations. This technique is based on applying the outer-approximation based algorithm iteratively over a reduced search space in order to identify regions that contain feasible solutions to the problem and discard others in which no feasible solution exists. As an example, we characterize the feasible enzyme activity changes that are compatible with an appropriate adaptive response of yeast Saccharomyces cerevisiae to heat shock Conclusion Our results show the utility of the

  10. Manual control of catalytic reactions: Reactions by an apoenzyme gel and a cofactor gel

    NASA Astrophysics Data System (ADS)

    Kobayashi, Yuichiro; Takashima, Yoshinori; Hashidzume, Akihito; Yamaguchi, Hiroyasu; Harada, Akira

    2015-11-01

    Enzymes play a vital role in catalysing almost all chemical reactions that occur in biological systems. Some enzymes must form complexes with non-protein molecules called cofactors to express catalytic activities. Although the control of catalytic reactions via apoenzyme-cofactor complexes has attracted significant attention, the reports have been limited to the microscale. Here, we report a system to express catalytic activity by adhesion of an apoenzyme gel and a cofactor gel. The apoenzyme and cofactor gels act as catalysts when they form a gel assembly, but they lose catalytic ability upon manual dissociation. We successfully construct a system with switchable catalytic activity via adhesion and separation of the apoenzyme gel with the cofactor gel. We expect that this methodology can be applied to regulate the functional activities of enzymes that bear cofactors in their active sites, such as the oxygen transport of haemoglobin or myoglobin and the electron transport of cytochromes.

  11. Mass spectrometry locates local and allosteric conformational changes that occur on cofactor binding

    PubMed Central

    Beveridge, Rebecca; Migas, Lukasz G.; Payne, Karl A. P.; Scrutton, Nigel S.; Leys, David; Barran, Perdita E.

    2016-01-01

    Fdc1 is a decarboxylase enzyme that requires the novel prenylated FMN cofactor for activity. Here, we use it as an exemplar system to show how native top-down and bottom-up mass spectrometry can measure the structural effect of cofactor binding by a protein. For Fdc1Ubix, the cofactor confers structural stability to the enzyme. IM–MS shows the holo protein to exist in four closely related conformational families, the populations of which differ in the apo form; the two smaller families are more populated in the presence of the cofactor and depopulated in its absence. These findings, supported by MD simulations, indicate a more open structure for the apo form. HDX-MS reveals that while the dominant structural changes occur proximal to the cofactor-binding site, rearrangements on cofactor binding are evident throughout the protein, predominantly attributable to allosteric conformational tightening, consistent with IM–MS data. PMID:27418477

  12. Mass spectrometry locates local and allosteric conformational changes that occur on cofactor binding

    NASA Astrophysics Data System (ADS)

    Beveridge, Rebecca; Migas, Lukasz G.; Payne, Karl A. P.; Scrutton, Nigel S.; Leys, David; Barran, Perdita E.

    2016-07-01

    Fdc1 is a decarboxylase enzyme that requires the novel prenylated FMN cofactor for activity. Here, we use it as an exemplar system to show how native top-down and bottom-up mass spectrometry can measure the structural effect of cofactor binding by a protein. For Fdc1Ubix, the cofactor confers structural stability to the enzyme. IM-MS shows the holo protein to exist in four closely related conformational families, the populations of which differ in the apo form; the two smaller families are more populated in the presence of the cofactor and depopulated in its absence. These findings, supported by MD simulations, indicate a more open structure for the apo form. HDX-MS reveals that while the dominant structural changes occur proximal to the cofactor-binding site, rearrangements on cofactor binding are evident throughout the protein, predominantly attributable to allosteric conformational tightening, consistent with IM-MS data.

  13. Non-enzymatic glycation reduces heparin cofactor II anti-thrombin activity.

    PubMed

    Ceriello, A; Marchi, E; Barbanti, M; Milani, M R; Giugliano, D; Quatraro, A; Lefebvre, P

    1990-04-01

    The effects of non-enzymatic glycation on heparin cofactor II activity, at glucose concentrations which might be expected in physiological or diabetic conditions have been evaluated in this study. Radiolabelled glucose incorporation was associated with a loss of heparin cofactor anti-thrombin activity. The heparin cofactor heparin and dermatan sulfate-dependent inhibition of thrombin was significantly reduced, showing a remarkable decrease of the maximum second order rate constant. This study shows that heparin cofactor can be glycated at glucose concentrations found in the blood, and that this phenomenon produces a loss of heparin cofactor-antithrombin activity. These data suggest, furthermore, a possible link between heparin cofactor glycation and the pathogenesis of thrombosis in diabetes mellitus.

  14. Manual control of catalytic reactions: Reactions by an apoenzyme gel and a cofactor gel

    PubMed Central

    Kobayashi, Yuichiro; Takashima, Yoshinori; Hashidzume, Akihito; Yamaguchi, Hiroyasu; Harada, Akira

    2015-01-01

    Enzymes play a vital role in catalysing almost all chemical reactions that occur in biological systems. Some enzymes must form complexes with non-protein molecules called cofactors to express catalytic activities. Although the control of catalytic reactions via apoenzyme–cofactor complexes has attracted significant attention, the reports have been limited to the microscale. Here, we report a system to express catalytic activity by adhesion of an apoenzyme gel and a cofactor gel. The apoenzyme and cofactor gels act as catalysts when they form a gel assembly, but they lose catalytic ability upon manual dissociation. We successfully construct a system with switchable catalytic activity via adhesion and separation of the apoenzyme gel with the cofactor gel. We expect that this methodology can be applied to regulate the functional activities of enzymes that bear cofactors in their active sites, such as the oxygen transport of haemoglobin or myoglobin and the electron transport of cytochromes. PMID:26537172

  15. Biocatalytic process optimization based on mechanistic modeling of cholic acid oxidation with cofactor regeneration.

    PubMed

    Braun, Michael; Link, Hannes; Liu, Luo; Schmid, Rolf D; Weuster-Botz, Dirk

    2011-06-01

    Reduction and oxidation of steroids in the human gut are catalyzed by hydroxysteroid dehydrogenases of microorganisms. For the production of 12-ketochenodeoxycholic acid (12-Keto-CDCA) from cholic acid the biocatalytic application of the 12α-hydroxysteroid dehydrogenase of Clostridium group P, strain C 48-50 (HSDH) is an alternative to chemical synthesis. However, due to the intensive costs the necessary cofactor (NADP(+) ) has to be regenerated. The alcohol dehydrogenase of Thermoanaerobacter ethanolicus (ADH-TE) was applied to catalyze the reduction of acetone while regenerating NADP(+) . A mechanistic kinetic model was developed for the process development of cholic acid oxidation using HSDH and ADH-TE. The process model was derived by identifying the parameters for both enzymatic models separately using progress curve measurements of batch processes over a broad range of concentrations and considering the underlying ordered bi-bi mechanism. Both independently derived kinetic models were coupled via mass balances to predict the production of 12-Keto-CDCA with HSDH and integrated cofactor regeneration with ADH-TE and acetone as co-substrate. The prediction of the derived model was suitable to describe the dynamics of the preparative 12-Keto-CDCA batch production with different initial reactant and enzyme concentrations. These datasets were used again for parameter identification. This led to a combined model which excellently described the reaction dynamics of biocatalytic batch processes over broad concentration ranges. Based on the identified process model batch process optimization was successfully performed in silico to minimize enzyme costs. By using 0.1 mM NADP(+) the HSDH concentration can be reduced to 3-4 µM and the ADH concentration to 0.4-0.6 µM to reach the maximal possible conversion of 100 mM cholic acid within 48 h. In conclusion, the identified mechanistic model offers a powerful tool for a cost-efficient process design. Copyright © 2010 Wiley

  16. In silico model-driven cofactor engineering strategies for improving the overall NADP(H) turnover in microbial cell factories.

    PubMed

    Lakshmanan, Meiyappan; Yu, Kai; Koduru, Lokanand; Lee, Dong-Yup

    2015-10-01

    Optimizing the overall NADPH turnover is one of the key challenges in various value-added biochemical syntheses. In this work, we first analyzed the NADPH regeneration potentials of common cell factories, including Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis, and Pichia pastoris across multiple environmental conditions and determined E. coli and glycerol as the best microbial chassis and most suitable carbon source, respectively. In addition, we identified optimal cofactor specificity engineering (CSE) enzyme targets, whose cofactors when switched from NAD(H) to NADP(H) improve the overall NADP(H) turnover. Among several enzyme targets, glyceraldehyde-3-phosphate dehydrogenase was recognized as a global candidate since its CSE improved the NADP(H) regeneration under most of the conditions examined. Finally, by analyzing the protein structures of all CSE enzyme targets via homology modeling, we established that the replacement of conserved glutamate or aspartate with serine in the loop region could change the cofactor dependence from NAD(H) to NADP(H).

  17. Catalase in peroxidase clothing: Interdependent cooperation of two cofactors in the catalytic versatility of KatG.

    PubMed

    Njuma, Olive J; Ndontsa, Elizabeth N; Goodwin, Douglas C

    2014-02-15

    Catalase-peroxidase (KatG) is found in eubacteria, archaea, and lower eukaryotae. The enzyme from Mycobacterium tuberculosis has received the greatest attention because of its role in activation of the antitubercular pro-drug isoniazid, and the high frequency with which drug resistance stems from mutations to the katG gene. Generally, the catalase activity of KatGs is striking. It rivals that of typical catalases, enzymes with which KatGs share no structural similarity. Instead, catalatic turnover is accomplished with an active site that bears a strong resemblance to a typical peroxidase (e.g., cytochrome c peroxidase). Yet, KatG is the only member of its superfamily with such capability. It does so using two mutually dependent cofactors: a heme and an entirely unique Met-Tyr-Trp (MYW) covalent adduct. Heme is required to generate the MYW cofactor. The MYW cofactor allows KatG to leverage heme intermediates toward a unique mechanism for H2O2 oxidation. This review evaluates the range of intermediates identified and their connection to the diverse catalytic processes KatG facilitates, including mechanisms of isoniazid activation.

  18. Simultaneous uncoupled expression and purification of the Dengue virus NS3 protease and NS2B co-factor domain.

    PubMed

    Shannon, A E; Chappell, K J; Stoermer, M J; Chow, S Y; Kok, W M; Fairlie, D P; Young, P R

    2016-03-01

    Dengue Virus (DENV) infection is responsible for the world's most significant insect-borne viral disease. Despite an increasing global impact, there are neither prophylactic nor therapeutic options available for the effective treatment of DENV infection. An attractive target for antiviral drugs is the virally encoded trypsin-like serine protease (NS3pro) and its associated cofactor (NS2B). The NS2B-NS3pro complex is responsible for cleaving the viral polyprotein into separate functional viral proteins, and is therefore essential for replication. Recombinant expression of an active NS2B-NS3 protease has primarily been based on constructs linking the C-terminus of the approximately 40 amino acid hydrophilic cofactor domain of NS2B to the N-terminus of NS3pro via a flexible glycine linker. The resulting complex can be expressed in high yield, is soluble and catalytically active and has been used for most in vitro screening, inhibitor, and X-ray crystallographic studies over the last 15 years. Despite extensive analysis, no inhibitor drug candidates have been identified yet. Moreover, the effect of the artificial linker introduced between the protease and its cofactor is unknown. Two alternate methods for bacterial expression of non-covalently linked, catalytically active, NS2B-NS3pro complex are described here along with a comparison of the kinetics of substrate proteolysis and binding affinities of substrate-based aldehyde inhibitors. Both expression methods produced high yields of soluble protein with improved substrate proteolysis kinetics and inhibitor binding compared to their glycine-linked equivalent. The non-covalent association between NS2B and NS3pro is predicted to be more relevant for examining inhibitors that target cofactor-protease interactions rather than the protease active site. Furthermore, these approaches offer alternative strategies for the high yield co-expression of other protein assemblies.

  19. Reduced Accumulation of ABA during Water Stress in a Molybdenum Cofactor Mutant of Barley 1

    PubMed Central

    Walker-Simmons, Mary; Kudrna, David A.; Warner, Robert L.

    1989-01-01

    A barley (Hordeum vulgare L.) mutant (Az34) has been identified with low basal levels of abscisic acid (ABA) and with reduced capacity for producing ABA in response to water stress. The mutation is in a gene controlling the molybdenum cofactor resulting in a pleiotropic deficiency in at least three molybdoenzymes, nitrate reductase, xanthine dehydrogenase, and aldehyde oxidase. The mutant was found to lack aldehyde oxidase activity with several substrates including: (a) ABA aldehyde, a putative precursor of ABA; (b) an acetylenic analog of ABA aldehyde; and (c) heptaldehyde. Elevating the growth temperature from 18 to 26°C caused mutant leaves to wilt and brown. Desiccation of mutant leaves was prevented by applying ABA. These results indicate that ABA biosynthesis at some developmental stages is dependent upon a molybdoenzyme which may be an aldehyde oxidase. Images Figure 5 PMID:16666835

  20. A Bombyx mandarina mutant exhibiting translucent larval skin is controlled by the molybdenum cofactor sulfurase gene.

    PubMed

    Fujii, Tsuguru; Ozaki, Masataka; Masamoto, Takaaki; Katsuma, Susumu; Abe, Hiroaki; Shimada, Toru

    2009-04-01

    During the maintenance of the wild silkworm, Bombyx mandarina, a mutant phenotype exhibiting translucent skin was identified. Based on the crossing experiments with the domesticated silkworm, Bombyx mori, we found that the mutant was controlled by molybdenum cofactor sulfurase (MoCoS) gene. We designated the mutant ''Ozaki's translucent'' (og(Z)). We found a 2.1-kb deletion containing the transcription initiation site, exons 1 and 2, and the 5' end of exon 3 of the MoCoS gene. The transcript of the MoCoS gene was not detected in the og(Z) homozygote. We concluded that og(Z) is a complete loss-of-function allele generated by a disruption of the MoCoS gene.

  1. Theoretical estimation of redox potential of biological quinone cofactors.

    PubMed

    Gillet, Natacha; Lévy, Bernard; Moliner, Vicent; Demachy, Isabelle; de la Lande, Aurélien

    2017-07-05

    Redox potentials are essential to understand biological cofactor reactivity and to predict their behavior in biological media. Experimental determination of redox potential in biological system is often difficult due to complexity of biological media but computational approaches can be used to estimate them. Nevertheless, the quality of the computational methodology remains a key issue to validate the results. Instead of looking to the best absolute results, we present here the calibration of theoretical redox potential for quinone derivatives in water coupling QM + MM or QM/MM scheme. Our approach allows using low computational cost theoretical level, ideal for long simulations in biological systems, and determination of the uncertainties linked to the calculations. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  2. mTOR Regulates Cellular Iron Homeostasis through Tristetraprolin

    PubMed Central

    Bayeva, Marina; Khechaduri, Arineh; Puig, Sergi; Chang, Hsiang-Chun; Patial, Sonika; Blackshear, Perry J.; Ardehali, Hossein

    2013-01-01

    SUMMARY Iron is an essential cofactor with unique redox properties. Iron regulatory proteins 1 and 2 (IRP1/2) have been established as important regulators of cellular iron homeostasis, but little is known about the role of other pathways in this process. Here we report that the mammalian target of rapamycin (mTOR) regulates iron homeostasis by modulating transferrin receptor 1 (TfR1) stability and altering cellular iron flux. Mechanistic studies identify tristetraprolin (TTP), a protein involved in anti-inflammatory response, as the downstream target of mTOR that binds to and enhances degradation of TfR1 mRNA. We also show that TTP is strongly induced by iron chelation, promotes downregulation of iron-requiring genes in both mammalian and yeast cells, and modulates survival in low-iron states. Taken together, our data uncover a link between metabolic, inflammatory, and iron regulatory pathways, and point towards the existence of a yeast-like TTP-mediated iron conservation program in mammals. PMID:23102618

  3. Daily magnesium fluxes regulate cellular timekeeping and energy balance.

    PubMed

    Feeney, Kevin A; Hansen, Louise L; Putker, Marrit; Olivares-Yañez, Consuelo; Day, Jason; Eades, Lorna J; Larrondo, Luis F; Hoyle, Nathaniel P; O'Neill, John S; van Ooijen, Gerben

    2016-04-21

    Circadian clocks are fundamental to the biology of most eukaryotes, coordinating behaviour and physiology to resonate with the environmental cycle of day and night through complex networks of clock-controlled genes. A fundamental knowledge gap exists, however, between circadian gene expression cycles and the biochemical mechanisms that ultimately facilitate circadian regulation of cell biology. Here we report circadian rhythms in the intracellular concentration of magnesium ions, [Mg(2+)]i, which act as a cell-autonomous timekeeping component to determine key clock properties both in a human cell line and in a unicellular alga that diverged from each other more than 1 billion years ago. Given the essential role of Mg(2+) as a cofactor for ATP, a functional consequence of [Mg(2+)]i oscillations is dynamic regulation of cellular energy expenditure over the daily cycle. Mechanistically, we find that these rhythms provide bilateral feedback linking rhythmic metabolism to clock-controlled gene expression. The global regulation of nucleotide triphosphate turnover by intracellular Mg(2+) availability has potential to impact upon many of the cell's more than 600 MgATP-dependent enzymes and every cellular system where MgNTP hydrolysis becomes rate limiting. Indeed, we find that circadian control of translation by mTOR is regulated through [Mg(2+)]i oscillations. It will now be important to identify which additional biological processes are subject to this form of regulation in tissues of multicellular organisms such as plants and humans, in the context of health and disease.

  4. Strain-dependent embryonic lethality and exaggerated vascular remodeling in heparin cofactor II–deficient mice

    PubMed Central

    Aihara, Ken-ichi; Azuma, Hiroyuki; Akaike, Masashi; Ikeda, Yasumasa; Sata, Masataka; Takamori, Nobuyuki; Yagi, Shusuke; Iwase, Takashi; Sumitomo, Yuka; Kawano, Hirotaka; Yamada, Takashi; Fukuda, Toru; Matsumoto, Takahiro; Sekine, Keisuke; Sato, Takashi; Nakamichi, Yuko; Yamamoto, Yoko; Yoshimura, Kimihiro; Watanabe, Tomoyuki; Nakamura, Takashi; Oomizu, Akimasa; Tsukada, Minoru; Hayashi, Hideki; Sudo, Toshiki; Kato, Shigeaki; Matsumoto, Toshio

    2007-01-01

    Heparin cofactor II (HCII) specifically inhibits thrombin action at sites of injured arterial wall, and patients with HCII deficiency exhibit advanced atherosclerosis. However, the in vivo effects and the molecular mechanism underlying the action of HCII during vascular remodeling remain elusive. To clarify the role of HCII in vascular remodeling, we generated HCII-deficient mice by gene targeting. In contrast to a previous report, HCII–/– mice were embryonically lethal. In HCII+/– mice, prominent intimal hyperplasia with increased cellular proliferation was observed after tube cuff and wire vascular injury. The number of protease-activated receptor–1–positive (PAR-1–positive) cells was increased in the thickened vascular wall of HCII+/– mice, suggesting enhanced thrombin action in this region. Cuff injury also increased the expression levels of inflammatory cytokines and chemokines in the vascular wall of HCII+/– mice. The intimal hyperplasia in HCII+/– mice with vascular injury was abrogated by human HCII supplementation. Furthermore, HCII deficiency caused acceleration of aortic plaque formation with increased PAR-1 expression and oxidative stress in apoE-KO mice. These results demonstrate that HCII protects against thrombin-induced remodeling of an injured vascular wall by inhibiting thrombin action and suggest that HCII is potentially therapeutic against atherosclerosis without causing coagulatory disturbance. PMID:17549254

  5. A Regulatory Role of NAD Redox Status on Flavin Cofactor Homeostasis in S. cerevisiae Mitochondria

    PubMed Central

    Giancaspero, Teresa Anna; Barile, Maria

    2013-01-01

    Flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD) are two redox cofactors of pivotal importance for mitochondrial functionality and cellular redox balance. Despite their relevance, the mechanism by which intramitochondrial NAD(H) and FAD levels are maintained remains quite unclear in Saccharomyces cerevisiae. We investigated here the ability of isolated mitochondria to degrade externally added FAD and NAD (in both its reduced and oxidized forms). A set of kinetic experiments demonstrated that mitochondrial FAD and NAD(H) destroying enzymes are different from each other and from the already characterized NUDIX hydrolases. We studied here, in some detail, FAD pyrophosphatase (EC 3.6.1.18), which is inhibited by NAD+ and NADH according to a noncompetitive inhibition, with Ki values that differ from each other by an order of magnitude. These findings, together with the ability of mitochondrial FAD pyrophosphatase to metabolize endogenous FAD, presumably deriving from mitochondrial holoflavoproteins destined to degradation, allow for proposing a novel possible role of mitochondrial NAD redox status in regulating FAD homeostasis and/or flavoprotein degradation in S. cerevisiae. PMID:24078860

  6. Enhanced drug delivery via hyperthermal membrane disruption using targeted gold nanoparticles with PEGylated Protein-G as a cofactor.

    PubMed

    Sun, Xinghua; Zhang, Guandong; Keynton, Robert S; O'Toole, Martin G; Patel, Dhruvinkumar; Gobin, Andre M

    2013-11-01

    Gold nanoparticles (GNPs) with near infrared (NIR) plasmon resonance have been promisingly used in photothermal cancer therapy as a less invasive treatment. Recombinant Protein-G (ProG) was PEGylated to act as a cofactor to immobilize immunoglobulins (IgGs) on GNPs by the Fc region, resulting in optimal orientation of IgGs for efficient cancer targeting. In-vitro studies showed that HER-2 overexpressing breast cancer cells, SK-BR-3, were efficiently targeted and ablated at a laser power of 900 J/cm(2) (5 W/cm(2) for 3 min). However, as a means of enhancing treatment efficacy by increasing cellular sensitivity to chemotherapeutic agents, we showed that GNP exposure to lower power laser resulted in small disruptions of cell membrane due to localized hyperthermia. This did not lead to cell death but provided a mechanism for killing cancer cells by providing enhanced uptake of drug molecules thus leading to a new avenue for hyperthermia-anticancer drug combined cancer therapeutics. PEGylated recombinant Protein-G was used as a cofactor to optimize the orientation of IgGs providing "target seeking" properties to gold nanoparticles used in photothermal cancer therapy. The system demonstrated excellent properties in cancer therapy, with the hope and expectation of future clinical translation. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. The non-enzymatic reduction of azo dyes by flavin and nicotinamide cofactors under varying conditions.

    PubMed

    Morrison, Jessica M; John, Gilbert H

    2013-10-01

    Azo dyes are ubiquitous in products and often become environmental pollutants due to their anthropogenic nature. Azoreductases are enzymes which are present within many bacteria and are capable of breaking down the azo dyes via reduction of the azo bond. Often, though, carcinogenic aromatic amines are formed as metabolites and are of concern to humans. Azoreductases function via an oxidation-reduction reaction and require cofactors (a nicotinamide cofactor and sometimes a flavin cofactor) to perform their function. Non-enzymatic reduction of azo dyes in the absence of an azoreductase enzyme has been suggested in previous studies, but has never been studied in detail in terms of varying cofactor combinations, different oxygen states or pHs, nor has the enzymatic reduction been compared to azoreduction in terms of dye reduction or metabolites produced, which was the aim of this study. Reduction of azo dyes by different cofactor combinations was found to occur under both aerobic and anaerobic conditions and under physiologically-relevant pHs to produce the same metabolites as an azoreductase. Our results show that, in some cases, the non-enzymatic reduction by the cofactors was found to be equal to that seen with the azoreductase, suggesting that all dye reduction in these cases is due to the cofactors themselves. This study details the importance of the use of a cofactor-only control when studying azoreductase enzymes.

  8. Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16

    PubMed Central

    Wenning, Leonie; Stöveken, Nadine; Wübbeler, Jan Hendrik

    2015-01-01

    Cysteine dioxygenases (Cdos), which catalyze the sulfoxidation of cysteine to cysteine sulfinic acid (CSA), have been extensively studied in eukaryotes because of their roles in several diseases. In contrast, only a few prokaryotic enzymes of this type have been investigated. In Ralstonia eutropha H16, two Cdo homologues (CdoA and CdoB) have been identified previously. In vivo studies showed that Escherichia coli cells expressing CdoA could convert 3-mercaptopropionate (3MP) to 3-sulfinopropionate (3SP), whereas no 3SP could be detected in cells expressing CdoB. The objective of this study was to confirm these findings and to study both enzymes in detail by performing an in vitro characterization. The proteins were heterologously expressed and purified to apparent homogeneity by immobilized metal chelate affinity chromatography (IMAC). Subsequent analysis of the enzyme activities revealed striking differences with regard to their substrate ranges and their specificities for the transition metal cofactor, e.g., CdoA catalyzed the sulfoxidation of 3MP to a 3-fold-greater extent than the sulfoxidation of cysteine, whereas CdoB converted only cysteine. Moreover, the dependency of the activities of the Cdos from R. eutropha H16 on the metal cofactor in the active center could be demonstrated. The importance of CdoA for the metabolism of the sulfur compounds 3,3′-thiodipropionic acid (TDP) and 3,3′-dithiodipropionic acid (DTDP) by further converting their degradation product, 3MP, was confirmed. Since 3MP can also function as a precursor for polythioester (PTE) synthesis in R. eutropha H16, deletion of cdoA might enable increased synthesis of PTEs. PMID:26590284

  9. Co‐immobilized Phosphorylated Cofactors and Enzymes as Self‐Sufficient Heterogeneous Biocatalysts for Chemical Processes

    PubMed Central

    Velasco‐Lozano, Susana; Benítez‐Mateos, Ana I.

    2016-01-01

    Abstract Enzyme cofactors play a major role in biocatalysis, as many enzymes require them to catalyze highly valuable reactions in organic synthesis. However, the cofactor recycling is often a hurdle to implement enzymes at the industrial level. The fabrication of heterogeneous biocatalysts co‐immobilizing phosphorylated cofactors (PLP, FAD+, and NAD+) and enzymes onto the same solid material is reported to perform chemical reactions without exogeneous addition of cofactors in aqueous media. In these self‐sufficient heterogeneous biocatalysts, the immobilized enzymes are catalytically active and the immobilized cofactors catalytically available and retained into the solid phase for several reaction cycles. Finally, we have applied a NAD+‐dependent heterogeneous biocatalyst to continuous flow asymmetric reduction of prochiral ketones, thus demonstrating the robustness of this approach for large scale biotransformations. PMID:28000978

  10. Radical S-Adenosyl-L-methionine Chemistry in the Synthesis of Hydrogenase and Nitrogenase Metal Cofactors

    DOE PAGES

    Byer, Amanda S.; Shepard, Eric M.; Peters, John W.; ...

    2014-12-04

    Nitrogenase, [FeFe]-hydrogenase, and [Fe]-hydrogenase enzymes perform catalysis at metal cofactors with biologically unusual non-protein ligands. Furthermore, the FeMo cofactor of nitrogenase has a MoFe7S9 cluster with a central carbon, whereas the H-cluster of [FeFe]-hydrogenase contains a 2Fe subcluster coordinated by cyanide and CO ligands as well as dithiomethylamine; the [Fe]-hydrogenase cofactor has CO and guanylylpyridinol ligands at a mononuclear iron site. Intriguingly, radical S-adenosyl-L-methionine enzymes are vital for the assembly of all three of these diverse cofactors. Here, in this minireview, we present and discuss the current state of knowledge of the radical S-adenosylmethionine enzymes required for synthesis of thesemore » remarkable metal cofactors.« less

  11. Beyond the Protein Matrix: Probing Cofactor Variants in a Baeyer-Villiger Oxygenation Reaction

    PubMed Central

    Martinoli, Christian; Dudek, Hanna M.; Orru, Roberto; Edmondson, Dale E.; Fraaije, Marco W.; Mattevi, Andrea

    2014-01-01

    A general question in biochemistry is the interplay between the chemical properties of cofactors and the surrounding protein matrix. Here, the functions of NADP+ and FAD are explored by investigation of a representative monooxygenase reconstituted with chemically-modified cofactor analogues. Like pieces of a jigsaw puzzle, the enzyme active site juxtaposes the flavin and nicotinamide rings, harnessing their H-bonding and steric properties to finely construct an oxygen-reacting center that restrains the flavin-peroxide intermediate in a catalytically-competent orientation. Strikingly, the regio- and stereoselectivities of the reaction are essentially unaffected by cofactor modifications. These observations indicate a remarkable robustness of this complex multi-cofactor active site, which has implications for enzyme design based on cofactor engineering approaches. PMID:24443704

  12. What's in a covalent bond? On the role and formation of covalently bound flavin cofactors.

    PubMed

    Heuts, Dominic P H M; Scrutton, Nigel S; McIntire, William S; Fraaije, Marco W

    2009-07-01

    Many enzymes use one or more cofactors, such as biotin, heme, or flavin. These cofactors may be bound to the enzyme in a noncovalent or covalent manner. Although most flavoproteins contain a noncovalently bound flavin cofactor (FMN or FAD), a large number have these cofactors covalently linked to the polypeptide chain. Most covalent flavin-protein linkages involve a single cofactor attachment via a histidyl, tyrosyl, cysteinyl or threonyl linkage. However, some flavoproteins contain a flavin that is tethered to two amino acids. In the last decade, many studies have focused on elucidating the mechanism(s) of covalent flavin incorporation (flavinylation) and the possible role(s) of covalent protein-flavin bonds. These endeavors have revealed that covalent flavinylation is a post-translational and self-catalytic process. This review presents an overview of the known types of covalent flavin bonds and the proposed mechanisms and roles of covalent flavinylation.

  13. Co-immobilized Phosphorylated Cofactors and Enzymes as Self-Sufficient Heterogeneous Biocatalysts for Chemical Processes.

    PubMed

    Velasco-Lozano, Susana; Benítez-Mateos, Ana I; López-Gallego, Fernando

    2017-01-16

    Enzyme cofactors play a major role in biocatalysis, as many enzymes require them to catalyze highly valuable reactions in organic synthesis. However, the cofactor recycling is often a hurdle to implement enzymes at the industrial level. The fabrication of heterogeneous biocatalysts co-immobilizing phosphorylated cofactors (PLP, FAD(+) , and NAD(+) ) and enzymes onto the same solid material is reported to perform chemical reactions without exogeneous addition of cofactors in aqueous media. In these self-sufficient heterogeneous biocatalysts, the immobilized enzymes are catalytically active and the immobilized cofactors catalytically available and retained into the solid phase for several reaction cycles. Finally, we have applied a NAD(+) -dependent heterogeneous biocatalyst to continuous flow asymmetric reduction of prochiral ketones, thus demonstrating the robustness of this approach for large scale biotransformations.

  14. Cofactors in allergic reactions to food: physical exercise and alcohol are the most important

    PubMed Central

    van Os‐Medendorp, Harmieke; Kruizinga, Astrid G.; Blom, W. Marty; Houben, Geert F.; Knulst, André C.

    2016-01-01

    Abstract Introduction Involvement of cofactors, like physical exercise, alcohol consumption and use of several types of medication, are associated with more severe food allergic symptoms. However, there is limited evidence on how often cofactors play a role in food allergic reactions. The study aimed to get more insight into the frequency of exposure to cofactors and how often cofactors are associated with more severe symptoms in food allergic patients. Methods A questionnaire was completed by patients visiting the Allergology outpatient clinic. Patients with food allergy were included. Outcome measures were the frequency of medication use of medication groups that might act as cofactor and the frequency that physical exercise, alcohol consumption and use of analgesics are associated with more severe food allergic symptoms. Results Four hundred ninety‐six patients were included in the study. The frequency with which patients used one or more types of medication that might act as cofactors was 7.7%: antacids/acid neutralizing medication (5%), NSAIDs (2%), beta blockers (0.6%), angiotensin‐converting enzyme inhibitors (0.6%), and angiotensin receptor blockers (0.2%). Of all patients, 13% reported more severe symptoms to food after involvement of one or more of the cofactors: physical exercise (10%), alcohol consumption (5%), and use of analgesics (0.6%). Sixty‐five percent did not know if these cofactors caused more severe symptoms; 22% reported that these cofactors had no effect. Conclusions Only a small percentage of patients (7.7%) used medication that might aggravate food allergic reactions. Physical exercise and alcohol consumption were the most frequently reported cofactors, but occurring still in only 10% or less. PMID:27980774

  15. Studying Nuclear Receptor Complexes in the Cellular Environment.

    PubMed

    Schaufele, Fred

    2016-01-01

    The ligand-regulated structure and biochemistry of nuclear receptor complexes are commonly determined by in vitro studies of isolated receptors, cofactors, and their fragments. However, in the living cell, the complexes that form are governed not just by the relative affinities of isolated cofactors for the receptor but also by the cell-specific sequestration or concentration of subsets of competing or cooperating cofactors, receptors, and other effectors into distinct subcellular domains and/or their temporary diversion into other cellular activities. Most methods developed to understand nuclear receptor function in the cellular environment involve the direct tagging of the nuclear receptor or its cofactors with fluorescent proteins (FPs) and the tracking of those FP-tagged factors by fluorescence microscopy. One of those approaches, Förster resonance energy transfer (FRET) microscopy, quantifies the transfer of energy from a higher energy "donor" FP to a lower energy "acceptor" FP attached to a single protein or to interacting proteins. The amount of FRET is influenced by the ligand-induced changes in the proximities and orientations of the FPs within the tagged nuclear receptor complexes, which is an indicator of the structure of the complexes, and by the kinetics of the interaction between FP-tagged factors. Here, we provide a guide for parsing information about the structure and biochemistry of nuclear receptor complexes from FRET measurements in living cells.

  16. Mechanism of pyranopterin ring formation in molybdenum cofactor biosynthesis

    DOE PAGES

    Hover, Bradley M.; Tonthat, Nam K.; Schumacher, Maria A.; ...

    2015-05-04

    The molybdenum cofactor (Moco) is essential for all kingdoms of life, plays central roles in various biological processes, and must be biosynthesized de novo. During Moco biosynthesis, the characteristic pyranopterin ring is constructed by a complex rearrangement of guanosine 5'-triphosphate (GTP) into cyclic pyranopterin (cPMP) through the action of two enzymes, MoaA and MoaC (molybdenum cofactor biosynthesis protein A and C, respectively). Conventionally, MoaA was considered to catalyze the majority of this transformation, with MoaC playing little or no role in the pyranopterin formation. Recently, this view was challenged by the isolation of 3',8-cyclo-7,8-dihydro-guanosine 5'-triphosphate (3',8-cH2GTP) as the product ofmore » in vitro MoaA reactions. To elucidate the mechanism of formation of Moco pyranopterin backbone, in this paper we performed biochemical characterization of 3',8-cH2GTP and functional and X-ray crystallographic characterizations of MoaC. These studies revealed that 3',8-cH2GTP is the only product of MoaA that can be converted to cPMP by MoaC. Our structural studies captured the specific binding of 3',8-cH2GTP in the active site of MoaC. These observations provided strong evidence that the physiological function of MoaA is the conversion of GTP to 3',8-cH2GTP (GTP 3',8-cyclase), and that of MoaC is to catalyze the rearrangement of 3',8-cH2GTP into cPMP (cPMP synthase). Furthermore, our structure-guided studies suggest that MoaC catalysis involves the dynamic motions of enzyme active-site loops as a way to control the timing of interaction between the reaction intermediates and catalytically essential amino acid residues. In conclusion, these results reveal the previously unidentified mechanism behind Moco biosynthesis and provide mechanistic and structural insights into how enzymes catalyze complex rearrangement reactions.« less

  17. Mechanism of pyranopterin ring formation in molybdenum cofactor biosynthesis

    SciTech Connect

    Hover, Bradley M.; Tonthat, Nam K.; Schumacher, Maria A.; Yokoyama, Kenichi

    2015-05-04

    The molybdenum cofactor (Moco) is essential for all kingdoms of life, plays central roles in various biological processes, and must be biosynthesized de novo. During Moco biosynthesis, the characteristic pyranopterin ring is constructed by a complex rearrangement of guanosine 5'-triphosphate (GTP) into cyclic pyranopterin (cPMP) through the action of two enzymes, MoaA and MoaC (molybdenum cofactor biosynthesis protein A and C, respectively). Conventionally, MoaA was considered to catalyze the majority of this transformation, with MoaC playing little or no role in the pyranopterin formation. Recently, this view was challenged by the isolation of 3',8-cyclo-7,8-dihydro-guanosine 5'-triphosphate (3',8-cH2GTP) as the product of in vitro MoaA reactions. To elucidate the mechanism of formation of Moco pyranopterin backbone, in this paper we performed biochemical characterization of 3',8-cH2GTP and functional and X-ray crystallographic characterizations of MoaC. These studies revealed that 3',8-cH2GTP is the only product of MoaA that can be converted to cPMP by MoaC. Our structural studies captured the specific binding of 3',8-cH2GTP in the active site of MoaC. These observations provided strong evidence that the physiological function of MoaA is the conversion of GTP to 3',8-cH2GTP (GTP 3',8-cyclase), and that of MoaC is to catalyze the rearrangement of 3',8-cH2GTP into cPMP (cPMP synthase). Furthermore, our structure-guided studies suggest that MoaC catalysis involves the dynamic motions of enzyme active-site loops as a way to control the timing of interaction between the reaction intermediates and catalytically essential amino acid residues. In conclusion, these results reveal the previously unidentified mechanism behind Moco biosynthesis and provide mechanistic and structural insights into how enzymes catalyze complex rearrangement reactions.

  18. Regulating cellular trace metal economy in algae

    DOE PAGES

    Blaby-Haas, Crysten E.; Merchant, Sabeeha S.

    2017-06-30

    As indispensable protein cofactors, Fe, Mn, Cu and Zn are at the center of multifaceted acclimation mechanisms that have evolved to ensure extracellular supply meets intracellular demand. In starting with selective transport at the plasma membrane and ending in protein metalation, metal homeostasis in algae involves regulated trafficking of metal ions across membranes, intracellular compartmentalization by proteins and organelles, and metal-sparing/recycling mechanisms to optimize metal-use efficiency. Overlaid on these processes are additional circuits that respond to the metabolic state as well as to the prior metal status of the cell. Here, we focus on recent progress made toward understanding themore » pathways by which the single-celled, green alga Chlamydomonas reinhardtii controls its cellular trace metal economy. We also compare these mechanisms to characterized and putative processes in other algal lineages. Photosynthetic microbes continue to provide insight into cellular regulation and handling of Cu, Fe, Zn and Mn as a function of the nutritional supply and cellular demand for metal cofactors. We found that new experimental tools such as RNA-Seq and subcellular metal imaging are bringing us closer to a molecular understanding of acclimation to supply dynamics in algae and beyond.« less

  19. Regulating cellular trace metal economy in algae.

    PubMed

    Blaby-Haas, Crysten E; Merchant, Sabeeha S

    2017-10-01

    As indispensable protein cofactors, Fe, Mn, Cu and Zn are at the center of multifaceted acclimation mechanisms that have evolved to ensure extracellular supply meets intracellular demand. Starting with selective transport at the plasma membrane and ending in protein metalation, metal homeostasis in algae involves regulated trafficking of metal ions across membranes, intracellular compartmentalization by proteins and organelles, and metal-sparing/recycling mechanisms to optimize metal-use efficiency. Overlaid on these processes are additional circuits that respond to the metabolic state as well as to the prior metal status of the cell. In this review, we focus on recent progress made toward understanding the pathways by which the single-celled, green alga Chlamydomonas reinhardtii controls its cellular trace metal economy. We also compare these mechanisms to characterized and putative processes in other algal lineages. Photosynthetic microbes continue to provide insight into cellular regulation and handling of Cu, Fe, Zn and Mn as a function of the nutritional supply and cellular demand for metal cofactors. New experimental tools such as RNA-Seq and subcellular metal imaging are bringing us closer to a molecular understanding of acclimation to supply dynamics in algae and beyond. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Sulphur shuttling across a chaperone during molybdenum cofactor maturation

    NASA Astrophysics Data System (ADS)

    Arnoux, Pascal; Ruppelt, Christian; Oudouhou, Flore; Lavergne, Jérôme; Siponen, Marina I.; Toci, René; Mendel, Ralf R.; Bittner, Florian; Pignol, David; Magalon, Axel; Walburger, Anne

    2015-02-01

    Formate dehydrogenases (FDHs) are of interest as they are natural catalysts that sequester atmospheric CO2, generating reduced carbon compounds with possible uses as fuel. FDHs activity in Escherichia coli strictly requires the sulphurtransferase EcFdhD, which likely transfers sulphur from IscS to the molybdenum cofactor (Mo-bisPGD) of FDHs. Here we show that EcFdhD binds Mo-bisPGD in vivo and has submicromolar affinity for GDP—used as a surrogate of the molybdenum cofactor’s nucleotide moieties. The crystal structure of EcFdhD in complex with GDP shows two symmetrical binding sites located on the same face of the dimer. These binding sites are connected via a tunnel-like cavity to the opposite face of the dimer where two dynamic loops, each harbouring two functionally important cysteine residues, are present. On the basis of structure-guided mutagenesis, we propose a model for the sulphuration mechanism of Mo-bisPGD where the sulphur atom shuttles across the chaperone dimer.

  1. Relocalization of human chromatin remodeling cofactor TIP48 in mitosis

    SciTech Connect

    Sigala, Barbara; Edwards, Mina; Puri, Teena; Tsaneva, Irina R. . E-mail: tsaneva@biochem.ucl.ac.uk

    2005-11-01

    TIP48 is a highly conserved eukaryotic AAA{sup +} protein which is an essential cofactor for several complexes involved in chromatin acetylation and remodeling, transcriptional and developmental regulation and nucleolar organization and trafficking. We show that TIP48 abundance in HeLa cells did not change during the cell cycle, nor did its distribution in various biochemical fractions. However, we observed distinct changes in the subcellular localization of TIP48 during M phase using immunofluorescence microscopy. Our studies demonstrate that in interphase cells TIP48 was found mainly in the nucleus and exhibited a distinct localization in the nuclear periphery. As the cells entered mitosis, TIP48 was excluded from the condensing chromosomes but showed association with the mitotic apparatus. During anaphase, some TIP48 was detected in the centrosome colocalizing with tubulin but the strongest staining appeared in the mitotic equator associated with the midzone central spindle. Accumulation of TIP48 in the midzone and the midbody was observed in late telophase and cytokinesis. This redeployment of TIP48 during anaphase and cytokinesis was independent of microtubule assembly. The relocation of endogenous TIP48 to the midzone/midbody under physiological conditions suggests a novel and distinct function for TIP48 in mitosis and possible involvement in the exit of mitosis.

  2. SAGA Is a General Cofactor for RNA Polymerase II Transcription.

    PubMed

    Baptista, Tiago; Grünberg, Sebastian; Minoungou, Nadège; Koster, Maria J E; Timmers, H T Marc; Hahn, Steve; Devys, Didier; Tora, László

    2017-10-05

    Prior studies suggested that SAGA and TFIID are alternative factors that promote RNA polymerase II transcription, with about 10% of genes in S. cerevisiae dependent on SAGA. We reassessed the role of SAGA by mapping its genome-wide location and role in global transcription in budding yeast. We find that SAGA maps to the UAS elements of most genes, overlapping with Mediator binding and irrespective of previous designations of SAGA- or TFIID-dominated genes. Disruption of SAGA through mutation or rapid subunit depletion reduces transcription from nearly all genes, measured by newly synthesized RNA. We also find that the acetyltransferase Gcn5 synergizes with Spt3 to promote global transcription and that Spt3 functions to stimulate TBP recruitment at all tested genes. Our data demonstrate that SAGA acts as a general cofactor required for essentially all RNA polymerase II transcription and is not consistent with the previous classification of SAGA- and TFIID-dominated genes. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Extracellular RNA constitutes a natural procoagulant cofactor in blood coagulation

    PubMed Central

    Kannemeier, Christian; Shibamiya, Aya; Nakazawa, Fumie; Trusheim, Heidi; Ruppert, Clemens; Markart, Philipp; Song, Yutong; Tzima, Eleni; Kennerknecht, Elisabeth; Niepmann, Michael; von Bruehl, Marie-Luise; Sedding, Daniel; Massberg, Steffen; Günther, Andreas; Engelmann, Bernd; Preissner, Klaus T.

    2007-01-01

    Upon vascular injury, locally controlled haemostasis prevents life-threatening blood loss and ensures wound healing. Intracellular material derived from damaged cells at these sites will become exposed to blood components and could contribute to blood coagulation and pathological thrombus formation. So far, the functional and mechanistic consequences of this concept are not understood. Here, we present in vivo and in vitro evidence that different forms of eukaryotic and prokaryotic RNA serve as promoters of blood coagulation. Extracellular RNA was found to augment (auto-)activation of proteases of the contact phase pathway of blood coagulation such as factors XII and XI, both exhibiting strong RNA binding. Moreover, administration of exogenous RNA provoked a significant procoagulant response in rabbits. In mice that underwent an arterial thrombosis model, extracellular RNA was found associated with fibrin-rich thrombi, and pretreatment with RNase (but not DNase) significantly delayed occlusive thrombus formation. Thus, extracellular RNA derived from damaged or necrotic cells particularly under pathological conditions or severe tissue damage represents the long sought natural “foreign surface” and provides a procoagulant cofactor template for the factors XII/XI-induced contact activation/amplification of blood coagulation. Extracellular RNA thereby reveals a yet unrecognized target for antithrombotic intervention, using RNase or related therapeutic strategies. PMID:17405864

  4. Multiple Quantitative Trait Loci Mapping With Cofactors and Application of Alternative Variants of the False Discovery Rate in an Enlarged Granddaughter Design

    PubMed Central

    Bennewitz, Jörn; Reinsch, Norbert; Guiard, Volker; Fritz, Sebastien; Thomsen, Hauke; Looft, Christian; Kühn, Christa; Schwerin, Manfred; Weimann, Christina; Erhardt, Georg; Reinhardt, Fritz; Reents, Reinhard; Boichard, Didier; Kalm, Ernst

    2004-01-01

    The experimental power of a granddaughter design to detect quantitative trait loci (QTL) in dairy cattle is often limited by the availability of progeny-tested sires, by the ignoring of already identified QTL in the statistical analysis, and by the application of stringent experimentwise significance levels. This study describes an experiment that addressed these points. A large granddaughter design was set up that included sires from two countries (Germany and France), resulting in almost 2000 sires. The animals were genotyped for markers on nine different chromosomes. The QTL analysis was done for six traits separately using a multimarker regression that included putative QTL on other chromosomes as cofactors in the model. Different variants of the false discovery rate (FDR) were applied. Two of them accounted for the proportion of truly null hypotheses, which were estimated to be 0.28 and 0.3, respectively, and were therefore tailored to the experiment. A total of 25 QTL could be mapped when cofactors were included in the model—7 more than without cofactors. Controlling the FDR at 0.05 revealed 31 QTL for the two FDR methods that accounted for the proportion of truly null hypotheses. The relatively high power of this study can be attributed to the size of the experiment, to the QTL analysis with cofactors, and to the application of an appropriate FDR. PMID:15514072

  5. Alteration of the specificity of the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase A.

    PubMed

    Banta, Scott; Swanson, Barbara A; Wu, Shan; Jarnagin, Alisha; Anderson, Stephen

    2002-02-01

    The NADPH-dependent 2,5-diketo-D-gluconic acid (2,5-DKG) reductase enzyme is a required component in some novel biosynthetic vitamin C production processes. This enzyme catalyzes the conversion of 2,5-DKG to 2-keto-L-gulonic acid, which is an immediate precursor to L-ascorbic acid. Forty unique site-directed mutations were made at five residues in the cofactor-binding pocket of 2,5-DKG reductase A in an attempt to improve its ability to use NADH as a cofactor. NADH is more stable, less expensive and more prevalent in the cell than is NADPH. To the best of our knowledge, this is the first focused attempt to alter the cofactor specificity of a member of the aldo-keto reductase superfamily by engineering improved activity with NADH into the enzyme. Activity of the mutants with NADH or NADPH was assayed using activity-stained native polyacrylamide gels. Eight of the mutants at three different sites were identified as having improved activity with NADH. These mutants were purified and subjected to a kinetic characterization with NADH as a cofactor. The best mutant obtained, R238H, produced an almost 7-fold improvement in catalysis with NADH compared with the wild-type enzyme. Surprisingly, most of this catalytic improvement appeared to be due to an improvement in the apparent kcat for the reaction rather than a large improvement in the affinity of the enzyme for NADH.

  6. Multiple quantitative trait loci mapping with cofactors and application of alternative variants of the false discovery rate in an enlarged granddaughter design.

    PubMed

    Bennewitz, Jörn; Reinsch, Norbert; Guiard, Volker; Fritz, Sebastien; Thomsen, Hauke; Looft, Christian; Kühn, Christa; Schwerin, Manfred; Weimann, Christina; Erhardt, Georg; Reinhardt, Fritz; Reents, Reinhard; Boichard, Didier; Kalm, Ernst

    2004-10-01

    The experimental power of a granddaughter design to detect quantitative trait loci (QTL) in dairy cattle is often limited by the availability of progeny-tested sires, by the ignoring of already identified QTL in the statistical analysis, and by the application of stringent experimentwise significance levels. This study describes an experiment that addressed these points. A large granddaughter design was set up that included sires from two countries (Germany and France), resulting in almost 2000 sires. The animals were genotyped for markers on nine different chromosomes. The QTL analysis was done for six traits separately using a multimarker regression that included putative QTL on other chromosomes as cofactors in the model. Different variants of the false discovery rate (FDR) were applied. Two of them accounted for the proportion of truly null hypotheses, which were estimated to be 0.28 and 0.3, respectively, and were therefore tailored to the experiment. A total of 25 QTL could be mapped when cofactors were included in the model-7 more than without cofactors. Controlling the FDR at 0.05 revealed 31 QTL for the two FDR methods that accounted for the proportion of truly null hypotheses. The relatively high power of this study can be attributed to the size of the experiment, to the QTL analysis with cofactors, and to the application of an appropriate FDR.

  7. Nanoparticle-supported multi-enzyme biocatalysis with in situ cofactor regeneration.

    PubMed

    Liu, Wenfang; Zhang, Songping; Wang, Ping

    2009-01-01

    Although there have been a long history of studying and using immobilized enzymes, little has been reported regarding the nature of immobilized cofactors. Herein we report that cofactor NAD(H) covalently attached to silica nanoparticles successfully coordinated with particle-immobilized enzymes and enabled multistep biotransformations. Specifically, silica nanoparticle-attached glutamate dehydrogenase (GLDH), lactate dehydrogenase (LDH) and NAD(H) were prepared and applied to catalyze the coupled reactions for production of alpha-ketoglutarate and lactate with the cofactor regenerated within the reaction cycle. It appeared that particle-particle collision driven by Brownian motion of the nanoparticles provided effective interactions among the catalytic components, and thus realized a dynamic shuttling of the particle-supported cofactor between the two enzymes to keep the reaction cycles continuing. Total turnover numbers (TTNs) as high as 20,000h(-1) were observed for the cofactor. It appeared to us that the use of particle-attached cofactor promises a new biochemical processing strategy for cofactor-dependent biotransformations.

  8. Enzyme-Mediated Conversion of Flavin Adenine Dinucleotide (FAD) to 8-Formyl FAD in Formate Oxidase Results in a Modified Cofactor with Enhanced Catalytic Properties.

    PubMed

    Robbins, John M; Souffrant, Michael G; Hamelberg, Donald; Gadda, Giovanni; Bommarius, Andreas S

    2017-07-25

    Flavins, including flavin adenine dinucleotide (FAD), are fundamental catalytic cofactors that are responsible for the redox functionality of a diverse set of proteins. Alternatively, modified flavin analogues are rarely found in nature as their incorporation typically results in inactivation of flavoproteins, thus leading to the disruption of important cellular pathways. Here, we report that the fungal flavoenzyme formate oxidase (FOX) catalyzes the slow conversion of noncovalently bound FAD to 8-formyl FAD and that this conversion results in a nearly 10-fold increase in formate oxidase activity. Although the presence of an enzyme-bound 8-formyl FMN has been reported previously as a result of site-directed mutagenesis studies of lactate oxidase, FOX is the first reported case of 8-formyl FAD in a wild-type enzyme. Therefore, the formation of the 8-formyl FAD cofactor in formate oxidase was investigated using steady-state kinetics, site-directed mutagenesis, ultraviolet-visible, circular dichroism, and fluorescence spectroscopy, liquid chromatography with mass spectrometry, and computational analysis. Surprisingly, the results from these studies indicate not only that 8-formyl FAD forms spontaneously and results in the active form of FOX but also that its autocatalytic formation is dependent on a nearby arginine residue, R87. Thus, this work describes a new enzyme cofactor and provides insight into the little-understood mechanism of enzyme-mediated 8α-flavin modifications.

  9. Biosynthesis of the iron-molybdenum cofactor and the molybdenum cofactor in Klebsiella pneumoniae: effect of sulfur source

    SciTech Connect

    Ugalde, R.A.; Imperial, J.; Shah, V.K.; Brill, W.J.

    1985-12-01

    NifQ/sup -/ and Mol/sup -/ mutants of Klebsiella pneumoniae show an elevated molybdenum requirement for nitrogen fixation. Substitution of cystine for sulfate as the sulfur source in the medium reduced the molybdenum requirement of these mutants to levels required by the wild type. Cystine also increased the intracellular molybdenum accumulation of NifQ/sup -/ and Mol/sup -/ mutants. Cystine did not affect the molybdenum requirement or accumulation in wild-type K. pneumoniae. Sulfate transport and metabolism in K. pneumoniae were repressed by cystine. However, the effect of cystine on the molybdenum requirement could not be explained by an interaction between sulfate and molybdate at the transport level. The data show that cystine does not have a generalized effect on molybdenum metabolism. Millimolar concentrations of molybdate inhibited nitrogenase and nitrate reductase derepression with sulfate as the sulfur source, but not with cystine. The inhibition was the result of a specific antagonism of sulfate metabolism by molybdate. This study suggests that a sulfur donor and molybdenum interact at an early step in the biosynthesis of the iron-molybdenum cofactor. This interaction might occur nonenzymatically when the levels of the reactants are high.

  10. Cofactor-mediated conformational control in the bifunctional kinase/RNase Ire1

    PubMed Central

    2011-01-01

    Background Ire1 is a signal transduction protein in the endoplasmic reticulum (ER) membrane that serves to adjust the protein-folding capacity of the ER according to the needs of the cell. Ire1 signals, in a transcriptional program, the unfolded protein response (UPR) via the coordinated action of its protein kinase and RNase domains. In this study, we investigated how the binding of cofactors to the kinase domain of Ire1 modulates its RNase activity. Results Our results suggest that the kinase domain of Ire1 initially binds cofactors without activation of the RNase domain. RNase is activated upon a subsequent conformational rearrangement of Ire1 governed by the chemical properties of bound cofactors. The conformational step can be selectively inhibited by chemical perturbations of cofactors. Substitution of a single oxygen atom in the terminal β-phosphate group of a potent cofactor ADP by sulfur results in ADPβS, a cofactor that binds to Ire1 as well as to ADP but does not activate RNase. RNase activity can be rescued by thiophilic metal ions such as Mn2+ and Cd2+, revealing a functional metal ion-phosphate interaction which controls the conformation and RNase activity of the Ire1 ADP complex. Mutagenesis of the kinase domain suggests that this rearrangement involves movement of the αC-helix, which is generally conserved among protein kinases. Using X-ray crystallography, we show that oligomerization of Ire1 is sufficient for placing the αC-helix in the active, cofactor-bound-like conformation, even in the absence of cofactors. Conclusions Our structural and biochemical evidence converges on a model that the cofactor-induced conformational change in Ire1 is coupled to oligomerization of the receptor, which, in turn, activates RNase. The data reveal that cofactor-Ire1 interactions occur in two independent steps: binding of a cofactor to Ire1 and subsequent rearrangement of Ire1 resulting in its self-association. The pronounced allosteric effect of cofactors on

  11. Cofactor Requirement of HpyAV Restriction Endonuclease

    PubMed Central

    Chan, Siu-Hong; Opitz, Lars; Higgins, Lauren; O'loane, Diana; Xu, Shuang-yong

    2010-01-01

    Background Helicobacter pylori is the etiologic agent of common gastritis and a risk factor for gastric cancer. It is also one of the richest sources of Type II restriction-modification (R-M) systems in microorganisms. Principal Findings We have cloned, expressed and purified a new restriction endonuclease HpyAV from H. pylori strain 26695. We determined the HpyAV DNA recognition sequence and cleavage site as CCTTC 6/5. In addition, we found that HpyAV has a unique metal ion requirement: its cleavage activity is higher with transition metal ions than in Mg++. The special metal ion requirement of HpyAV can be attributed to the presence of a HNH catalytic site similar to ColE9 nuclease instead of the canonical PD-X-D/EXK catalytic site found in many other REases. Site-directed mutagenesis was carried out to verify the catalytic residues of HpyAV. Mutation of the conserved metal-binding Asn311 and His320 to alanine eliminated cleavage activity. HpyAV variant H295A displayed approximately 1% of wt activity. Conclusions/Significance Some HNH-type endonucleases have unique metal ion cofactor requirement for optimal activities. Homology modeling and site-directed mutagenesis confirmed that HpyAV is a member of the HNH nuclease family. The identification of catalytic residues in HpyAV paved the way for further engineering of the metal binding site. A survey of sequenced microbial genomes uncovered 10 putative R-M systems that show high sequence similarity to the HpyAV system, suggesting lateral transfer of a prototypic HpyAV-like R-M system among these microorganisms. PMID:20140205

  12. HIV-1 evades innate immune recognition through specific cofactor recruitment

    NASA Astrophysics Data System (ADS)

    Rasaiyaah, Jane; Tan, Choon Ping; Fletcher, Adam J.; Price, Amanda J.; Blondeau, Caroline; Hilditch, Laura; Jacques, David A.; Selwood, David L.; James, Leo C.; Noursadeghi, Mahdad; Towers, Greg J.

    2013-11-01

    Human immunodeficiency virus (HIV)-1 is able to replicate in primary human macrophages without stimulating innate immunity despite reverse transcription of genomic RNA into double-stranded DNA, an activity that might be expected to trigger innate pattern recognition receptors. We reasoned that if correctly orchestrated HIV-1 uncoating and nuclear entry is important for evasion of innate sensors then manipulation of specific interactions between HIV-1 capsid and host factors that putatively regulate these processes should trigger pattern recognition receptors and stimulate type 1 interferon (IFN) secretion. Here we show that HIV-1 capsid mutants N74D and P90A, which are impaired for interaction with cofactors cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and cyclophilins (Nup358 and CypA), respectively, cannot replicate in primary human monocyte-derived macrophages because they trigger innate sensors leading to nuclear translocation of NF-κB and IRF3, the production of soluble type 1 IFN and induction of an antiviral state. Depletion of CPSF6 with short hairpin RNA expression allows wild-type virus to trigger innate sensors and IFN production. In each case, suppressed replication is rescued by IFN-receptor blockade, demonstrating a role for IFN in restriction. IFN production is dependent on viral reverse transcription but not integration, indicating that a viral reverse transcription product comprises the HIV-1 pathogen-associated molecular pattern. Finally, we show that we can pharmacologically induce wild-type HIV-1 infection to stimulate IFN secretion and an antiviral state using a non-immunosuppressive cyclosporine analogue. We conclude that HIV-1 has evolved to use CPSF6 and cyclophilins to cloak its replication, allowing evasion of innate immune sensors and induction of a cell-autonomous innate immune response in primary human macrophages.

  13. A cofactor approach to copper-dependent catalytic antibodies

    PubMed Central

    Nicholas, Kenneth M.; Wentworth, Paul; Harwig, Curtis W.; Wentworth, Anita D.; Shafton, Asher; Janda, Kim D.

    2002-01-01

    A strategy for the preparation of semisynthetic copper(II)-based catalytic metalloproteins is described in which a metal-binding bis-imidazole cofactor is incorporated into the combining site of the aldolase antibody 38C2. Antibody 38C2 features a large hydrophobic-combining site pocket with a highly nucleophilic lysine residue, LysH93, that can be covalently modified. A comparison of several lactone and anhydride reagents shows that the latter are the most effective and general derivatizing agents for the 38C2 Lys residue. A bis-imidazole anhydride (5) was efficiently prepared from N-methyl imidazole. The 38C2–5-Cu conjugate was prepared by either (i) initial derivatization of 38C2 with 5 followed by metallation with CuCl2, or (ii) precoordination of 5 with CuCl2 followed by conjugation with 38C2. The resulting 38C2–5-Cu conjugate was an active catalyst for the hydrolysis of the coordinating picolinate ester 11, following Michaelis–Menten kinetics [kcat(11) = 2.3 min−1 and Km(11) 2.2 mM] with a rate enhancement [kcat(11)kuncat(11)] of 2.1 × 105. Comparison of the second-order rate constants of the modified 38C2 and the Cu(II)-bis-imidazolyl complex k(6-CuCl2) gives a rate enhancement of 3.5 × 104 in favor of the antibody complex with an effective molarity of 76.7 M, revealing a significant catalytic benefit to the binding of the bis-imidazolyl ligand into 38C2. PMID:11880619

  14. Cofactor engineering of ketol-acid reductoisomerase (IlvC) and alcohol dehydrogenase (YqhD) improves the fusel alcohol yield in algal protein anaerobic fermentation

    DOE PAGES

    Wu, Weihua; Tran-Gyamfi, Mary Bao; Jaryenneh, James Dekontee; ...

    2016-08-24

    Recently the feasibility of conversion of algal protein to mixed alcohols has been demonstrated with an engineered E.coli strain, enabling comprehensive utilization of the biomass for biofuel applications. However, the yield and titers of mixed alcohol production must be improved for market adoption. A major limiting factor for achieving the necessary yield and titer improvements is cofactor imbalance during the fermentation of algal protein. To resolve this problem, a directed evolution approach was applied to modify the cofactor specificity of two key enzymes (IlvC and YqhD) from NADPH to NADH in the mixed alcohol metabolic pathway. Using high throughput screening,more » more than 20 YqhD mutants were identified to show activity on NADH as a cofactor. Of these 20 mutants, the top five of YqhD mutants were selected for combination with two IlvC mutants with NADH as a cofactor for the modification of the protein conversion strain. The combination of the IlvC and YqhD mutants yielded a refined E.coli strain, subtype AY3, with increased fusel alcohol yield of ~60% compared to wild type under anaerobic fermentation on amino acid mixtures. When applied to real algal protein hydrolysates, the strain AY3 produced 100% and 38% more total mixed alcohols than the wild type strain on two different algal hydrolysates, respectively. The results indicate that cofactor engineering is a promising approach to improve the feasibility of bioconversion of algal protein into mixed alcohols as advanced biofuels.« less

  15. Decoding a Signature-Based Model of Transcription Cofactor Recruitment Dictated by Cardinal Cis-Regulatory Elements in Proximal Promoter Regions

    PubMed Central

    Benner, Christopher; Hutt, Kasey R.; Stunnenberg, Rieka; Garcia-Bassets, Ivan

    2013-01-01

    Genome-wide maps of DNase I hypersensitive sites (DHSs) reveal that most human promoters contain perpetually active cis-regulatory elements between −150 bp and +50 bp (−150/+50 bp) relative to the transcription start site (TSS). Transcription factors (TFs) recruit cofactors (chromatin remodelers, histone/protein-modifying enzymes, and scaffold proteins) to these elements in order to organize the local chromatin structure and coordinate the balance of post-translational modifications nearby, contributing to the overall regulation of transcription. However, the rules of TF-mediated cofactor recruitment to the −150/+50 bp promoter regions remain poorly understood. Here, we provide evidence for a general model in which a series of cis-regulatory elements (here termed ‘cardinal’ motifs) prefer acting individually, rather than in fixed combinations, within the −150/+50 bp regions to recruit TFs that dictate cofactor signatures distinctive of specific promoter subsets. Subsequently, human promoters can be subclassified based on the presence of cardinal elements and their associated cofactor signatures. In this study, furthermore, we have focused on promoters containing the nuclear respiratory factor 1 (NRF1) motif as the cardinal cis-regulatory element and have identified the pervasive association of NRF1 with the cofactor lysine-specific demethylase 1 (LSD1/KDM1A). This signature might be distinctive of promoters regulating nuclear-encoded mitochondrial and other particular genes in at least some cells. Together, we propose that decoding a signature-based, expanded model of control at proximal promoter regions should lead to a better understanding of coordinated regulation of gene transcription. PMID:24244184

  16. Influence of the molybdenum cofactor biosynthesis on anaerobic respiration, biofilm formation and motility in Burkholderia thailandensis.

    PubMed

    Andreae, Clio A; Titball, Richard W; Butler, Clive S

    2014-01-01

    Burkholderia thailandensis is closely related to Burkholderia pseudomallei, a bacterial pathogen and the causative agent of melioidosis. B. pseudomallei can survive and persist within a hypoxic environment for up to one year and has been shown to grow anaerobically in the presence of nitrate. Currently, little is known about the role of anaerobic respiration in pathogenesis of melioidosis. Using B. thailandensis as a model, a library of 1344 transposon mutants was created to identify genes required for anaerobic nitrate respiration. One transposon mutant (CA01) was identified with an insertion in BTH_I1704 (moeA), a gene required for the molybdopterin biosynthetic pathway. This pathway is involved in the synthesis of a molybdopterin cofactor required for a variety of molybdoenzymes, including nitrate reductase. Disruption of molybdopterin biosynthesis prevented growth under anaerobic conditions, when using nitrate as the sole terminal electron acceptor. Defects in anaerobic respiration, nitrate reduction, motility and biofilm formation were observed for CA01. Mutant complementation with pDA-17:BTH_I1704 was able to restore anaerobic growth on nitrate, nitrate reductase activity and biofilm formation, but did not restore motility. This study highlights the potential importance of molybdoenzyme-dependent anaerobic respiration in the survival and virulence of B. thailandensis. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  17. Dynamic interplay between nitration and phosphorylation of tubulin cofactor B in the control of microtubule dynamics

    PubMed Central

    Rayala, Suresh K.; Martin, Emil; Sharina, Iraida G.; Molli, Poonam R.; Wang, Xiaoping; Jacobson, Raymond; Murad, Ferid; Kumar, Rakesh

    2007-01-01

    Tubulin cofactor B (TCoB) plays an important role in microtubule dynamics by facilitating the dimerization of α- and β-tubulin. Recent evidence suggests that p21-activated kinase 1 (Pak1), a major signaling nodule in eukaryotic cells, phosphorylates TCoB on Ser-65 and Ser-128 and plays an essential role in microtubule regrowth. However, to date, no upstream signaling molecules have been identified to antagonize the functions of TCoB, which might help in maintaining the equilibrium of microtubules. Here, we discovered that TCoB is efficiently nitrated, mainly on Tyr-64 and Tyr-98, and nitrated-TCoB attenuates the synthesis of new microtubules. In addition, we found that nitration of TCoB antagonizes signaling-dependent phosphorylation of TCoB, whereas optimal nitration of TCoB requires the presence of functional Pak1 phosphorylation sites, thus providing a feedback mechanism to regulate phosphorylation-dependent MT regrowth. Together these findings identified TCoB as the third cytoskeleton protein to be nitrated and suggest a previously undescribed mechanism, whereby growth factor signaling may coordinately integrate nitric oxide signaling in the regulation of microtubule dynamics. PMID:18048340

  18. Age-group Differences in Human Papillomavirus Types and Cofactors for Cervical Intraepithelial Neoplasia 3 Among Women Referred to Colposcopy

    PubMed Central

    Gargano, Julia W.; Nisenbaum, Rosane; Lee, Daisy R.; Ruffin, Mack T.; Steinau, Martin; Horowitz, Ira R.; Flowers, Lisa C.; Tadros, Talaat S.; Birdsong, George; Unger, Elizabeth R.

    2011-01-01

    Background Recommendations for high risk human papillomavirus (HR-HPV) testing as an adjunct to cytology for cervical cancer screening differ by age group, because HR-HPV tests lack adequate specificity in women aged <30. Here, we assess age-group differences in HPV types and other risk factors for cervical intraepithelial neoplasia (CIN) grade 3 or worse (CIN3+) versus CIN0–2 in women from four colposcopy clinics. Methods Women ages 18–69 (n=1658) were enrolled and completed structured interviews to elicit data on behavioral risk factors prior to their examinations. HPV genotyping was performed on exfoliated cervical cell samples. We estimated relative risks (RR) for HPV types and cofactors for CIN3+, overall and stratified by age group. Results After 2 years of follow-up, we identified 178 CIN3+, 1305 CIN0–2, and 175 indeterminate outcomes. Non-vaccine HR-HPV types were only associated with CIN3+ among women ≥30 (RR=2.3, 95% CI 1.5–3.4; <30: RR=0.9). Among all HR-HPV positive women, adjusting for age, significant cofactors for CIN3+ included current smoking (RR=1.5), former smoking (RR=1.8), regular Pap screening (RR=0.7), current regular condom use (RR=0.5), and parity ≥5 (RR=1.6, p-trend for increasing parity=.07). However, the parity association differed by age group (≥30: RR=1.8, p-trend=.008; <30: RR=0.9, p-trend=.55). Conclusions Subgroup variation by age in the risk of CIN3+ points to the importance of the timing of exposures in relation to CIN3+ detection. Impact Future screening strategies need to consider natural history and secular trends in cofactor prevalence in the pursuit of appropriately sensitive and specific screening tools applied to appropriate age groups. PMID:22028398

  19. Cofactor metals and antioxidant enzymes in cisplatin-treated rats: effect of antioxidant intervention.

    PubMed

    Sabuncuoglu, Suna; Eken, Ayse; Aydin, Ahmet; Ozgunes, Hilal; Orhan, Hilmi

    2015-10-01

    We explored the association between the activities of antioxidant enzymes and their metallic cofactors in rats treated with cisplatin. The antioxidant effects of aminoguanidine, and a combination of vitamins E and C were investigated. Plasma platin was significantly lower than liver and kidney. Cisplatin treatment caused significant increase in plasma Se-glutathione peroxidase activity. Activities of Se-glutathione peroxidase, glutathione S-transferase, catalase and Cu,Zn-superoxide dismutase have been found to be significantly decreased in liver and kidney compared to controls. Zn levels in these organs were diminished upon cisplatin treatment, while levels of Cu were unaffected. Interestingly, levels of iron, the cofactor of catalase, were found to be significantly increased in liver and kidney. Intervention with aminoguanidine or vitamins was generally prevented cisplatin-caused changes in the activity of enzymes and in the tissue levels of cofactor metals. These observations suggest that relation between activities of enzymes and levels of cofactor metals is multifactorial.

  20. The effect of ribosome assembly cofactors on in vitro 30S subunit reconstitution.

    PubMed

    Bunner, Anne E; Nord, Stefan; Wikström, P Mikael; Williamson, James R

    2010-04-23

    Ribosome biogenesis is facilitated by a growing list of assembly cofactors, including helicases, GTPases, chaperones, and other proteins, but the specific functions of many of these assembly cofactors are still unclear. The effect of three assembly cofactors on 30S ribosome assembly was determined in vitro using a previously developed mass-spectrometry-based method that monitors the rRNA binding kinetics of ribosomal proteins. The essential GTPase Era caused several late-binding proteins to bind rRNA faster when included in a 30S reconstitution. RimP enabled faster binding of S9 and S19 and inhibited the binding of S12 and S13, perhaps by blocking those proteins' binding sites. RimM caused proteins S5 and S12 to bind dramatically faster. These quantitative kinetic data provide important clues about the roles of these assembly cofactors in the mechanism of 30S biogenesis. (c) 2010 Elsevier Ltd. All rights reserved.

  1. Understanding Dermatan Sulfate-Heparin Cofactor II Interaction through Virtual Library Screening.

    PubMed

    Raghuraman, Arjun; Mosier, Philip D; Desai, Umesh R

    2010-09-09

    Dermatan sulfate, an important member of the glycosaminoglycan family, interacts with heparin cofactor II, a member of the serpin family of proteins, to modulate antithrombotic response. Yet, the nature of this interaction remains poorly understood at a molecular level. We report the genetic algorithm-based combinatorial virtual library screening study of a natural, high-affinity dermatan sulfate hexasaccharide with heparin cofactor II. Of the 192 topologies possible for the hexasaccharide, only 16 satisfied the "high-specificity" criteria used in computational study. Of these, 13 topologies were predicted to bind in the heparin-binding site of heparin cofactor II at a ∼60° angle to helix D, a novel binding mode. This new binding geometry satisfies all known solution and mutagenesis data and supports thrombin ternary complexation through a template mechanism. The study is expected to facilitate the design of allosteric agonists of heparin cofactor II as antithrombotic agents.

  2. The glmS Ribozyme Cofactor is a General Acid-Base Catalyst

    PubMed Central

    Viladoms, Julia; Fedor, Martha J.

    2012-01-01

    The glmS ribozyme is the first natural self-cleaving ribozyme known to require a cofactor. The D-glucosamine-6-phosphate (GlcN6P) cofactor has been proposed to serve as a general acid, but its role in the catalytic mechanism has not been established conclusively. We surveyed GlcN6P-like molecules for their ability to support self-cleavage of the glmS ribozyme and found a strong correlation between the pH dependence of the cleavage reaction and the intrinsic acidity of the cofactors. For cofactors with low binding affinities the contribution to rate enhancement was proportional to their intrinsic acidity. This linear free-energy relationship between cofactor efficiency and acid dissociation constants is consistent with a mechanism in which the cofactors participate directly in the reaction as general acid-base catalysts. A high value for the Brønsted coefficient (β ~ 0.7) indicates that a significant amount of proton transfer has already occurred in the transition state. The glmS ribozyme is the first self-cleaving RNA to use an exogenous acid-base catalyst. PMID:23113700

  3. A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases.

    PubMed

    Cahn, Jackson K B; Werlang, Caroline A; Baumschlager, Armin; Brinkmann-Chen, Sabine; Mayo, Stephen L; Arnold, Frances H

    2017-02-17

    The ability to control enzymatic nicotinamide cofactor utilization is critical for engineering efficient metabolic pathways. However, the complex interactions that determine cofactor-binding preference render this engineering particularly challenging. Physics-based models have been insufficiently accurate and blind directed evolution methods too inefficient to be widely adopted. Building on a comprehensive survey of previous studies and our own prior engineering successes, we present a structure-guided, semirational strategy for reversing enzymatic nicotinamide cofactor specificity. This heuristic-based approach leverages the diversity and sensitivity of catalytically productive cofactor binding geometries to limit the problem to an experimentally tractable scale. We demonstrate the efficacy of this strategy by inverting the cofactor specificity of four structurally diverse NADP-dependent enzymes: glyoxylate reductase, cinnamyl alcohol dehydrogenase, xylose reductase, and iron-containing alcohol dehydrogenase. The analytical components of this approach have been fully automated and are available in the form of an easy-to-use web tool: Cofactor Specificity Reversal-Structural Analysis and Library Design (CSR-SALAD).

  4. The glmS ribozyme cofactor is a general acid-base catalyst.

    PubMed

    Viladoms, Júlia; Fedor, Martha J

    2012-11-21

    The glmS ribozyme is the first natural self-cleaving ribozyme known to require a cofactor. The d-glucosamine-6-phosphate (GlcN6P) cofactor has been proposed to serve as a general acid, but its role in the catalytic mechanism has not been established conclusively. We surveyed GlcN6P-like molecules for their ability to support self-cleavage of the glmS ribozyme and found a strong correlation between the pH dependence of the cleavage reaction and the intrinsic acidity of the cofactors. For cofactors with low binding affinities, the contribution to rate enhancement was proportional to their intrinsic acidity. This linear free-energy relationship between cofactor efficiency and acid dissociation constants is consistent with a mechanism in which the cofactors participate directly in the reaction as general acid-base catalysts. A high value for the Brønsted coefficient (β ~ 0.7) indicates that a significant amount of proton transfer has already occurred in the transition state. The glmS ribozyme is the first self-cleaving RNA to use an exogenous acid-base catalyst.

  5. Non-DNA-binding cofactors enhance DNA-binding specificity of a transcriptional regulatory complex

    PubMed Central

    Siggers, Trevor; Duyzend, Michael H; Reddy, Jessica; Khan, Sidra; Bulyk, Martha L

    2011-01-01

    Recruitment of cofactors to specific DNA sites is integral for specificity in gene regulation. As a model system, we examined how targeting and transcriptional control of the sulfur metabolism genes in Saccharomyces cerevisiae is governed by recruitment of the transcriptional co-activator Met4. We developed genome-scale approaches to measure transcription factor (TF) DNA-binding affinities and cofactor recruitment to >1300 genomic binding site sequences. We report that genes responding to the TF Cbf1 and cofactor Met28 contain a novel ‘recruitment motif' (RYAAT), adjacent to Cbf1 binding sites, which enhances the binding of a Met4–Met28–Cbf1 regulatory complex, and that abrogation of this motif significantly reduces gene induction under low-sulfur conditions. Furthermore, we show that correct recognition of this composite motif requires both non-DNA-binding cofactors Met4 and Met28. Finally, we demonstrate that the presence of an RYAAT motif next to a Cbf1 site, rather than Cbf1 binding affinity, specifies Cbf1-dependent sulfur metabolism genes. Our results highlight the need to examine TF/cofactor complexes, as novel specificity can result from cofactors that lack intrinsic DNA-binding specificity. PMID:22146299

  6. Cofactor self-sufficient whole-cell biocatalysts for the production of 2-phenylethanol.

    PubMed

    Wang, Pengchao; Yang, Xinwei; Lin, Baixue; Huang, Jianzhong; Tao, Yong

    2017-09-22

    The efficiency of biocatalysis is often affected by an insufficient supply and regeneration of cofactors and redox equivalents. To alleviate this shortcoming, a cofactor self-sufficient system was developed for enhanced production of 2-phenylethanol (2-PE) in E. coli. A "bridge" between the amino acid and its corresponding alcohol was designed in the system using glutamate dehydrogenase. By coupling glutamate dehydrogenase with transaminase and alcohol dehydrogenase, the cosubstrate (2-oxoglutarate) and redox equivalents (NAD(P)H) were regenerated simultaneously, so that no external cofactor or redox source was required. Thus, a cofactor self-sufficient system was developed, which improved the biocatalyst efficiency 3.8-fold. The ammonium generated in this process was removed using zeolite, which further improved the biosynthetic efficiency and resulted in a cleaner system. To the best of our knowledge, this system yielded the highest titer of 2-PE ever obtained in E. coli. Additionally, the wider applicability of this self-sufficient strategy was demonstrated in the production of D-phenyllactic acid. This study thus offers a new method to resolve the cofactor/redox imbalance problem and demonstrates the feasibility of the cofactor self-sufficient strategy for enhanced production of diverse chemicals. Copyright © 2017. Published by Elsevier Inc.

  7. Effects of the cofactor binding sites on the activities of secondary alcohol dehydrogenase (SADH).

    PubMed

    Wang, Tao; Chen, Xiangjun; Han, Jun; Ma, Sichun; Wang, Jianmei; Li, Xufeng; Zhang, Hui; Liu, Zhibin; Yang, Yi

    2016-07-01

    SADHs from Thermoanaerobacter ethanolicus are enzymes that, together with various cofactors, catalyze the reversible reduction of carbonyl compounds to their corresponding alcohols. To explore how cofactors bind to SADH, TeSADH was cloned in this study, and Ser(199) and Arg(200) were replaced by Tyr and Asp, respectively. Both sites were expected to be inside or adjacent to the cofactor-binding domain according to computational a prediction. Analysis of TeSADH activities revealed that the enzymatic efficiency (kcat/Km) of the S199Y mutant was noticeably enhanced using by NADH, NADPH as cofactors, and similar with that of wild-type using by NADP(+), NAD(+). Conversely, the activity of the R200D mutant significantly decreased with all cofactors. Furthermore, in yeast, the S199Y mutant substantially elevated the ethanol concentration compared with the wild type. Molecular dynamics simulation results indicated the H-bonding network between TeSADH and the cofactors was stronger for the S199Y mutant and the binding energy was simultaneously increased. Moreover, the fluorescence results indicated the S199Y mutant exhibited an increased preference for NAD(P)H, binding with NAD(P)H more compactly compared with wild type. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Serine 1179 Phosphorylation of Endothelial Nitric Oxide Synthase Increases Superoxide Generation and Alters Cofactor Regulation.

    PubMed

    Peng, Hu; Zhuang, Yugang; Harbeck, Mark C; He, Donghong; Xie, Lishi; Chen, Weiguo

    2015-01-01

    Endothelial nitric oxide synthase (eNOS) is responsible for maintaining systemic blood pressure, vascular remodeling and angiogenesis. In addition to producing NO, eNOS can also generate superoxide (O2-.) in the absence of the cofactor tetrahydrobiopterin (BH4). Previous studies have shown that bovine eNOS serine 1179 (Serine 1177/human) phosphorylation critically modulates NO synthesis. However, the effect of serine 1179 phosphorylation on eNOS superoxide generation is unknown. Here, we used the phosphomimetic form of eNOS (S1179D) to determine the effect of S1179 phosphorylation on superoxide generating activity, and its sensitivity to regulation by BH4, Ca2+, and calmodulin (CAM). S1179D eNOS exhibited significantly increased superoxide generating activity and NADPH consumption compared to wild-type eNOS (WT eNOS). The superoxide generating activities of S1179D eNOS and WT eNOS did not differ significantly in their sensitivity to regulation by either Ca2+ or CaM. The sensitivity of the superoxide generating activity of S1179D eNOS to inhibition by BH4 was significantly reduced compared to WT eNOS. In eNOS-overexpressing 293 cells, BH4 depletion with 10mM DAHP for 48 hours followed by 50ng/ml VEGF for 30 min to phosphorylate eNOS S1179 increased ROS accumulation compared to DAHP-only treated cells. Meanwhile, MTT assay indicated that overexpression of eNOS in HEK293 cells decreased cellular viability compared to control cells at BH4 depletion condition (P<0.01). VEGF-mediated Serine 1179 phosphorylation further decreased the cellular viability in eNOS-overexpressing 293 cells (P<0.01). Our data demonstrate that eNOS serine 1179 phosphorylation, in addition to enhancing NO production, also profoundly affects superoxide generation: S1179 phosphorylation increases superoxide production while decreasing sensitivity to the inhibitory effect of BH4 on this activity.

  9. Properties of a microtubule-associated cofactor-independent protein kinase from pig brain.

    PubMed Central

    Scott, C W; Caputo, C B; Salama, A I

    1989-01-01

    A protein kinase activity was identified in pig brain that co-purified with microtubules through repeated cycles of temperature-dependent assembly and disassembly. The microtubule-associated protein kinase (MTAK) phosphorylated histone H1; this activity was not stimulated by cyclic nucleotides. Ca2+ plus calmodulin, phospholipids or polyamines. MTAK did not phosphorylate synthetic peptides which are substrates for cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase. Ca2+/calmodulin-dependent protein kinase II, protein kinase C or casein kinase II. MTAK activity was inhibited by trifluoperazine [IC50 (median inhibitory concn.) = 600 microM] in a Ca2+-independent fashion. Ca2+ alone was inhibitory [IC50 = 4 mM). MTAK was not inhibited by heparin, a potent inhibitor of casein kinase II, nor a synthetic peptide inhibitor of cyclic AMP-dependent protein kinase. MTAK demonstrated a broad pH maximum (7.5-8.5) and an apparent Km for ATP of 45 microM. Mg2+ was required for enzyme activity and could not be replaced by Mn2+. MTAK phosphorylated serine and threonine residues on histone H1. MTAK is a unique cofactor-independent protein kinase that binds to microtubule structures. Images Fig. 1. Fig. 6. Fig. 7. PMID:2557823

  10. A novel cofactor-binding mode in bacterial IMP dehydrogenases explains inhibitor selectivity

    DOE PAGES

    Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; ...

    2015-01-09

    The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5'-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD+, which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes withmore » different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD+ and XMP/NAD+. In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD+ adenosine moiety. More importantly, this new NAD+-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD+-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. As a result, these findings offer a potential strategy for further ligand optimization.« less

  11. Molybdenum enzymes, their maturation and molybdenum cofactor biosynthesis in Escherichia coli.

    PubMed

    Iobbi-Nivol, Chantal; Leimkühler, Silke

    2013-01-01

    Molybdenum cofactor (Moco) biosynthesis is an ancient, ubiquitous, and highly conserved pathway leading to the biochemical activation of molybdenum. Moco is the essential component of a group of redox enzymes, which are diverse in terms of their phylogenetic distribution and their architectures, both at the overall level and in their catalytic geometry. A wide variety of transformations are catalyzed by these enzymes at carbon, sulfur and nitrogen atoms, which include the transfer of an oxo group or two electrons to or from the substrate. More than 50 molybdoenzymes were identified in bacteria to date. In molybdoenzymes Mo is coordinated to a dithiolene group on the 6-alkyl side chain of a pterin called molybdopterin (MPT). The biosynthesis of Moco can be divided into four general steps in bacteria: 1) formation of the cyclic pyranopterin monophosphate, 2) formation of MPT, 3) insertion of molybdenum into molybdopterin to form Moco, and 4) additional modification of Moco with the attachment of GMP or CMP to the phosphate group of MPT, forming the dinucleotide variant of Moco. This review will focus on molybdoenzymes, the biosynthesis of Moco, and its incorporation into specific target proteins focusing on Escherichia coli. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. A novel cofactor-binding mode in bacterial IMP dehydrogenases explains inhibitor selectivity.

    PubMed

    Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Osipiuk, Jerzy; Gu, Minyi; Zhang, Minjia; Mandapati, Kavitha; Gollapalli, Deviprasad R; Gorla, Suresh Kumar; Hedstrom, Lizbeth; Joachimiak, Andrzej

    2015-02-27

    The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5'-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+), which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD(+) and XMP/NAD(+). In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD(+) adenosine moiety. More importantly, this new NAD(+)-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD(+)-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Lipoprotein cofactors located in the outer membrane activate bacterial cell wall polymerases.

    PubMed

    Paradis-Bleau, Catherine; Markovski, Monica; Uehara, Tsuyoshi; Lupoli, Tania J; Walker, Suzanne; Kahne, Daniel E; Bernhardt, Thomas G

    2010-12-23

    Most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by polysaccharide polymerases called penicillin-binding proteins (PBPs). Because they are the targets of penicillin and related antibiotics, the structure and biochemical functions of the PBPs have been extensively studied. Despite this, we still know surprisingly little about how these enzymes build the PG layer in vivo. Here, we identify the Escherichia coli outer-membrane lipoproteins LpoA and LpoB as essential PBP cofactors. We show that LpoA and LpoB form specific trans-envelope complexes with their cognate PBP and are critical for PBP function in vivo. We further show that LpoB promotes PG synthesis by its partner PBP in vitro and that it likely does so by stimulating glycan chain polymerization. Overall, our results indicate that PBP accessory proteins play a central role in PG biogenesis, and like the PBPs they work with, these factors are attractive targets for antibiotic development. Copyright © 2010 Elsevier Inc. All rights reserved.

  14. A Novel Cofactor-binding Mode in Bacterial IMP Dehydrogenases Explains Inhibitor Selectivity*

    PubMed Central

    Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Osipiuk, Jerzy; Gu, Minyi; Zhang, Minjia; Mandapati, Kavitha; Gollapalli, Deviprasad R.; Gorla, Suresh Kumar; Hedstrom, Lizbeth; Joachimiak, Andrzej

    2015-01-01

    The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5′-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD+, which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD+ and XMP/NAD+. In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD+ adenosine moiety. More importantly, this new NAD+-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD+-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization. PMID:25572472

  15. Novel Cofactors and TFIIA Mediate Functional Core Promoter Selectivity by the Human TAFII150-Containing TFIID Complex

    PubMed Central

    Martinez, Ernest; Ge, Hui; Tao, Yong; Yuan, Chao-Xing; Palhan, Vikas; Roeder, Robert G.

    1998-01-01

    TATA-binding protein-associated factors (TAFIIs) within TFIID control differential gene transcription through interactions with both activators and core promoter elements. In particular, TAFII150 contributes to initiator-dependent transcription through an unknown mechanism. Here, we address whether TAFIIs within TFIID are sufficient, in conjunction with highly purified general transcription factors (GTFs), for differential core promoter-dependent transcription by RNA polymerase II and whether additional cofactors are required. We identify the human homologue of Drosophila TAFII150 through cognate cDNA cloning and show that it is a tightly associated component of human TFIID. More importantly, we demonstrate that the human TAFII150-containing TFIID complex is not sufficient, in the context of all purified GTFs and RNA polymerase II, to mediate transcription synergism between TATA and initiator elements and initiator-directed transcription from a TAFII-dependent TATA-less promoter. Therefore, TAFII-promoter interactions are not sufficient for the productive core promoter-selective functions of TFIID. Consistent with this finding, we have partially purified novel cofactor activities (TICs) that potentiate the TAFII-mediated synergism between TATA and initiator elements (TIC-1) and TAFII-dependent transcription from TATA-less promoters (TIC-2 and -3). Furthermore, we demonstrate an essential function for TFIIA in TIC- and TAFII-dependent basal transcription from a TATA-less promoter. Our results reveal a parallel between the basal transcription activity of TAFIIs through core promoter elements and TAFII-dependent activator function. PMID:9774672

  16. Engineering redox cofactor utilization for detoxification of glycolaldehyde, a key inhibitor of bioethanol production, in yeast Saccharomyces cerevisiae.

    PubMed

    Jayakody, Lahiru N; Horie, Kenta; Hayashi, Nobuyuki; Kitagaki, Hiroshi

    2013-07-01

    Hot-compressed water treatment of lignocellulose liberates numerous inhibitors that prevent ethanol fermentation of yeast Saccharomyces cerevisiae. Glycolaldehyde is one of the strongest fermentation inhibitors and we developed a tolerant strain by overexpressing ADH1 encoding an NADH-dependent reductase; however, its recovery was partial. In this study, to overcome this technical barrier, redox cofactor preference of glycolaldehyde detoxification was investigated. Glycolaldehyde-reducing activity of the ADH1-overexpressing strain was NADH-dependent but not NADPH-dependent. Moreover, genes encoding components of the pentose phosphate pathway, which generates intracellular NADPH, was upregulated in response to high concentrations of glycolaldehyde. Mutants defective in pentose phosphate pathways were sensitive to glycolaldehyde. Genome-wide survey identified GRE2 encoding a NADPH-dependent reductase as the gene that confers tolerance to glycolaldehyde. Overexpression of GRE2 in addition to ADH1 further improved the tolerance to glycolaldehyde. NADPH-dependent glycolaldehyde conversion to ethylene glycol and NADP+ content of the strain overexpressing both ADH1 and GRE2 were increased at 5 mM glycolaldehyde. Expression of GRE2 was increased in response to glycolaldehyde. Carbon metabolism of the strain was rerouted from glycerol to ethanol. Thus, it was concluded that the overexpression of GRE2 together with ADH1 restores glycolaldehyde tolerance by augmenting the NADPH-dependent reduction pathway in addition to NADH-dependent reduction pathway. The redox cofactor control for detoxification of glycolaldehyde proposed in this study could influence strategies for improving the tolerance of other fermentation inhibitors.

  17. Novel cofactors and TFIIA mediate functional core promoter selectivity by the human TAFII150-containing TFIID complex.

    PubMed

    Martinez, E; Ge, H; Tao, Y; Yuan, C X; Palhan, V; Roeder, R G

    1998-11-01

    TATA-binding protein-associated factors (TAFIIs) within TFIID control differential gene transcription through interactions with both activators and core promoter elements. In particular, TAFII150 contributes to initiator-dependent transcription through an unknown mechanism. Here, we address whether TAFIIs within TFIID are sufficient, in conjunction with highly purified general transcription factors (GTFs), for differential core promoter-dependent transcription by RNA polymerase II and whether additional cofactors are required. We identify the human homologue of Drosophila TAFII150 through cognate cDNA cloning and show that it is a tightly associated component of human TFIID. More importantly, we demonstrate that the human TAFII150-containing TFIID complex is not sufficient, in the context of all purified GTFs and RNA polymerase II, to mediate transcription synergism between TATA and initiator elements and initiator-directed transcription from a TAFII-dependent TATA-less promoter. Therefore, TAFII-promoter interactions are not sufficient for the productive core promoter-selective functions of TFIID. Consistent with this finding, we have partially purified novel cofactor activities (TICs) that potentiate the TAFII-mediated synergism between TATA and initiator elements (TIC-1) and TAFII-dependent transcription from TATA-less promoters (TIC-2 and -3). Furthermore, we demonstrate an essential function for TFIIA in TIC- and TAFII-dependent basal transcription from a TATA-less promoter. Our results reveal a parallel between the basal transcription activity of TAFIIs through core promoter elements and TAFII-dependent activator function.

  18. The human papillomavirus type 11 and 16 E6 proteins modulate the cell-cycle regulator and transcription cofactor TRIP-Br1.

    PubMed

    Gupta, Sanjay; Takhar, Param Parkash S; Degenkolbe, Roland; Koh, Choon Heng; Zimmermann, Holger; Yang, Christopher Maolin; Guan Sim, Khe; Hsu, Stephen I-Hong; Bernard, Hans-Ulrich

    2003-12-05

    The genital human papillomaviruses (HPVs) are a taxonomic group including HPV types that preferentially cause genital and laryngeal warts ("low-risk types"), such as HPV-6 and HPV-11, or cancer of the cervix and its precursor lesions ("high-risk types"), such as HPV-16. The transforming processes induced by these viruses depend on the proteins E5, E6, and E7. Among these oncoproteins, the E6 protein stands out because it supports a particularly large number of functions and interactions with cellular proteins, some of which are specific for the carcinogenic HPVs, while others are shared among low- and high-risk HPVs. Here we report yeast two-hybrid screens with HPV-6 and -11 E6 proteins that identified TRIP-Br1 as a novel cellular target. TRIP-Br1 was recently detected by two research groups, which described two separate functions, namely that of a transcriptional integrator of the E2F1/DP1/RB cell-cycle regulatory pathway (and then named TRIP-Br1), and that of an antagonist of the cyclin-dependent kinase suppression of p16INK4a (and then named p34SEI-1). We observed that TRIP-Br1 interacts with low- and high-risk HPV E6 proteins in yeast, in vitro and in mammalian cell cultures. Transcription activation of a complex consisting of E2F1, DP1, and TRIP-Br1 was efficiently stimulated by both E6 proteins. TRIP-Br1 has an LLG E6 interaction motif, which contributed to the binding of E6 proteins. Apparently, E6 does not promote degradation of TRIP-Br1. Our observations imply that the cell-cycle promoting transcription factor E2F1/DP1 is dually targeted by HPV oncoproteins, namely (i) by interference of the E7 protein with repression by RB, and (ii) by the transcriptional cofactor function of the E6 protein. Our data reveal the natural context of the transcription activator function of E6, which has been predicted without knowledge of the E2F1/DP1/TRIP-Br/E6 complex by studying chimeric constructs, and add a function to the limited number of transforming properties shared

  19. Investigation into the nature of substrate binding to the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase

    SciTech Connect

    Warren, M.J.; Jordan, P.M.

    1988-12-13

    The formation of the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase was shown to depend on the presence of 5-aminolevulinic acid. A hemA/sup -/ mutant formed inactive deaminase when grown in the absence of 5-aminolevulinic acid since this strain was unable to biosynthesize the dipyrromethane cofactor. The mutant formed normal levels of deaminase, however, when grown in the presence of 5-aminolevulinic acid. Porphobilinogen, the substrate, interacts with the free ..cap alpha..-position of the dipyrromethane cofactor to give stable enzyme-intermediate complexes. Experiments with regiospecifically labeled intermediate complexes have shown that, in the absence of further substrate molecules, the complexes are interconvertible by the exchange of the terminal pyrrole ring of each complex. The formation of enzyme-intermediate complexes is accompanied by the exposure of a cysteine residue, suggesting that substantial conformational changes occur on binding substrate. Specific labeling of the dipyrromethane cofactor by growth of the E. coli in the presence of 5-amino(5-/sup 14/C)levulinic acid has confirmed that the cofactor is not subject to catalytic turnover. Experiments with the ..cap alpha..-substituted substrate analogue ..cap alpha..-bromoporphobilinogen have provided further evidence that the cofactor is responsible for the covalent binding of the substrate at the catalytic site. On the basis of these cummulative findings, it has been possible to construct a mechanistic scheme for the deaminase reaction involving a single catalytic site which is able to catalyze the addition or removal of either NH/sub 3/ or H/sub 2/O. The role of the cofactor both as a primer and as a means for regulating the number of substrates bound in each catalytic cycle is discussed.

  20. Genome-scale consequences of cofactor balancing in engineered pentose utilization pathways in Saccharomyces cerevisiae.

    PubMed

    Ghosh, Amit; Zhao, Huimin; Price, Nathan D

    2011-01-01

    Biofuels derived from lignocellulosic biomass offer promising alternative renewable energy sources for transportation fuels. Significant effort has been made to engineer Saccharomyces cerevisiae to efficiently ferment pentose sugars such as D-xylose and L-arabinose into biofuels such as ethanol through heterologous expression of the fungal D-xylose and L-arabinose pathways. However, one of the major bottlenecks in these fungal pathways is that the cofactors are not balanced, which contributes to inefficient utilization of pentose sugars. We utilized a genome-scale model of S. cerevisiae to predict the maximal achievable growth rate for cofactor balanced and imbalanced D-xylose and L-arabinose utilization pathways. Dynamic flux balance analysis (DFBA) was used to simulate batch fermentation of glucose, D-xylose, and L-arabinose. The dynamic models and experimental results are in good agreement for the wild type and for the engineered D-xylose utilization pathway. Cofactor balancing the engineered D-xylose and L-arabinose utilization pathways simulated an increase in ethanol batch production of 24.7% while simultaneously reducing the predicted substrate utilization time by 70%. Furthermore, the effects of cofactor balancing the engineered pentose utilization pathways were evaluated throughout the genome-scale metabolic network. This work not only provides new insights to the global network effects of cofactor balancing but also provides useful guidelines for engineering a recombinant yeast strain with cofactor balanced engineered pathways that efficiently co-utilizes pentose and hexose sugars for biofuels production. Experimental switching of cofactor usage in enzymes has been demonstrated, but is a time-consuming effort. Therefore, systems biology models that can predict the likely outcome of such strain engineering efforts are highly useful for motivating which efforts are likely to be worth the significant time investment.

  1. Genome-Scale Consequences of Cofactor Balancing in Engineered Pentose Utilization Pathways in Saccharomyces cerevisiae

    PubMed Central

    Ghosh, Amit; Zhao, Huimin; Price, Nathan D.

    2011-01-01

    Biofuels derived from lignocellulosic biomass offer promising alternative renewable energy sources for transportation fuels. Significant effort has been made to engineer Saccharomyces cerevisiae to efficiently ferment pentose sugars such as D-xylose and L-arabinose into biofuels such as ethanol through heterologous expression of the fungal D-xylose and L-arabinose pathways. However, one of the major bottlenecks in these fungal pathways is that the cofactors are not balanced, which contributes to inefficient utilization of pentose sugars. We utilized a genome-scale model of S. cerevisiae to predict the maximal achievable growth rate for cofactor balanced and imbalanced D-xylose and L-arabinose utilization pathways. Dynamic flux balance analysis (DFBA) was used to simulate batch fermentation of glucose, D-xylose, and L-arabinose. The dynamic models and experimental results are in good agreement for the wild type and for the engineered D-xylose utilization pathway. Cofactor balancing the engineered D-xylose and L-arabinose utilization pathways simulated an increase in ethanol batch production of 24.7% while simultaneously reducing the predicted substrate utilization time by 70%. Furthermore, the effects of cofactor balancing the engineered pentose utilization pathways were evaluated throughout the genome-scale metabolic network. This work not only provides new insights to the global network effects of cofactor balancing but also provides useful guidelines for engineering a recombinant yeast strain with cofactor balanced engineered pathways that efficiently co-utilizes pentose and hexose sugars for biofuels production. Experimental switching of cofactor usage in enzymes has been demonstrated, but is a time-consuming effort. Therefore, systems biology models that can predict the likely outcome of such strain engineering efforts are highly useful for motivating which efforts are likely to be worth the significant time investment. PMID:22076150

  2. Basic residues within the ebolavirus VP35 protein are required for its viral polymerase cofactor function.

    PubMed

    Prins, Kathleen C; Binning, Jennifer M; Shabman, Reed S; Leung, Daisy W; Amarasinghe, Gaya K; Basler, Christopher F

    2010-10-01

    The ebolavirus (EBOV) VP35 protein binds to double-stranded RNA (dsRNA), inhibits host alpha/beta interferon (IFN-α/β) production, and is an essential component of the viral polymerase complex. Structural studies of the VP35 C-terminal IFN inhibitory domain (IID) identified specific structural features, including a central basic patch and a hydrophobic pocket, that are important for dsRNA binding and IFN inhibition. Several other conserved basic residues bordering the central basic patch and a separate cluster of basic residues, called the first basic patch, were also identified. Functional analysis of alanine substitution mutants indicates that basic residues outside the central basic patch are not required for dsRNA binding or for IFN inhibition. However, minigenome assays, which assess viral RNA polymerase complex function, identified these other basic residues to be critical for viral RNA synthesis. Of these, a subset located within the first basic patch is important for VP35-nucleoprotein (NP) interaction, as evidenced by the inability of alanine substitution mutants to coimmunoprecipitate with NP. Therefore, first basic patch residues are likely critical for replication complex formation through interactions with NP. Coimmunoprecipitation studies further demonstrate that the VP35 IID is sufficient to interact with NP and that dsRNA can modulate VP35 IID interactions with NP. Other basic residue mutations that disrupt the VP35 polymerase cofactor function do not affect interaction with NP or with the amino terminus of the viral polymerase. Collectively, these results highlight the importance of conserved basic residues from the EBOV VP35 C-terminal IID and validate the VP35 IID as a potential therapeutic target.

  3. Dsc E3 ligase localization to the Golgi requires the ATPase Cdc48 and cofactor Ufd1 for activation of sterol regulatory element-binding protein in fission yeast.

    PubMed

    Burr, Risa; Ribbens, Diedre; Raychaudhuri, Sumana; Stewart, Emerson V; Ho, Jason; Espenshade, Peter J

    2017-09-29

    Sterol regulatory element-binding proteins (SREBPs) in the fission yeast Schizosaccharomyces pombe regulate lipid homeostasis and the hypoxic response under conditions of low sterol or oxygen availability. SREBPs are cleaved in the Golgi through the combined action of the Dsc E3 ligase complex, the rhomboid protease Rbd2, and the essential ATPases associated with diverse cellular activities (AAA(+)) ATPase Cdc48. The soluble SREBP N-terminal transcription factor domain is then released into the cytosol to enter the nucleus and regulate gene expression. Previously, we reported that Cdc48 binding to Rbd2 is required for Rbd2-mediated SREBP cleavage. Here, using affinity chromatography and mass spectrometry experiments, we identified Cdc48-binding proteins in S. pombe, generating a list of many previously unknown potential Cdc48-binding partners. We show that the established Cdc48 cofactor Ufd1 is required for SREBP cleavage but does not interact with the Cdc48-Rbd2 complex. Cdc48-Ufd1 is instead required at a step prior to Rbd2 function, during Golgi localization of the Dsc E3 ligase complex. Together, these findings demonstrate that two distinct Cdc48 complexes, Cdc48-Ufd1 and Cdc48-Rbd2, are required for SREBP activation and low-oxygen adaptation in S. pombe. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Daily magnesium fluxes regulate cellular timekeeping and energy balance

    PubMed Central

    Feeney, Kevin A.; Hansen, Louise L.; Putker, Marrit; Olivares-Yañez, Consuelo; Day, Jason; Eades, Lorna J.; Larrondo, Luis F.; Hoyle, Nathaniel P.; O'Neill, John S.; van Ooijen, Gerben

    2016-01-01

    Circadian clocks are fundamental to the biology of most eukaryotes, coordinating behavior and physiology to resonate with the environmental cycle of day and night through complex networks of clock-controlled genes1–3. A fundamental knowledge gap exists however, between circadian gene expression cycles and the biochemical mechanisms that ultimately facilitate circadian regulation of cell biology4,5. Here we report circadian rhythms in the intracellular concentration of magnesium ions, [Mg2+]i, which act as a cell-autonomous timekeeping component to determine key clock properties in both a human cell line and a unicellular alga that diverged from metazoans more than 1 billion years ago6. Given the essential role of Mg2+ as a cofactor for ATP, a functional consequence of [Mg2+]i oscillations is dynamic regulation of cellular energy expenditure over the daily cycle. Mechanistically, we find that these rhythms provide bilateral feedback linking rhythmic metabolism to clock-controlled gene expression. The global regulation of nucleotide triphosphate turnover by intracellular Mg2+ availability has potential to impact upon many of the cell’s >600 MgATP-dependent enzymes7 and every cellular system where MgNTP hydrolysis becomes rate limiting. Indeed, we find that circadian control of translation by mTOR8 is regulated through [Mg2+]i oscillations. It will now be important to identify which additional biological processes are subject to this form of regulation in tissues of multicellular organisms such as plants and humans, in the context of health and disease. PMID:27074515

  5. A new cofactor in prokaryotic enzyme: Tryptophan tryptophylquinone as the redox prosthetic group in methylamine dehydrogenase

    SciTech Connect

    McIntire, W.S. Univ. of California, San Francisco ); Wemmer, D.E. ); Chistoserdov, A.; Lidstrom, M.E. )

    1991-05-10

    Methylamine dehydrogenase (MADH), an {alpha}{sub 2}{beta}{sub 2} enzyme from numerous methylotrophic soil bacteria, contains a novel quinonoid redox prosthetic group that is covalently bound to its small {beta} subunit through two amino acyl residues. A comparison of the amino acid sequence deduced from the gene sequence of the small subunit for the enzyme from Methylobacterium extorquens AM1 with the published amino acid sequence obtained by Edman degradation method, allowed the identification of the amino acyl constituents of the cofactor as two tryptophyl residues. This information was crucial for interpreting {sup 1}H and {sup 13}C nuclear magnetic resonance, and mass spectral data collected for the semicarbazide- and carboxymethyl-derivatized bis(tripeptidyl)-cofactor of MADH from bacterium W3A1. The cofactor is composed of two cross-linked tryptophyl residues. Although there are many possible isomers, only one is consistent with all the data: The first tryptophyl residue in the peptide sequence exists as an indole-6,7-dione, and is attached at its 4 position to the 2 position of the second, otherwise unmodified, indole side group. Contrary to earlier reports, the cofactor of MADH is not 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), a derivative thereof, of pro-PQQ. This appears to be the only example of two cross-linked, modified amino acyl residues having a functional role in the active site of an enzyme, in the absence of other cofactors or metal ions.

  6. Cofactor-specific photochemical function resolved by ultrafast spectroscopy in photosynthetic reaction center crystals.

    PubMed

    Huang, Libai; Ponomarenko, Nina; Wiederrecht, Gary P; Tiede, David M

    2012-03-27

    High-resolution mapping of cofactor-specific photochemistry in photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides was achieved by polarization selective ultrafast spectroscopy in single crystals at cryogenic temperature. By exploiting the fixed orientation of cofactors within crystals, we isolated a single transition within the multicofactor manifold, and elucidated the site-specific photochemical functions of the cofactors associated with the symmetry-related active A and inactive B branches. Transient spectra associated with the initial excited states were found to involve a set of cofactors that differ depending upon whether the monomeric bacteriochlorophylls, BChl(A), BChl(B), or the special pair bacteriochlorophyll dimer, P, was chosen for excitation. Proceeding from these initial excited states, characteristic photochemical functions were resolved. Specifically, our measurements provide direct evidence for an alternative charge separation pathway initiated by excitation of BChl(A) that does not involve P*. Conversely, the initial excited state produced by excitation of BChl(B) was found to decay by energy transfer to P. A clear sequential kinetic resolution of BChl(A) and the A-side bacteriopheophytin, BPh(A), in the electron transfer proceeding from P* was achieved. These experiments demonstrate the opportunity to resolve photochemical function of individual cofactors within the multicofactor RC complexes using single crystal spectroscopy.

  7. Cofactor dependence and isotype distribution of anticardiolipin antibodies in viral infections

    PubMed Central

    Guglielmone, H; Vitozzi, S; Elbarcha, O; Fernandez, E

    2001-01-01

    BACKGROUND—Antibodies to cardiolipin (aCLs) are often detected in patients with autoimmune disorders or infectious diseases.
OBJECTIVE—To investigate the distribution of aCL isotypes and requirement of protein cofactor in viral infections in order to establish the importance, if any, of these antibodies in these infectious diseases.
PATIENTS AND METHODS—The isotype distribution of aCLs in the sera from 160 patients with infection caused by HIV-1 (n=40), hepatitis A virus (n=40), hepatitis B virus (n=40), or hepatitis C virus (n=40) was studied by standardised enzyme linked immunosorbent assay (ELISA) in the presence and absence of protein cofactor (mainly β2-glycoprotein I). Serum samples from healthy volunteers and patients with syphilis and antiphospholipid syndrome were also included and served as negative and positive control groups respectively.
RESULTS—The prevalence of one or more aCL isotypes in serum of patients with HIV-1, hepatitis A virus, hepatitis B virus, or hepatitis C virus infection was 47%, 92%, 42%, and 17% respectively (principally IgM and/or IgA). Most of these antibodies were mainly cofactor independent.
CONCLUSIONS—The presence of aCLs in viral infections is principally cofactor independent, suggesting that cofactor dependence of the aCLs should be assessed to distinguish subjects most likely to suffer from clinical symptoms observed in the presence of these antibodies.

 PMID:11302873

  8. Cellular multitasking: the dual role of human Cu-ATPases in cofactor delivery and intracellular copper balance.

    PubMed

    Lutsenko, Svetlana; Gupta, Arnab; Burkhead, Jason L; Zuzel, Vesna

    2008-08-01

    The human copper-transporting ATPases (Cu-ATPases) are essential for dietary copper uptake, normal development and function of the CNS, and regulation of copper homeostasis in the body. In a cell, Cu-ATPases maintain the intracellular concentration of copper by transporting copper into intracellular exocytic vesicles. In addition, these P-type ATPases mediate delivery of copper to copper-dependent enzymes in the secretory pathway and in specialized cell compartments such as secretory granules or melanosomes. The multiple functions of human Cu-ATPase necessitate complex regulation of these transporters that is mediated through the presence of regulatory domains in their structure, posttranslational modification and intracellular trafficking, as well as interactions with the copper chaperone Atox1 and other regulatory molecules. In this review, we summarize the current information on the function and regulatory mechanisms acting on human Cu-ATPases ATP7A and ATP7B. Brief comparison with the Cu-ATPase orthologs from other species is included.

  9. Identification of a novel cellular target and a co-factor for norovirus infection – B cells & commensal bacteria

    PubMed Central

    Karst, Stephanie M

    2015-01-01

    Human noroviruses are a leading cause of gastroenteritis worldwide but research on these important enteric pathogens has long been restricted by their uncultivability. Extensive efforts to infect intestinal epithelial cells with murine and human noroviruses in vitro have been thus far unsuccessful while murine noroviruses efficiently and lytically infect innate immune cells including macrophages and dendritic cells. We have recently discovered that murine and human noroviruses infect B cells in vitro. The nature of B cell infection was distinct from innate immune cell infection in that mature B cells were infected noncytopathically in contrast to the lytic infection of macrophages and dendritic cells. Human norovirus infection of B cells was facilitated by commensal bacteria expressing an appropriate histo-blood group antigen. Importantly, we used the mouse model of norovirus infection to confirm that Peyer's patch B cells are infected, and that commensal bacteria stimulate infection, in vivo. PMID:25997033

  10. Identification of a novel cellular target and a co-factor for norovirus infection - B cells & commensal bacteria.

    PubMed

    Karst, Stephanie M

    2015-07-04

    Human noroviruses are a leading cause of gastroenteritis worldwide but research on these important enteric pathogens has long been restricted by their uncultivability. Extensive efforts to infect intestinal epithelial cells with murine and human noroviruses in vitro have been thus far unsuccessful while murine noroviruses efficiently and lytically infect innate immune cells including macrophages and dendritic cells. We have recently discovered that murine and human noroviruses infect B cells in vitro. The nature of B cell infection was distinct from innate immune cell infection in that mature B cells were infected noncytopathically in contrast to the lytic infection of macrophages and dendritic cells. Human norovirus infection of B cells was facilitated by commensal bacteria expressing an appropriate histo-blood group antigen. Importantly, we used the mouse model of norovirus infection to confirm that Peyer's patch B cells are infected, and that commensal bacteria stimulate infection, in vivo.

  11. Cytosolic iron chaperones: Proteins delivering iron cofactors in the cytosol of mammalian cells.

    PubMed

    Philpott, Caroline C; Ryu, Moon-Suhn; Frey, Avery; Patel, Sarju

    2017-08-04

    Eukaryotic cells contain hundreds of metalloproteins that are supported by intracellular systems coordinating the uptake and distribution of metal cofactors. Iron cofactors include heme, iron-sulfur clusters, and simple iron ions. Poly(rC)-binding proteins are multifunctional adaptors that serve as iron ion chaperones in the cytosolic/nuclear compartment, binding iron at import and delivering it to enzymes, for storage (ferritin) and export (ferroportin). Ferritin iron is mobilized by autophagy through the cargo receptor, nuclear co-activator 4. The monothiol glutaredoxin Glrx3 and BolA2 function as a [2Fe-2S] chaperone complex. These proteins form a core system of cytosolic iron cofactor chaperones in mammalian cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Catalytic reduction of CN−, CO and CO2 by nitrogenase cofactors in lanthanide-driven reactions**

    PubMed Central

    Lee, Chi Chung

    2014-01-01

    Nitrogenase cofactors can be extracted into an organic solvent and added in an adenosine triphosphate (ATP)-free, organic solvent-based reaction medium to catalyze the reduction of cyanide (CN−), carbon monoxide (CO) and carbon dioxide (CO2) when samarium (II) iodide (SmI2) and 2,6-lutidinium triflate (Lut-H) are supplied as a reductant and a proton source, respectively. Driven by SmI2, the cofactors not only catalytically reduce CN− or CO to C1-C4 hydrocarbons, but also catalytically reduce CO2 to CO and C1-C3 hydrocarbons. The observation of C-C coupling from CO2 reveals a unique, Fischer-Tropsch-like reaction with an atypical carbonaceous substrate; whereas the achievement of catalytic turnover of CN−, CO and CO2 by isolated cofactors suggests the possibility to develop nitrogenase-based electrocatalysts for hydrocarbon production from these carbon-containing compounds. PMID:25420957

  13. Cofactor regeneration in phototrophic cyanobacteria applied for asymmetric reduction of ketones.

    PubMed

    Havel, Jan; Weuster-Botz, Dirk

    2007-07-01

    The obligate photoautotrophic cyanobacterium Synechococcus PCC7942 and the photoheterotrophic heterocystous cyanobacterium Noctoc muscorum are able to reduce prochiral ketones asymmetrically to optical pure chiral alcohols without light. An example is the synthesis of S-pentafluoro(phenyl-)ethanol with an enantiomeric excess >99% if 2'-3'-4'-5'-6'-pentafluoroacetophenone is used as substrate. If no light is available for regeneration of the cofactor nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH), glucose is used as cosubstrate. Membrane disintegration during asymmetric reduction promotes cytosolic energy generating metabolic pathways. Observed regulatory effects depicted by an adenosine triphosphate (ATP) to nicotinamide adenine dinucleotide phosphate (oxidized form) (NADP(+)) ratio of 3:1 for efficient cofactor recycling indicate a metabolization via glycolisis. The stoichiometric formation of the by-product acetate (1 mol acetate/1 mol chiral alcohol) indicates homoacetic acid fermentation for cofactor regeneration including the obligate photoautotrophic cyanobacterium Synechococcus PCC7942.

  14. Anthocyanin copigmentation and color of wine: The effect of naturally obtained hydroxycinnamic acids as cofactors.

    PubMed

    Bimpilas, Andreas; Panagopoulou, Marilena; Tsimogiannis, Dimitrios; Oreopoulou, Vassiliki

    2016-04-15

    Copigmentation of anthocyanins accounts for over 30% of fresh red wine color, while during storage, the color of polymeric pigments formed between anthocyanins and proanthocyanidins predominates. Rosmarinic acid and natural extracts rich in hydroxycinnamic acids, obtained from aromatic plants (Origanum vulgare and Satureja thymbra), were examined as cofactors to fresh Merlot wine and the effect on anthocyanin copigmentation and wine color was studied during storage for 6months. An increase of the copigmented anthocyanins that enhanced color intensity by 15-50% was observed, confirming the ability of complex hydroxycinnamates to form copigments. The samples with added cofactors retained higher percentages of copigmented anthocyanins and higher color intensity, compared to the control wine, up to 3 months. However, the change in the equilibrium between monomeric and copigmented anthocyanins that was induced by added cofactors, did not affect the rate of polymerization reactions during storage. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Chemomimetic biocatalysis: exploiting the synthetic potential of cofactor-dependent enzymes to create new catalysts.

    PubMed

    Prier, Christopher K; Arnold, Frances H

    2015-11-11

    Despite the astonishing breadth of enzymes in nature, no enzymes are known for many of the valuable catalytic transformations discovered by chemists. Recent work in enzyme design and evolution, however, gives us good reason to think that this will change. We describe a chemomimetic biocatalysis approach that draws from small-molecule catalysis and synthetic chemistry, enzymology, and molecular evolution to discover or create enzymes with non-natural reactivities. We illustrate how cofactor-dependent enzymes can be exploited to promote reactions first established with related chemical catalysts. The cofactors can be biological, or they can be non-biological to further expand catalytic possibilities. The ability of enzymes to amplify and precisely control the reactivity of their cofactors together with the ability to optimize non-natural reactivity by directed evolution promises to yield exceptional catalysts for challenging transformations that have no biological counterparts.

  16. The Fe-V Cofactor of Vanadium Nitrogenase Contains an Interstitial Carbon Atom.

    PubMed

    Rees, Julian A; Bjornsson, Ragnar; Schlesier, Julia; Sippel, Daniel; Einsle, Oliver; DeBeer, Serena

    2015-11-02

    The first direct evidence is provided for the presence of an interstitial carbide in the Fe-V cofactor of Azotobacter vinelandii vanadium nitrogenase. As for our identification of the central carbide in the Fe-Mo cofactor, we employed Fe Kβ valence-to-core X-ray emission spectroscopy and density functional theory calculations, and herein report the highly similar spectra of both variants of the cofactor-containing protein. The identification of an analogous carbide, and thus an atomically homologous active site in vanadium nitrogenase, highlights the importance and influence of both the interstitial carbide and the identity of the heteroatom on the electronic structure and catalytic activity of the enzyme.

  17. The Fe–V Cofactor of Vanadium Nitrogenase Contains an Interstitial Carbon Atom

    PubMed Central

    Rees, Julian A; Bjornsson, Ragnar; Schlesier, Julia; Sippel, Daniel; Einsle, Oliver; DeBeer, Serena

    2015-01-01

    The first direct evidence is provided for the presence of an interstitial carbide in the Fe–V cofactor of Azotobacter vinelandii vanadium nitrogenase. As for our identification of the central carbide in the Fe–Mo cofactor, we employed Fe Kβ valence-to-core X-ray emission spectroscopy and density functional theory calculations, and herein report the highly similar spectra of both variants of the cofactor-containing protein. The identification of an analogous carbide, and thus an atomically homologous active site in vanadium nitrogenase, highlights the importance and influence of both the interstitial carbide and the identity of the heteroatom on the electronic structure and catalytic activity of the enzyme. PMID:26376620

  18. Formulating Fluorogenic Assay to Evaluate S-adenosyl-L-methionine Analogues as Protein Methyltransferase Cofactors

    PubMed Central

    Wang, Rui; Ibáñez, Glorymar; Islam, Kabirul; Zheng, Weihong; Blum, Gil; Sengelaub, Caitlin; Luo, Minkui

    2013-01-01

    Protein methyltransferases (PMTs) catalyze arginine and lysine methylation of diverse histone and nonhistone targets. These posttranslational modifications play essential roles in regulating multiple cellular events in an epigenetic manner. In the recent process of defining PMT targets, S-adenosyl-L-methionine (SAM) analogues have emerged as powerful small molecule probes to label and profile PMT targets. To examine efficiently the reactivity of PMTs and their variants on SAM analogues, we transformed a fluorogenic PMT assay into a ready high throughput screening (HTS) format. The reformulated fluorogenic assay is featured by its uncoupled but more robust character with the first step of accumulation of the commonly-shared reaction byproduct S-adenosyl-L-homocysteine (SAH), followed by SAH-hydrolyase-mediated fluorogenic quantification. The HTS readiness and robustness of the assay were demonstrated by its excellent Z′ values of 0.83–0.95 for the so-far-examined 8 human PMTs with SAM as a cofactor (PRMT1, PRMT3, CARM1, SUV39H2, SET7/9, SET8, G9a and GLP1). The fluorogenic assay was further implemented to screen the PMTs against five SAM analogues (allyl-SAM, propargyl-SAM, (E)-pent-2-en-4-ynyl-SAM (EnYn-SAM), (E)-hex-2-en-5-ynyl-SAM (Hey-SAM) and 4-propargyloxy-but-2-enyl-SAM (Pob-SAM)). Among the examined 8×5 pairs of PMTs and SAM analogues, native SUV39H2, G9a and GLP1 showed promiscuous activity on allyl-SAM. In contrast, the bulky SAM analogues, such as EnYn-SAM, Hey-SAM and Pob-SAM are inert toward the panel of human PMTs. These findings therefore provide the useful structure-activity guidance to further evolve PMTs and SAM analogues for substrate labeling. The current assay format is ready to screen methyltransferase variants on structurally-diverse SAM analogues. PMID:21866297

  19. ModE-dependent molybdate regulation of the molybdenum cofactor operon moa in Escherichia coli.

    PubMed

    Anderson, L A; McNairn, E; Lubke, T; Pau, R N; Boxer, D H; Leubke, T

    2000-12-01

    The expression of the moa locus, which encodes enzymes required for molybdopterin biosynthesis, is enhanced under anaerobiosis but repressed when the bacterium is able to synthesize active molybdenum cofactor. In addition, moa expression exhibits a strong requirement for molybdate. The molybdate enhancement of moa transcription is fully dependent upon the molybdate-binding protein, ModE, which also mediates molybdate repression of the mod operon encoding the high-affinity molybdate uptake system. Due to the repression of moa in molybdenum cofactor-sufficient strains, the positive molybdate regulation of moa is revealed only in strains unable to make the active cofactor. Transcription of moa is controlled at two sigma-70-type promoters immediately upstream of the moaA gene. Deletion mutations covering the region upstream of moaA have allowed each of the promoters to be studied in isolation. The distal promoter is the site of the anaerobic enhancement which is Fnr-dependent. The molybdate induction of moa is exerted at the proximal promoter. Molybdate-ModE binds adjacent to the -35 region of this promoter, acting as a direct positive regulator of moa. The molybdenum cofactor repression also appears to act at the proximal transcriptional start site, but the mechanism remains to be established. Tungstate in the growth medium affects moa expression in two ways. Firstly, it can act as a functional molybdate analogue for the ModE-mediated regulation. Secondly, tungstate brings about the loss of the molybdenum cofactor repression of moa. It is proposed that the tungsten derivative of the molybdenum cofactor, which is known to be formed under such conditions, is ineffective in bringing about repression of moa. The complex control of moa is discussed in relation to the synthesis of molybdoenzymes in the bacterium.

  20. Identification of a Bis-molybdopterin Intermediate in Molybdenum Cofactor Biosynthesis in Escherichia coli*

    PubMed Central

    Reschke, Stefan; Sigfridsson, Kajsa G. V.; Kaufmann, Paul; Leidel, Nils; Horn, Sebastian; Gast, Klaus; Schulzke, Carola; Haumann, Michael; Leimkühler, Silke

    2013-01-01

    The molybdenum cofactor is an important cofactor, and its biosynthesis is essential for many organisms, including humans. Its basic form comprises a single molybdopterin (MPT) unit, which binds a molybdenum ion bearing three oxygen ligands via a dithiolene function, thus forming Mo-MPT. In bacteria, this form is modified to form the bis-MPT guanine dinucleotide cofactor with two MPT units coordinated at one molybdenum atom, which additionally contains GMPs bound to the terminal phosphate group of the MPTs (bis-MGD). The MobA protein catalyzes the nucleotide addition to MPT, but the mechanism of the biosynthesis of the bis-MGD cofactor has remained enigmatic. We have established an in vitro system for studying bis-MGD assembly using purified compounds. Quantification of the MPT/molybdenum and molybdenum/phosphorus ratios, time-dependent assays for MPT and MGD detection, and determination of the numbers and lengths of Mo–S and Mo–O bonds by X-ray absorption spectroscopy enabled identification of a novel bis-Mo-MPT intermediate on MobA prior to nucleotide attachment. The addition of Mg-GTP to MobA loaded with bis-Mo-MPT resulted in formation and release of the final bis-MGD product. This cofactor was fully functional and reconstituted the catalytic activity of apo-TMAO reductase (TorA). We propose a reaction sequence for bis-MGD formation, which involves 1) the formation of bis-Mo-MPT, 2) the addition of two GMP units to form bis-MGD on MobA, and 3) the release and transfer of the mature cofactor to the target protein TorA, in a reaction that is supported by the specific chaperone TorD, resulting in an active molybdoenzyme. PMID:24003231

  1. ModE-Dependent Molybdate Regulation of the Molybdenum Cofactor Operon moa in Escherichia coli

    PubMed Central

    Anderson, Lisa A.; McNairn, Elizabeth; Leubke, Torben; Pau, Richard N.; Boxer, David H.

    2000-01-01

    The expression of the moa locus, which encodes enzymes required for molybdopterin biosynthesis, is enhanced under anaerobiosis but repressed when the bacterium is able to synthesize active molybdenum cofactor. In addition, moa expression exhibits a strong requirement for molybdate. The molybdate enhancement of moa transcription is fully dependent upon the molybdate-binding protein, ModE, which also mediates molybdate repression of the mod operon encoding the high-affinity molybdate uptake system. Due to the repression of moa in molybdenum cofactor-sufficient strains, the positive molybdate regulation of moa is revealed only in strains unable to make the active cofactor. Transcription of moa is controlled at two sigma-70-type promoters immediately upstream of the moaA gene. Deletion mutations covering the region upstream of moaA have allowed each of the promoters to be studied in isolation. The distal promoter is the site of the anaerobic enhancement which is Fnr-dependent. The molybdate induction of moa is exerted at the proximal promoter. Molybdate-ModE binds adjacent to the −35 region of this promoter, acting as a direct positive regulator of moa. The molybdenum cofactor repression also appears to act at the proximal transcriptional start site, but the mechanism remains to be established. Tungstate in the growth medium affects moa expression in two ways. Firstly, it can act as a functional molybdate analogue for the ModE-mediated regulation. Secondly, tungstate brings about the loss of the molybdenum cofactor repression of moa. It is proposed that the tungsten derivative of the molybdenum cofactor, which is known to be formed under such conditions, is ineffective in bringing about repression of moa. The complex control of moa is discussed in relation to the synthesis of molybdoenzymes in the bacterium. PMID:11092866

  2. Identification of a bis-molybdopterin intermediate in molybdenum cofactor biosynthesis in Escherichia coli.

    PubMed

    Reschke, Stefan; Sigfridsson, Kajsa G V; Kaufmann, Paul; Leidel, Nils; Horn, Sebastian; Gast, Klaus; Schulzke, Carola; Haumann, Michael; Leimkühler, Silke

    2013-10-11

    The molybdenum cofactor is an important cofactor, and its biosynthesis is essential for many organisms, including humans. Its basic form comprises a single molybdopterin (MPT) unit, which binds a molybdenum ion bearing three oxygen ligands via a dithiolene function, thus forming Mo-MPT. In bacteria, this form is modified to form the bis-MPT guanine dinucleotide cofactor with two MPT units coordinated at one molybdenum atom, which additionally contains GMPs bound to the terminal phosphate group of the MPTs (bis-MGD). The MobA protein catalyzes the nucleotide addition to MPT, but the mechanism of the biosynthesis of the bis-MGD cofactor has remained enigmatic. We have established an in vitro system for studying bis-MGD assembly using purified compounds. Quantification of the MPT/molybdenum and molybdenum/phosphorus ratios, time-dependent assays for MPT and MGD detection, and determination of the numbers and lengths of Mo-S and Mo-O bonds by X-ray absorption spectroscopy enabled identification of a novel bis-Mo-MPT intermediate on MobA prior to nucleotide attachment. The addition of Mg-GTP to MobA loaded with bis-Mo-MPT resulted in formation and release of the final bis-MGD product. This cofactor was fully functional and reconstituted the catalytic activity of apo-TMAO reductase (TorA). We propose a reaction sequence for bis-MGD formation, which involves 1) the formation of bis-Mo-MPT, 2) the addition of two GMP units to form bis-MGD on MobA, and 3) the release and transfer of the mature cofactor to the target protein TorA, in a reaction that is supported by the specific chaperone TorD, resulting in an active molybdoenzyme.

  3. Structural Basis for Oxygen Activation at a Heterodinuclear Manganese/Iron Cofactor.

    PubMed

    Griese, Julia J; Kositzki, Ramona; Schrapers, Peer; Branca, Rui M M; Nordström, Anders; Lehtiö, Janne; Haumann, Michael; Högbom, Martin

    2015-10-16

    Two recently discovered groups of prokaryotic di-metal carboxylate proteins harbor a heterodinuclear Mn/Fe cofactor. These are the class Ic ribonucleotide reductase R2 proteins and a group of oxidases that are found predominantly in pathogens and extremophiles, called R2-like ligand-binding oxidases (R2lox). We have recently shown that the Mn/Fe cofactor of R2lox self-assembles from Mn(II) and Fe(II) in vitro and catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold (Griese, J. J., Roos, K., Cox, N., Shafaat, H. S., Branca, R. M., Lehtiö, J., Gräslund, A., Lubitz, W., Siegbahn, P. E., and Högbom, M. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 17189-17194). Here, we present a detailed structural analysis of R2lox in the nonactivated, reduced, and oxidized resting Mn/Fe- and Fe/Fe-bound states, as well as the nonactivated Mn/Mn-bound state. X-ray crystallography and x-ray absorption spectroscopy demonstrate that the active site ligand configuration of R2lox is essentially the same regardless of cofactor composition. Both the Mn/Fe and the diiron cofactor activate oxygen and catalyze formation of the ether cross-link, whereas the dimanganese cluster does not. The structures delineate likely routes for gated oxygen and substrate access to the active site that are controlled by the redox state of the cofactor. These results suggest that oxygen activation proceeds via similar mechanisms at the Mn/Fe and Fe/Fe center and that R2lox proteins might utilize either cofactor in vivo based on metal availability. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Structural Basis for Oxygen Activation at a Heterodinuclear Manganese/Iron Cofactor*

    PubMed Central

    Griese, Julia J.; Kositzki, Ramona; Schrapers, Peer; Branca, Rui M. M.; Nordström, Anders; Lehtiö, Janne; Haumann, Michael; Högbom, Martin

    2015-01-01

    Two recently discovered groups of prokaryotic di-metal carboxylate proteins harbor a heterodinuclear Mn/Fe cofactor. These are the class Ic ribonucleotide reductase R2 proteins and a group of oxidases that are found predominantly in pathogens and extremophiles, called R2-like ligand-binding oxidases (R2lox). We have recently shown that the Mn/Fe cofactor of R2lox self-assembles from MnII and FeII in vitro and catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold (Griese, J. J., Roos, K., Cox, N., Shafaat, H. S., Branca, R. M., Lehtiö, J., Gräslund, A., Lubitz, W., Siegbahn, P. E., and Högbom, M. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 17189–17194). Here, we present a detailed structural analysis of R2lox in the nonactivated, reduced, and oxidized resting Mn/Fe- and Fe/Fe-bound states, as well as the nonactivated Mn/Mn-bound state. X-ray crystallography and x-ray absorption spectroscopy demonstrate that the active site ligand configuration of R2lox is essentially the same regardless of cofactor composition. Both the Mn/Fe and the diiron cofactor activate oxygen and catalyze formation of the ether cross-link, whereas the dimanganese cluster does not. The structures delineate likely routes for gated oxygen and substrate access to the active site that are controlled by the redox state of the cofactor. These results suggest that oxygen activation proceeds via similar mechanisms at the Mn/Fe and Fe/Fe center and that R2lox proteins might utilize either cofactor in vivo based on metal availability. PMID:26324712

  5. Disruption of rimP-SC, encoding a ribosome assembly cofactor, markedly enhances the production of several antibiotics in Streptomyces coelicolor.

    PubMed

    Pan, Yuanyuan; Lu, Cheng; Dong, Hailing; Yu, Lingjun; Liu, Gang; Tan, Huarong

    2013-07-02

    Ribosome assembly cofactor RimP is one of the auxiliary proteins required for maturation of the 30S subunit in Escherichia coli. Although RimP in protein synthesis is important, its role in secondary metabolites biosynthesis has not been reported so far. Considering the close relationship between protein synthesis and the production of secondary metabolites, the function of ribosome assembly cofactor RimP on antibiotics production was studied in Streptomyces coelicolor and Streptomyces venezuelae. In this study, the rimP homologue rimP-SC was identified and cloned from Streptomyces coelicolor. Disruption of rimP-SC led to enhanced production of actinorhodin and calcium-dependent antibiotics by promoting the transcription of actII-ORF4 and cdaR. Further experiments demonstrated that MetK was one of the reasons for the increment of antibiotics production. In addition, rimP-SC disruption mutant could be used as a host to produce more peptidyl nucleoside antibiotics (polyoxin or nikkomycin) than the wild-type strain. Likewise, disruption of rimP-SV of Streptomyces venezuelae also significantly stimulated jadomycin production, suggesting that enhanced antibiotics production might be widespread in many other Streptomyces species. These results established an important relationship between ribosome assembly cofactor and secondary metabolites biosynthesis and provided an approach for yield improvement of secondary metabolites in Streptomyces.

  6. Lipids as cofactors in protein folding: Stereo-specific lipid–protein interactions are required to form HAMLET (human α-lactalbumin made lethal to tumor cells)

    PubMed Central

    Svensson, Malin; Mossberg, Ann-Kristin; Pettersson, Jenny; Linse, Sara; Svanborg, Catharina

    2003-01-01

    Proteins can adjust their structure and function in response to shifting environments. Functional diversity is created not only by the sequence but by changes in tertiary structure. Here we present evidence that lipid cofactors may enable otherwise unstable protein folding variants to maintain their conformation and to form novel, biologically active complexes. We have identified unsaturated C18 fatty acids in the cis conformation as the cofactors that bind apo α-lactalbumin and form HAMLET (human α-lactalbumin made lethal to tumor cells). The complexes were formed on an ion exchange column, were stable in a molten globule-like conformation, and had attained the novel biological activity. The protein–fatty acid interaction was specific, as saturated C18 fatty acids, or unsaturated C18:1trans conformers were unable to form complexes with apo α-lactalbumin, as were fatty acids with shorter or longer carbon chains. Unsaturated cis fatty acids other than C18:1:9cis were able to form stable complexes, but these were not active in the apoptosis assay. The results demonstrate that stereo-specific lipid–protein interactions can stabilize partially unfolded conformations and form molecular complexes with novel biological activity. The results offer a new mechanism for the functional diversity of proteins, by exploiting lipids as essential, tissue-specific cofactors in this process. PMID:14627740

  7. Cellular Restriction Factors of Feline Immunodeficiency Virus

    PubMed Central

    Zielonka, Jörg; Münk, Carsten

    2011-01-01

    Lentiviruses are known for their narrow cell- and species-tropisms, which are determined by cellular proteins whose absence or presence either support viral replication (dependency factors, cofactors) or inhibit viral replication (restriction factors). Similar to Human immunodeficiency virus type 1 (HIV-1), the cat lentivirus Feline immunodeficiency virus (FIV) is sensitive to recently discovered cellular restriction factors from non-host species that are able to stop viruses from replicating. Of particular importance are the cellular proteins APOBEC3, TRIM5α and tetherin/BST-2. In general, lentiviruses counteract or escape their species’ own variant of the restriction factor, but are targeted by the orthologous proteins of distantly related species. Most of the knowledge regarding lentiviral restriction factors has been obtained in the HIV-1 system; however, much less is known about their effects on other lentiviruses. We describe here the molecular mechanisms that explain how FIV maintains its replication in feline cells, but is largely prevented from cross-species infections by cellular restriction factors. PMID:22069525

  8. Redirecting metabolic flux in Saccharomyces cerevisiae through regulation of cofactors in UMP production.

    PubMed

    Chen, Yong; Liu, Qingguo; Chen, Xiaochun; Wu, Jinglan; Guo, Ting; Zhu, Chenjie; Ying, Hanjie

    2015-04-01

    Although it is generally known that cofactors play a major role in the production of different fermentation products, their role has not been thoroughly and systematically studied. To understand the impact of cofactors on physiological functions, a systematic approach was applied, which involved redox state analysis, energy charge analysis, and metabolite analysis. Using uridine 5'-monophosphate metabolism in Saccharomyces cerevisiae as a model, we demonstrated that regulation of intracellular the ratio of NADPH to NADP(+) not only redistributed the carbon flux between the glycolytic and pentose phosphate pathways, but also regulated the redox state of NAD(H), resulting in a significant change of ATP, and a significantly altered spectrum of metabolic products.

  9. A global transcription cofactor bound to juxtaposed strands of unwound DNA.

    PubMed

    Werten, Sebastiaan; Moras, Dino

    2006-02-01

    The 1.74-A crystal structure of the human transcription cofactor PC4 in complex with a single-stranded 20-mer oligonucleotide reveals how symmetry-related beta-surfaces of the protein homodimer interact with juxtaposed 5-nucleotide DNA regions running in opposite directions. The structure explains high-affinity binding of PC4 to the complementary strands of unwinding duplex DNA, and it suggests the cofactor may have a role in relaxing negative supercoils or exposing unpaired bases for sequence-specific recognition by other biomolecules.

  10. Targeting RNA for processing or destruction by the eukaryotic RNA exosome and its cofactors

    PubMed Central

    Zinder, John C.

    2017-01-01

    The eukaryotic RNA exosome is an essential and conserved protein complex that can degrade or process RNA substrates in the 3′-to-5′ direction. Since its discovery nearly two decades ago, studies have focused on determining how the exosome, along with associated cofactors, achieves the demanding task of targeting particular RNAs for degradation and/or processing in both the nucleus and cytoplasm. In this review, we highlight recent advances that have illuminated roles for the RNA exosome and its cofactors in specific biological pathways, alongside studies that attempted to dissect these activities through structural and biochemical characterization of nuclear and cytoplasmic RNA exosome complexes. PMID:28202538

  11. An ancient oxidoreductase making differential use of its cofactors.

    PubMed

    Blüher, Doreen; Reinhardt-Tews, Annekathrin; Hey, Martin; Lilie, Hauke; Golbik, Ralph; Breunig, Karin D; Anders, Alexander

    2014-07-01

    Abstract Many transcription factors contribute to cellular homeostasis by integrating multiple signals. Signaling via the yeast Gal80 protein, a negative regulator of the prototypic transcription activator Gal4, is primarily regulated by galactose. ScGal80 from Saccharomyces cerevisiae has been reported to bind NAD(P). Here, we show that the ability to bind these ligands is conserved in KlGal80, a Gal80 homolog from the distantly related yeast Kluyveromyces lactis. However, the homologs apparently have diverged with respect to response to the dinucleotide. Strikingly, ScGal80 binds NAD(P) and NAD(P)H with more than 50-fold higher affinity than KlGal80. In contrast to ScGal80, where NAD is neutral, NAD and NADP have a negative effect in KlGal80 on its interaction with a KlGal4-peptide in vitro. Swapping a loop in the NAD(P) binding Rossmann-fold of ScGal80 into KlGal80 increases the affinity for NAD(P) and has a significant impact on KlGal4 regulation in vivo. Apparently, dinucleotide binding allows coupling of the metabolic state of the cell to regulation of the GAL/LAC genes. The particular sequences involved in binding determine how exactly the metabolic state is sensed and integrated by Gal80 to regulate Gal4.

  12. A Hexasaccharide Containing Rare 2‐O‐Sulfate‐Glucuronic Acid Residues Selectively Activates Heparin Cofactor II

    PubMed Central

    Sankarayanarayanan, Nehru Viji; Strebel, Tamara R.; Boothello, Rio S.; Sheerin, Kevin; Raghuraman, Arjun; Sallas, Florence; Mosier, Philip D.; Watermeyer, Nicholas D.

    2017-01-01

    Abstract Glycosaminoglycan (GAG) sequences that selectively target heparin cofactor II (HCII), a key serpin present in human plasma, remain unknown. Using a computational strategy on a library of 46 656 heparan sulfate hexasaccharides we identified a rare sequence consisting of consecutive glucuronic acid 2‐O‐sulfate residues as selectively targeting HCII. This and four other unique hexasaccharides were chemically synthesized. The designed sequence was found to activate HCII ca. 250‐fold, while leaving aside antithrombin, a closely related serpin, essentially unactivated. This group of rare designed hexasaccharides will help understand HCII function. More importantly, our results show for the first time that rigorous use of computational techniques can lead to discovery of unique GAG sequences that can selectively target GAG‐binding protein(s), which may lead to chemical biology or drug discovery tools. PMID:28124818

  13. VAPB/ALS8 interacts with FFAT-like proteins including the p97 cofactor FAF1 and the ASNA1 ATPase

    PubMed Central

    2014-01-01

    Background FAF1 is a ubiquitin-binding adaptor for the p97 ATPase and belongs to the UBA-UBX family of p97 cofactors. p97 converts the energy derived from ATP hydrolysis into conformational changes of the p97 hexamer, which allows the dissociation of its targets from cellular structures or from larger protein complexes to facilitate their ubiquitin-dependent degradation. VAPB and the related protein VAPA form homo- and heterodimers that are anchored in the endoplasmic reticulum membrane and can interact with protein partners carrying a FFAT motif. Mutations in either VAPB or p97 can cause amyotrophic lateral sclerosis, a neurodegenerative disorder that affects upper and lower motor neurons. Results We show that FAF1 contains a non-canonical FFAT motif that allows it to interact directly with the MSP domain of VAPB and, thereby, to mediate VAPB interaction with p97. This finding establishes a link between two proteins that can cause amyotrophic lateral sclerosis when mutated, VAPB/ALS8 and p97/ALS14. Subsequently, we identified a similar FFAT-like motif in the ASNA1 subunit of the transmembrane-domain recognition complex (TRC), which in turn mediates ASNA1 interaction with the MSP domain of VAPB. Proteasome inhibition leads to the accumulation of ubiquitinated species in VAPB immunoprecipitates and this correlates with an increase in FAF1 and p97 binding. We found that VAPB interaction with ubiquitinated proteins is strongly reduced in cells treated with FAF1 siRNA. Our efforts to determine the identity of the ubiquitinated targets common to VAPB and FAF1 led to the identification of RPN2, a subunit of an oligosaccharyl-transferase located at the endoplasmic reticulum, which may be regulated by ubiquitin-mediated degradation. Conclusions The FFAT-like motifs we identified in FAF1 and ASNA1 demonstrate that sequences containing a single phenylalanine residue with the consensus (D/E)(D/E)FEDAx(D/E) are also proficient to mediate interaction with VAPB. Our findings

  14. A periplasmic aldehyde oxidoreductase represents the first molybdopterin cytosine dinucleotide cofactor containing molybdo-flavoenzyme from Escherichia coli.

    PubMed

    Neumann, Meina; Mittelstädt, Gerd; Iobbi-Nivol, Chantal; Saggu, Miguel; Lendzian, Friedhelm; Hildebrandt, Peter; Leimkühler, Silke

    2009-05-01

    Three DNA regions carrying genes encoding putative homologs of xanthine dehydrogenases were identified in Escherichia coli, named xdhABC, xdhD, and yagTSRQ. Here, we describe the purification and characterization of gene products of the yagTSRQ operon, a molybdenum-containing iron-sulfur flavoprotein from E. coli, which is located in the periplasm. The 135 kDa enzyme comprised a noncovalent (alpha beta gamma) heterotrimer with a large (78.1 kDa) molybdenum cofactor (Moco)-containing YagR subunit, a medium (33.9 kDa) FAD-containing YagS subunit, and a small (21.0 kDa) 2 x [2Fe2S]-containing YagT subunit. YagQ is not a subunit of the mature enzyme, and the protein is expected to be involved in Moco modification and insertion into YagTSR. Analysis of the form of Moco present in YagTSR revealed the presence of the molybdopterin cytosine dinucleotide cofactor. Two different [2Fe2S] clusters, typical for this class of enzyme, were identified by EPR. YagTSR represents the first example of a molybdopterin cytosine dinucleotide-containing protein in E. coli. Kinetic characterization of the enzyme revealed that YagTSR converts a broad spectrum of aldehydes, with a preference for aromatic aldehydes. Ferredoxin instead of NAD(+) or molecular oxygen was used as terminal electron acceptor. Complete growth inhibition of E. coli cells devoid of genes from the yagTSRQ operon was observed by the addition of cinnamaldehyde to a low-pH medium. This finding shows that YagTSR might have a role in the detoxification of aromatic aldehydes for E. coli under certain growth conditions.

  15. Identification of a protein-protein interaction network downstream of molybdenum cofactor biosynthesis in Arabidopsis thaliana.

    PubMed

    Kaufholdt, David; Baillie, Christin-Kirsty; Meyer, Martin H; Schwich, Oliver D; Timmerer, Ulrike L; Tobias, Lydia; van Thiel, Daniela; Hänsch, Robert; Mendel, Ralf R

    2016-12-01

    The molybdenum cofactor (Moco) is ubiquitously present in all kingdoms of life and vitally important for survival. Among animals, loss of the Moco-containing enzyme (Mo-enzyme) sulphite oxidase is lethal, while for plants the loss of nitrate reductase prohibits nitrogen assimilation. Moco is highly oxygen-sensitive, which obviates a freely diffusible pool and necessitates protein-mediated distribution. During the highly conserved Moco biosynthesis pathway, intermediates are channelled through a multi-protein complex facilitating protected transport. However, the mechanism by which Moco is subsequently transferred to apo-enzymes is still unclear. Moco user enzymes can be divided into two families: the sulphite oxidase (SO) and the xanthine oxidoreductase (XOR) family. The latter requires a final sulphurisation of Moco catalysed via ABA3. To examine Moco transfer towards apo-Mo-enzymes, two different and independent protein-protein interaction assays were performed in vivo: bimolecular fluorescence complementation and split luciferase. The results revealed a direct contact between Moco producer molybdenum insertase CNX1, which represents the last biosynthesis step, and members of the SO family. However, no protein contact was observed between Moco producer CNX1 and apo-enzymes of the XOR family or between CNX1 and the Moco sulphurase ABA3. Instead, the Moco-binding protein MOBP2 was identified as a mediator between CNX1 and ABA3. This interaction was followed by contact between ABA3 and enzymes of the XOR family. These results allow to describe an interaction matrix of proteins beyond Moco biosynthesis and to demonstrate the complexity of transferring a prosthetic group after biosynthesis. Copyright © 2016 Elsevier GmbH. All rights reserved.

  16. A novel cofactor-binding mode in bacterial IMP dehydrogenases explains inhibitor selectivity

    SciTech Connect

    Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Osipiuk, Jerzy; Gu, Minyi; Zhang, Minjia; Mandapati, Kavitha; Gollapalli, Deviprasad R.; Gorla, Suresh Kumar; Hedstrom, Lizbeth; Joachimiak, Andrzej

    2015-01-09

    The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5'-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD+, which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD+ and XMP/NAD+. In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD+ adenosine moiety. More importantly, this new NAD+-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD+-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. As a result, these findings offer a potential strategy for further ligand optimization.

  17. Dual posttranscriptional regulation via a cofactor-responsive mRNA leader.

    PubMed

    Patterson-Fortin, Laura M; Vakulskas, Christopher A; Yakhnin, Helen; Babitzke, Paul; Romeo, Tony

    2013-10-09

    Riboswitches are cis-acting mRNA elements that regulate gene expression in response to ligand binding. Recently, a class of riboswitches was proposed to respond to the molybdenum cofactor (Moco), which serves as a redox center for metabolic enzymes. The 5' leader of the Escherichia coli moaABCDE transcript exemplifies this candidate riboswitch class. This mRNA encodes enzymes for Moco biosynthesis, and moaA expression is feedback inhibited by Moco. Previous RNA-seq analyses showed that moaA mRNA copurified with the RNA binding protein CsrA (carbon storage regulator), suggesting that CsrA binds to this RNA in vivo. Among its global regulatory roles, CsrA represses stationary phase metabolism and activates central carbon metabolism. Here, we used gel mobility shift analysis to determine that CsrA binds specifically and with high affinity to the moaA 5' mRNA leader. Northern blotting and studies with a series of chromosomal lacZ reporter fusions showed that CsrA posttranscriptionally activates moaA expression without altering moaA mRNA levels, indicative of translation control. Deletion analyses, nucleotide replacement studies and footprinting with CsrA-FeBABE identified two sites for CsrA binding. Toeprinting assays suggested that CsrA binding causes changes in moaA RNA structure. We propose that the moaA mRNA leader forms an aptamer, which serves as a target of posttranscriptional regulation by at least two different factors, Moco and the protein CsrA. While we are not aware of similar dual posttranscriptional regulatory mechanisms, additional examples are likely to emerge. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Dual Posttranscriptional Regulation via a Cofactor-Responsive mRNA Leader

    PubMed Central

    Patterson-Fortin, Laura M.; Vakulskas, Christopher A.; Yakhnin, Helen; Babitzke, Paul; Romeo, Tony

    2013-01-01

    Riboswitches are cis-acting mRNA elements that regulate gene expression in response to ligand binding. Recently, a class of riboswitches was proposed to respond to the molybdenum cofactor (Moco), which serves as a redox center for metabolic enzymes. The 5′ leader of the Escherichia coli moaABCDE transcript exemplifies this candidate riboswitch class. This mRNA encodes enzymes for Moco biosynthesis, and moaA expression is feedback inhibited by Moco. Previous RNA-seq analyses showed that moaA mRNA copurified with the RNA binding protein CsrA (carbon storage regulator), suggesting that CsrA binds to this RNA in vivo. Among its global regulatory roles, CsrA represses stationary phase metabolism and activates central carbon metabolism. Here, we used gel mobility shift analysis to determine that CsrA binds specifically and with high affinity to the moaA 5′ mRNA leader. Northern blotting and studies with a series of chromosomal lacZ reporter fusions showed that CsrA posttranscriptionally activates moaA expression without altering moaA mRNA levels, indicative of translation control. Deletion analyses, nucleotide replacement studies and footprinting with CsrA-FeBABE identified two sites for CsrA binding. Toeprinting assays suggested that CsrA binding causes changes in moaA RNA structure. We propose that the moaA mRNA leader forms an aptamer, which serves as a target of posttranscriptional regulation by at least two different factors, Moco and the protein CsrA. While we are not aware of similar dual posttranscriptional regulatory mechanisms, additional examples are likely to emerge. PMID:23274138

  19. A novel high-throughput screening assay for discovery of molecules that increase cellular tetrahydrobiopterin.

    PubMed

    Li, Li; Du, Yuhong; Chen, Wei; Fu, Haian; Harrison, David G

    2011-09-01

    Tetrahydrobiopterin (BH(4)) is an essential cofactor for the nitric oxide (NO) synthases and the aromatic amino acid hydroxylases. Insufficient BH(4) has been implicated in various cardiovascular and neurological disorders. GTP cyclohydrolase 1 (GTPCH-1) is the rate-limiting enzyme for de novo biosynthesis of BH(4). The authors have recently shown that the interaction of GTPCH-1 with GTP cyclohydrolase feedback regulatory protein (GFRP) inhibits endothelial GTPCH-1 enzyme activity, BH(4) levels, and NO production. They propose that agents that disrupt the GTPCH-1/GFRP interaction can increase cellular GTPCH-1 activity, BH(4) levels, and NO production. They developed and optimized a novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor the interaction of GTPCH-1 and GFRP. This assay is highly sensitive and stable and has a signal-to-background ratio (S/B) greater than 12 and a Z' factor greater than 0.8. This assay was used in an ultra-high-throughput screening (uHTS) format to screen the Library of Pharmacologically Active Compounds. Using independent protein-protein interaction and cellular activity assays, the authors identified compounds that disrupt GTPCH-1/GFRP binding and increase endothelial cell biopterin levels. Thus, this TR-FRET assay could be applied in future uHTS of additional libraries to search for molecules that increase GTPCH-1 activity and BH(4) levels.

  20. Cell Fusion as a Cofactor in Prostate Cancer Metastasis

    DTIC Science & Technology

    2010-01-01

    fusion. However, our findings indicated a different and unexpected to us mechanism. We found that PC-3 released one or more infectious viruses that...and MLV are, we could not identify the virus secreted by PC-3 cells unambiguously. While were we conducting our studies , several reports questioned...vesicular stomatitis virus (VSV), to be reversibly activated by a brief incubation in mildly acidic medium. The advantages of this method are that it is not

  1. Promoter and Cofactor Requirements for SERM-ER Activity

    DTIC Science & Technology

    2006-05-01

    Rett syndrome . Nat. Genet. 37, 31–40. keda, K., Sato, M., Tsutsumi, O., Tsuchiya, F., Tsuneizumi, M., Emi, ., Imoto, I., Inazawa, J., Muramatsu, M., and...already found a number of novel ER associated proteins that appear to be essential for ER-mediated transcription 14. SUBJECT TERMS 15. NUMBER OF...identify long distance cis-regulatory elements, which proved successful in two of the three assessed cases, including TFF-1 and NRIP-1. This for the first

  2. New cofactor supports α,β-unsaturated acid decarboxylation via 1,3-dipolar cycloaddition.

    PubMed

    Payne, Karl A P; White, Mark D; Fisher, Karl; Khara, Basile; Bailey, Samuel S; Parker, David; Rattray, Nicholas J W; Trivedi, Drupad K; Goodacre, Royston; Beveridge, Rebecca; Barran, Perdita; Rigby, Stephen E J; Scrutton, Nigel S; Hay, Sam; Leys, David

    2015-06-25

    The bacterial ubiD and ubiX or the homologous fungal fdc1 and pad1 genes have been implicated in the non-oxidative reversible decarboxylation of aromatic substrates, and play a pivotal role in bacterial ubiquinone (also known as coenzyme Q) biosynthesis or microbial biodegradation of aromatic compounds, respectively. Despite biochemical studies on individual gene products, the composition and cofactor requirement of the enzyme responsible for in vivo decarboxylase activity remained unclear. Here we show that Fdc1 is solely responsible for the reversible decarboxylase activity, and that it requires a new type of cofactor: a prenylated flavin synthesized by the associated UbiX/Pad1. Atomic resolution crystal structures reveal that two distinct isomers of the oxidized cofactor can be observed, an isoalloxazine N5-iminium adduct and a N5 secondary ketimine species with markedly altered ring structure, both having azomethine ylide character. Substrate binding positions the dipolarophile enoic acid group directly above the azomethine ylide group. The structure of a covalent inhibitor-cofactor adduct suggests that 1,3-dipolar cycloaddition chemistry supports reversible decarboxylation in these enzymes. Although 1,3-dipolar cycloaddition is commonly used in organic chemistry, we propose that this presents the first example, to our knowledge, of an enzymatic 1,3-dipolar cycloaddition reaction. Our model for Fdc1/UbiD catalysis offers new routes in alkene hydrocarbon production or aryl (de)carboxylation.

  3. Mycofactocin-associated mycobacterial dehydrogenases with non-exchangeable NAD cofactors

    PubMed Central

    Haft, Daniel H.; Pierce, Phillip G.; Mayclin, Stephen J.; Sullivan, Amy; Gardberg, Anna S.; Abendroth, Jan; Begley, Darren W.; Phan, Isabelle Q.; Staker, Bart L.; Myler, Peter J.; Marathias, Vasilios M.; Lorimer, Donald D.; Edwards, Thomas E.

    2017-01-01

    During human infection, Mycobacterium tuberculosis (Mtb) survives the normally bacteriocidal phagosome of macrophages. Mtb and related species may be able to combat this harsh acidic environment which contains reactive oxygen species due to the mycobacterial genomes encoding a large number of dehydrogenases. Typically, dehydrogenase cofactor binding sites are open to solvent, which allows NAD/NADH exchange to support multiple turnover. Interestingly, mycobacterial short chain dehydrogenases/reductases (SDRs) within family TIGR03971 contain an insertion at the NAD binding site. Here we present crystal structures of 9 mycobacterial SDRs in which the insertion buries the NAD cofactor except for a small portion of the nicotinamide ring. Line broadening and STD-NMR experiments did not show NAD or NADH exchange on the NMR timescale. STD-NMR demonstrated binding of the potential substrate carveol, the potential product carvone, the inhibitor tricyclazol, and an external redox partner 2,6-dichloroindophenol (DCIP). Therefore, these SDRs appear to contain a non-exchangeable NAD cofactor and may rely on an external redox partner, rather than cofactor exchange, for multiple turnover. Incidentally, these genes always appear in conjunction with the mftA gene, which encodes the short peptide MftA, and with other genes proposed to convert MftA into the external redox partner mycofactocin. PMID:28120876

  4. Artificial photosynthesis on a chip: microfluidic cofactor regeneration and photoenzymatic synthesis under visible light.

    PubMed

    Lee, Joon Seok; Lee, Sahng Ha; Kim, Jae Hong; Park, Chan Beum

    2011-07-21

    We present a microfluidic artificial photosynthetic platform that incorporates quantum dots and redox enzymes for photoenzymatic synthesis of fine chemicals under visible light. Similar to natural photosynthesis, photochemical cofactor regeneration takes place in the light-dependent reaction zone, which is then coupled with the light-independent, enzymatic synthesis in the downstream of the microchannel.

  5. New cofactor supports α,β-unsaturated acid decarboxylation via 1,3-dipolar cycloaddition

    PubMed Central

    Payne, Karl A.P.; White, Mark D.; Fisher, Karl; Khara, Basile; Bailey, Samuel S.; Parker, David; Rattray, Nicholas J.W.; Trivedi, Drupad K.; Goodacre, Royston; Beveridge, Rebecca; Barran, Perdita; Rigby, Stephen E.J.; Scrutton, Nigel S.; Hay, Sam; Leys, David

    2016-01-01

    The ubiD/ubiX or the homologous fdc/pad genes have been implicated in the non-oxidative reversible decarboxylation of aromatic substrates, and play a pivotal role in bacterial ubiquinone biosynthesis1–3 or microbial biodegradation of aromatic compounds4–6 respectively. Despite biochemical studies on individual gene products, the composition and co-factor requirement of the enzyme responsible for in vivo decarboxylase activity remained unclear7–9. We show Fdc is solely responsible for (de)carboxylase activity, and that it requires a new type of cofactor: a prenylated flavin synthesised by the associated UbiX/Pad10. Atomic resolution crystal structures reveal two distinct isomers of the oxidized cofactor can be observed: an isoalloxazine N5-iminium adduct and a N5 secondary ketimine species with drastically altered ring structure, both having azomethine ylide character. Substrate binding positions the dipolarophile enoic acid group directly above the azomethine ylide group. The structure of a covalent inhibitor-cofactor adduct suggests 1,3-dipolar cycloaddition chemistry supports reversible decarboxylation in these enzymes. While 1,3-dipolar cycloaddition is commonly used in organic chemistry11–12, we propose this presents the first example of an enzymatic 1,3-dipolar cycloaddition reaction. Our model for Fdc/UbiD catalysis offers new routes in alkene hydrocarbon production or aryl (de)carboxylation. PMID:26083754

  6. Tetrahydropterin as a possible natural cofactor in the drosophila phenylalanine hydroxylation system

    SciTech Connect

    Bel, Y.; Jacobson, K.B.; Ferre, J. . Dept. of Genetics; Oak Ridge National Lab., TN; Valencia Univ. . Dept. of Genetics)

    1989-01-01

    The aim of the present work is the study of phenylalanine hydroxylase (PH) activity of Drosophila melanogaster wild type with different cofactors: the two natural occurring tetrahydropteridines (BH{sub 4} and PH{sub 4}) and the synthetic 6,7-dimethyltetrahydropterin (DMPH{sub 4}), as well as the determination of this activity at different developmental stages. 7 refs., 2 figs.

  7. The archaeal cofactor F0 is a light-harvesting antenna chromophore in eukaryotes.

    PubMed

    Glas, Andreas F; Maul, Melanie J; Cryle, Max; Barends, Thomas R M; Schneider, Sabine; Kaya, Emine; Schlichting, Ilme; Carell, Thomas

    2009-07-14

    Archae possess unique biochemical systems quite distinct from the pathways present in eukaryotes and eubacteria. 7,8-Dimethyl-8-hydroxy-5deazaflavin (F(0)) and F(420) are unique deazaflavin-containing coenzyme and methanogenic signature molecules, essential for a variety of biochemical transformations associated with methane biosynthesis and light-dependent DNA repair. The deazaflavin cofactor system functions during methane biosynthesis as a low-potential hydrid shuttle F(420)/F(420)H(2). In DNA photolyase repair proteins, the deazaflavin cofactor is in the deprotonated state active as a light-collecting energy transfer pigment. As such, it converts blue sunlight into energy used by the proteins to drive an essential repair process. Analysis of a eukaryotic (6-4) DNA photolyase from Drosophila melanogaster revealed a binding pocket, which tightly binds F(0). Residues in the pocket activate the cofactor by deprotonation so that light absorption and energy transfer are switched on. The crystal structure of F(0) in complex with the D. melanogaster protein shows the atomic details of F(0) binding and activation, allowing characterization of the residues involved in F(0) activation. The results show that the F(0)/F(420) coenzyme system, so far believed to be strictly limited to the archael kingdom of life, is far more widespread than anticipated. Analysis of a D. melanogaster extract and of a DNA photolyase from the primitive eukaryote Ostreococcus tauri provided direct proof for the presence of the F(0) cofactor also in higher eukaryotes.

  8. Prevalence and Gene Characteristics of Antibodies with Cofactor-induced HIV-1 Specificity*

    PubMed Central

    Lecerf, Maxime; Scheel, Tobias; Pashov, Anastas D.; Jarossay, Annaelle; Ohayon, Delphine; Planchais, Cyril; Mesnage, Stephane; Berek, Claudia; Kaveri, Srinivas V.; Lacroix-Desmazes, Sébastien; Dimitrov, Jordan D.

    2015-01-01

    The healthy immune repertoire contains a fraction of antibodies that bind to various biologically relevant cofactors, including heme. Interaction of heme with some antibodies results in induction of new antigen binding specificities and acquisition of binding polyreactivity. In vivo, extracellular heme is released as a result of hemolysis or tissue damage; hence the post-translational acquisition of novel antigen specificities might play an important role in the diversification of the immunoglobulin repertoire and host defense. Here, we demonstrate that seronegative immune repertoires contain antibodies that gain reactivity to HIV-1 gp120 upon exposure to heme. Furthermore, a panel of human recombinant antibodies was cloned from different B cell subpopulations, and the prevalence of antibodies with cofactor-induced specificity for gp120 was determined. Our data reveal that upon exposure to heme, ∼24% of antibodies acquired binding specificity for divergent strains of HIV-1 gp120. Sequence analyses reveal that heme-sensitive antibodies do not differ in their repertoire of variable region genes and in most of the molecular features of their antigen-binding sites from antibodies that do not change their antigen binding specificity. However, antibodies with cofactor-induced gp120 specificity possess significantly lower numbers of somatic mutations in their variable region genes. This study contributes to the understanding of the significance of cofactor-binding antibodies in immunoglobulin repertoires and of the influence that the tissue microenvironment might have in shaping adaptive immune responses. PMID:25564611

  9. Refolding of horseradish peroxidase is enhanced in presence of metal cofactors and ionic liquids.

    PubMed

    Bae, Sang-Woo; Eom, Doyoung; Mai, Ngoc Lan; Koo, Yoon-Mo

    2016-03-01

    The effects of various refolding additives, including metal cofactors, organic co-solvents, and ionic liquids, on the refolding of horseradish peroxidase (HRP), a well-known hemoprotein containing four disulfide bonds and two different types of metal centers, a ferrous ion-containing heme group and two calcium atoms, which provide a stabilizing effect on protein structure and function, were investigated. Both metal cofactors (Ca(2+) and hemin) and ionic liquids have positive impact on the refolding of HRP. For instance, the HRP refolding yield remarkably increased by over 3-fold upon addition of hemin and calcium chloride to the refolding buffer as compared to that in the conventional urea-containing refolding buffer. Moreover, the addition of ionic liquids [EMIM][Cl] to the hemin and calcium cofactor-containing refolding buffer further enhanced the HRP refolding yield up to 80% as compared to 12% in conventional refolding buffer at relatively high initial protein concentration (5 mg/ml). These results indicated that refolding method utilizing metal cofactors and ionic liquids could enhance the yield and efficiency for metalloprotein.

  10. Different dynamic movements of wild-type and pathogenic VCPs and their cofactors to damaged mitochondria in a Parkin-mediated mitochondrial quality control system.

    PubMed

    Kimura, Yoko; Fukushi, Junpei; Hori, Seiji; Matsuda, Noriyuki; Okatsu, Kei; Kakiyama, Yukie; Kawawaki, Junko; Kakizuka, Akira; Tanaka, Keiji

    2013-12-01

    VCP/p97 is a hexameric ring-shaped AAA(+) ATPase that participates in various ubiquitin-associated cellular functions. Mis-sense mutations in VCP gene are associated with the pathogenesis of two inherited diseases: inclusion body myopathy associated with Paget's disease of the bone and front-temporal dementia (IBMPFD) and familial amyotrophic lateral sclerosis (ALS). These pathogenic VCPs have higher affinities for several cofactors, including Npl4, Ufd1 and p47. In Parkin-dependent mitochondrial quality control systems, VCP migrates to damaged mitochondria (e.g., those treated with uncouplers) to aid in the degradation of mitochondrial outer membrane proteins and to eliminate mitochondria. We showed that endogenous Npl4 and p47 also migrate to mitochondria after uncoupler treatment, and Npl4, Ufd1 or p47 silencing causes defective mitochondria clearance after uncoupler treatment. Moreover, pathogenic VCPs show impaired migration to mitochondria, and the exogenous pathogenic VCP expression partially inhibits Npl4 and p47 localization to mitochondria. These results suggest that the increased affinities of pathogenic VCPs for these cofactors cause the impaired movement of pathogenic VCPs. In adult flies, exogenous expression of wild-type VCP, but not pathogenic VCPs, reduces the number of abnormal mitochondria in muscles. Failure of pathogenic VCPs to function on damaged mitochondria may be related to the pathogenesis of IBMPFD and ALS.

  11. Cofactor binding modulates the conformational stabilities and unfolding patterns of NAD(+)-dependent DNA ligases from Escherichia coli and Thermus scotoductus.

    PubMed

    Georlette, Daphné; Blaise, Vinciane; Dohmen, Christophe; Bouillenne, Fabrice; Damien, Benjamin; Depiereux, Eric; Gerday, Charles; Uversky, Vladimir N; Feller, Georges

    2003-12-12

    DNA ligases are important enzymes required for cellular processes such as DNA replication, recombination, and repair. NAD(+)-dependent DNA ligases are essentially restricted to eubacteria, thus constituting an attractive target in the development of novel antibiotics. Although such a project might involve the systematic testing of a vast number of chemical compounds, it can essentially gain from the preliminary deciphering of the conformational stability and structural perturbations associated with the formation of the catalytically active adenylated enzyme. We have, therefore, investigated the adenylation-induced conformational changes in the mesophilic Escherichia coli and thermophilic Thermus scotoductus NAD(+)-DNA ligases, and the resistance of these enzymes to thermal and chemical (guanidine hydrochloride) denaturation. Our results clearly demonstrate that anchoring of the cofactor induces a conformational rearrangement within the active site of both mesophilic and thermophilic enzymes accompanied by their partial compaction. Furthermore, the adenylation of enzymes increases their resistance to thermal and chemical denaturation, establishing a thermodynamic link between cofactor binding and conformational stability enhancement. Finally, guanidine hydrochloride-induced unfolding of NAD(+)-dependent DNA ligases is shown to be a complex process that involves accumulation of at least two equilibrium intermediates, the molten globule and its precursor.

  12. Metabolic Impact of Redox Cofactor Perturbations on the Formation of Aroma Compounds in Saccharomyces cerevisiae

    PubMed Central

    Sanchez, Isabelle; Dequin, Sylvie; Camarasa, Carole

    2015-01-01

    Redox homeostasis is a fundamental requirement for the maintenance of metabolism, energy generation, and growth in Saccharomyces cerevisiae. The redox cofactors NADH and NADPH are among the most highly connected metabolites in metabolic networks. Changes in their concentrations may induce widespread changes in metabolism. Redox imbalances were achieved with a dedicated biological tool overexpressing native NADH-dependent or engineered NADPH-dependent 2,3-butanediol dehydrogenase, in the presence of acetoin. We report that targeted perturbation of the balance of cofactors (NAD+/NADH or, to a lesser extent, NADP+/NADPH) significantly affected the production of volatile compounds. In most cases, variations in the redox state of yeasts modified the formation of all compounds from the same biochemical pathway (isobutanol, isoamyl alcohol, and their derivatives) or chemical class (ethyl esters), irrespective of the cofactors. These coordinated responses were found to be closely linked to the impact of redox status on the availability of intermediates of central carbon metabolism. This was the case for α-keto acids and acetyl coenzyme A (acetyl-CoA), which are precursors for the synthesis of many volatile compounds. We also demonstrated that changes in the availability of NADH selectively affected the synthesis of some volatile molecules (e.g., methionol, phenylethanol, and propanoic acid), reflecting the specific cofactor requirements of the dehydrogenases involved in their formation. Our findings indicate that both the availability of precursors from central carbon metabolism and the accessibility of reduced cofactors contribute to cell redox status modulation of volatile compound formation. PMID:26475113

  13. Selective androgen receptor modulator activity of a steroidal antiandrogen TSAA-291 and its cofactor recruitment profile.

    PubMed

    Hikichi, Yukiko; Yamaoka, Masuo; Kusaka, Masami; Hara, Takahito

    2015-10-15

    Selective androgen receptor modulators (SARMs) specifically bind to the androgen receptor and exert agonistic or antagonistic effects on target organs. In this study, we investigated the SARM activity of TSAA-291, previously known as a steroidal antiandrogen, in mice because TSAA-291 was found to possess partial androgen receptor agonist activity in reporter assays. In addition, to clarify the mechanism underlying its tissue selectivity, we performed comprehensive cofactor recruitment analysis of androgen receptor using TSAA-291 and dihydrotestosterone (DHT), an endogenous androgen. The androgen receptor agonistic activity of TSAA-291 was more obvious in reporter assays using skeletal muscle cells than in those using prostate cells. In castrated mice, TSAA-291 increased the weight of the levator ani muscle without increasing the weight of the prostate and seminal vesicle. Comprehensive cofactor recruitment analysis via mammalian two-hybrid methods revealed that among a total of 112 cofactors, 12 cofactors including the protein inhibitor of activated STAT 1 (PIAS1) were differently recruited to androgen receptor in the presence of TSAA-291 and DHT. Prostate displayed higher PIAS1 expression than skeletal muscle. Forced expression of the PIAS1 augmented the transcriptional activity of the androgen receptor, and silencing of PIAS1 by siRNAs suppressed the secretion of prostate-specific antigen, an androgen responsive marker. Our results demonstrate that TSAA-291 has SARM activity and suggest that TSAA-291 may induce different conformational changes of the androgen receptor and recruitment profiles of cofactors such as PIAS1, compared with DHT, to exert tissue-specific activity. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. The Structure and Competitive Substrate Inhibition of Dihydrofolate Reductase from Enterococcus faecalis Reveal Restrictions to Cofactor Docking

    PubMed Central

    2015-01-01

    We are addressing bacterial resistance to antibiotics by repurposing a well-established classic antimicrobial target, the dihydrofolate reductase (DHFR) enzyme. In this work, we have focused on Enterococcus faecalis, a nosocomial pathogen that frequently harbors antibiotic resistance determinants leading to complicated and difficult-to-treat infections. An inhibitor series with a hydrophobic dihydrophthalazine heterocycle was designed from the anti-folate trimethoprim. We have examined the potency of this inhibitor series based on inhibition of DHFR enzyme activity and bacterial growth, including in the presence of the exogenous product analogue folinic acid. The resulting preferences were rationalized using a cocrystal structure of the DHFR from this organism with a propyl-bearing series member (RAB-propyl). In a companion apo structure, we identify four buried waters that act as placeholders for a conserved hydrogen-bonding network to the substrate and indicate an important role in protein stability during catalytic cycling. In these structures, the nicotinamide of the nicotinamide adenine dinucleotide phosphate cofactor is visualized outside of its binding pocket, which is exacerbated by RAB-propyl binding. Finally, homology models of the TMPR sequences dfrK and dfrF were constructed. While the dfrK-encoded protein shows clear sequence changes that would be detrimental to inhibitor binding, the dfrF-encoded protein model suggests the protein would be relatively unstable. These data suggest a utility for anti-DHFR compounds for treating infections arising from E. faecalis. They also highlight a role for water in stabilizing the DHFR substrate pocket and for competitive substrate inhibitors that may gain advantages in potency by the perturbation of cofactor dynamics. PMID:24495113

  15. The structure and competitive substrate inhibition of dihydrofolate reductase from Enterococcus faecalis reveal restrictions to cofactor docking.

    PubMed

    Bourne, Christina R; Wakeham, Nancy; Webb, Nicole; Nammalwar, Baskar; Bunce, Richard A; Berlin, K Darrell; Barrow, William W

    2014-02-25

    We are addressing bacterial resistance to antibiotics by repurposing a well-established classic antimicrobial target, the dihydrofolate reductase (DHFR) enzyme. In this work, we have focused on Enterococcus faecalis, a nosocomial pathogen that frequently harbors antibiotic resistance determinants leading to complicated and difficult-to-treat infections. An inhibitor series with a hydrophobic dihydrophthalazine heterocycle was designed from the anti-folate trimethoprim. We have examined the potency of this inhibitor series based on inhibition of DHFR enzyme activity and bacterial growth, including in the presence of the exogenous product analogue folinic acid. The resulting preferences were rationalized using a cocrystal structure of the DHFR from this organism with a propyl-bearing series member (RAB-propyl). In a companion apo structure, we identify four buried waters that act as placeholders for a conserved hydrogen-bonding network to the substrate and indicate an important role in protein stability during catalytic cycling. In these structures, the nicotinamide of the nicotinamide adenine dinucleotide phosphate cofactor is visualized outside of its binding pocket, which is exacerbated by RAB-propyl binding. Finally, homology models of the TMP(R) sequences dfrK and dfrF were constructed. While the dfrK-encoded protein shows clear sequence changes that would be detrimental to inhibitor binding, the dfrF-encoded protein model suggests the protein would be relatively unstable. These data suggest a utility for anti-DHFR compounds for treating infections arising from E. faecalis. They also highlight a role for water in stabilizing the DHFR substrate pocket and for competitive substrate inhibitors that may gain advantages in potency by the perturbation of cofactor dynamics.

  16. Structural Basis of Thermal Stability of the Tungsten Cofactor Synthesis Protein MoaB from Pyrococcus furiosus

    PubMed Central

    Havarushka, Nastassia; Fischer-Schrader, Katrin; Lamkemeyer, Tobias; Schwarz, Guenter

    2014-01-01

    Molybdenum and tungsten cofactors share a similar pterin-based scaffold, which hosts an ene-dithiolate function being essential for the coordination of either molybdenum or tungsten. The biosynthesis of both cofactors involves a multistep pathway, which ends with the activation of the metal binding pterin (MPT) by adenylylation before the respective metal is incorporated. In the hyperthermophilic organism Pyrococcus furiosus, the hexameric protein MoaB (PfuMoaB) has been shown to catalyse MPT-adenylylation. Here we determined the crystal structure of PfuMoaB at 2.5 Å resolution and identified key residues of α3-helix mediating hexamer formation. Given that PfuMoaB homologues from mesophilic organisms form trimers, we investigated the impact on PfuMoaB hexamerization on thermal stability and activity. Using structure-guided mutagenesis, we successfully disrupted the hexamer interface in PfuMoaB. The resulting PfuMoaB-H3 variant formed monomers, dimers and trimers as determined by size exclusion chromatography. Circular dichroism spectroscopy as well as chemical cross-linking coupled to mass spectrometry confirmed a wild-type-like fold of the protomers as well as inter-subunits contacts. The melting temperature of PfuMoaB-H3 was found to be reduced by more than 15°C as determined by differential scanning calorimetry, thus demonstrating hexamerization as key determinant for PfuMoaB thermal stability. Remarkably, while a loss of activity at temperatures higher than 50°C was observed in the PfuMoaB-H3 variant, at lower temperatures, we determined a significantly increased catalytic activity. The latter suggests a gain in conformational flexibility caused by the disruption of the hexamerization interface. PMID:24465852

  17. Molybdenum cofactor deficiency causes translucent integument, male-biased lethality, and flaccid paralysis in the silkworm Bombyx mori.

    PubMed

    Fujii, Tsuguru; Yamamoto, Kimiko; Banno, Yutaka

    2016-06-01

    Uric acid accumulates in the epidermis of Bombyx mori larvae and renders the larval integument opaque and white. Yamamoto translucent (oya) is a novel spontaneous mutant with a translucent larval integument and unique phenotypic characteristics, such as male-biased lethality and flaccid larval paralysis. Xanthine dehydrogenase (XDH) that requires a molybdenum cofactor (MoCo) for its activity is a key enzyme for uric acid synthesis. It has been observed that injection of a bovine xanthine oxidase, which corresponds functionally to XDH and contains its own MoCo activity, changes the integuments of oya mutants from translucent to opaque and white. This finding suggests that XDH/MoCo activity might be defective in oya mutants. Our linkage analysis identified an association between the oya locus and chromosome 23. Because XDH is not linked to chromosome 23 in B. mori, MoCo appears to be defective in oya mutants. In eukaryotes, MoCo is synthesized by a conserved biosynthesis pathway governed by four loci (MOCS1, MOCS2, MOCS3, and GEPH). Through a candidate gene approach followed by sequence analysis, a 6-bp deletion was detected in an exon of the B. mori molybdenum cofactor synthesis-step 1 gene (BmMOCS1) in the oya strain. Moreover, recombination was not observed between the oya and BmMOCS1 loci. These results indicate that the BmMOCS1 locus is responsible for the oya locus. Finally, we discuss the potential cause of male-biased lethality and flaccid paralysis observed in the oya mutants.

  18. Cell-free activation of phagocyte NADPH-oxidase: tissue and differentiation-specific expression of cytosolic cofactor activity.

    PubMed

    Parkinson, J F; Akard, L P; Schell, M J; Gabig, T G

    1987-06-30

    We examined a variety of tissues for the presence of cytosolic cofactor activity that would support arachidonate-dependent cell-free activation of NADPH-oxidase in isolated human neutrophil membranes. Cofactor activity was not found in cytosol isolated from erythrocytes, lymphocytes, placenta, brain, liver, or the human promyelocytic leukemic cell line HL-60. Induction of differentiation in HL-60 cells led to expression of cytosolic cofactor activity. In dimethylsulphoxide-induced HL-60 cells the level of cytosolic cofactor activity was closely correlated with phorbol myristate acetate-stimulated whole cell superoxide production. These results strongly suggest that the cytosolic cofactor is a phagocyte-specific regulatory protein of physiologic importance in NADPH-oxidase activation.

  19. Radical S-Adenosyl-L-methionine Chemistry in the Synthesis of Hydrogenase and Nitrogenase Metal Cofactors

    SciTech Connect

    Byer, Amanda S.; Shepard, Eric M.; Peters, John W.; Broderick, Joan B.

    2014-12-04

    Nitrogenase, [FeFe]-hydrogenase, and [Fe]-hydrogenase enzymes perform catalysis at metal cofactors with biologically unusual non-protein ligands. Furthermore, the FeMo cofactor of nitrogenase has a MoFe7S9 cluster with a central carbon, whereas the H-cluster of [FeFe]-hydrogenase contains a 2Fe subcluster coordinated by cyanide and CO ligands as well as dithiomethylamine; the [Fe]-hydrogenase cofactor has CO and guanylylpyridinol ligands at a mononuclear iron site. Intriguingly, radical S-adenosyl-L-methionine enzymes are vital for the assembly of all three of these diverse cofactors. Here, in this minireview, we present and discuss the current state of knowledge of the radical S-adenosylmethionine enzymes required for synthesis of these remarkable metal cofactors.

  20. Cellular disulfide bond formation in bioactive peptides and proteins.

    PubMed

    Patil, Nitin A; Tailhades, Julien; Hughes, Richard Anthony; Separovic, Frances; Wade, John D; Hossain, Mohammed Akhter

    2015-01-14

    Bioactive peptides play important roles in metabolic regulation and modulation and many are used as therapeutics. These peptides often possess disulfide bonds, which are important for their structure, function and stability. A systematic network of enzymes--a disulfide bond generating enzyme, a disulfide bond donor enzyme and a redox cofactor--that function inside the cell dictates the formation and maintenance of disulfide bonds. The main pathways that catalyze disulfide bond formation in peptides and proteins in prokaryotes and eukaryotes are remarkably similar and share several mechanistic features. This review summarizes the formation of disulfide bonds in peptides and proteins by cellular and recombinant machinery.

  1. Inhibitors – cellular aspects and novel approaches for tolerance

    PubMed Central

    SCOTT, D. W.

    2014-01-01

    Summary The immune response against therapeutic clotting factors VIII and IX (FVIII and FIX) is a major adverse event that can effectively thwart their effectiveness in correcting bleeding disorders. Thus, a significant number of haemophilia patients form antibodies, called inhibitors, which neutralize the procoagulant functions of therapeutic cofactors FVIII (haemophilia A) or FIX (haemophilia B). Understanding the cellular and molecular aspects of inhibitor formation is critical to designing tolerogenic therapies for clinical use. This review will focus on the basis of the immune response to FVIII, in particular, and will discuss emerging efforts to not only reduce immunogenicity but also to prevent and/or reverse inhibitor formation. PMID:24762281

  2. Targeted cofactor quantification in metabolically engineered E. coli using solid phase extraction and hydrophilic interaction liquid chromatography-mass spectrometry.

    PubMed

    Li, Zhucui; Yang, Afang; Li, Yujing; Liu, Pingping; Zhang, Zhidan; Zhang, Xueli; Shui, Wenqing

    2016-03-01

    Quantification of energy and redox cofactors is of great value to synthetic biologists to infer the balance of energy metabolism in engineered microbial strains and assess each strain's potential for further improvement. Most currently used methods for intracellular cofactor measurement suffer from incomplete coverage, low reproducibility, suboptimal sensitivity or specificity. In this study, we described an SPE-HILIC/MS approach for simultaneous determination of six cofactor targets (ATP, ADP, NAD, NADH, NADP, NADPH) in Escherichia coli cells. Sufficient linearity, precision and metabolite recoveries of this new approach justified its reliability in targeted cofactor quantification. Our approach was then compared with conventional enzymatic assays to demonstrate its superior performance. We applied the SPE-HILIC/MS approach to profile shift of cofactor balances in several engineered E. coli strains with varying isobutanol production. Our cofactor analysis clearly revealed that optimal energy fitness was achieved in the highest-yield strain through combined modulation of a transhydrogenase and a NAD(+) kinase. Apart from the targeted cofactors, the SPE enrichment procedure also allowed for confident identification of 39 groups of polar metabolites mainly involved in central carbon metabolism in E. coli cells. Copyright © 2016. Published by Elsevier B.V.

  3. A transcriptional cofactor YAP regulates IFNT expression via transcription factor TEAD in bovine conceptuses.

    PubMed

    Kusama, K; Bai, R; Sakurai, T; Bai, H; Ideta, A; Aoyagi, Y; Imakawa, K

    2016-10-01

    Interferon tau (IFNT) is the pregnancy recognition protein in all ruminants, and its expression is restricted to trophoblast cells. Interferon tau production increases as the conceptus elongates; however, its expression is downregulated soon after the initiation of conceptus attachment to the uterine epithelium. Our previous study identified that among 8 bovine IFNT genes, only 2 forms of IFNTs, IFNT2 and IFN-tau-c1, were expressed by the conceptuses during the periattachment period. To characterize whether Hippo signaling including a transcription cofactor yes-associated protein (YAP) was involved in the IFNT regulation, we examined the expression and effects of YAP and/or TEAD in human choriocarcinoma JEG3 and bovine trophoblast CT-1 cells, and in bovine conceptuses obtained from day 17, 20 or 22 pregnant animals (pregnant day 19.5 = day of conceptus attachment to the endometrium). YAP was expressed in bovine conceptuses and transfection of YAP or TEAD4, a transcription factor partner of YAP, expression plasmid increased the luciferase activity of IFNT2 and IFN-tau-c1 reporter plasmids in JEG3 cells. In the presence of YAP expression plasmid, TEAD2 or TEAD4 expression plasmid further upregulated transcriptional activity of IFNT2 or IFN-tau-c1 constructs, which were substantially reduced in the absence of the TEAD-binding site on IFNT2 or IFN-tau-c1 promoter region in JEG3 cells. In CT-1 cells, treatment with TEAD2, TEAD4, or YAP small-interfering RNA downregulated endogenous IFNT expression. It should be noted that TEAD2 and TEAD4 were predominantly localized in the nuclei of trophectoderm of Day 17 conceptuses, but nuclear localization appeared to be lower in those cells of conceptuses on days 20 and 22 of pregnancy. Moreover, the binding of TEAD4 to the TEAD-binding site of the IFN-tau-c1 promoter region in day 17 conceptuses was less in day 20 and 22 conceptuses. Furthermore, the level of YAP phosphorylation increased in day 20 and 22 conceptuses. These

  4. Ligand binding to the FeMo-cofactor: structures of CO-bound and reactivated nitrogenase

    PubMed Central

    Spatzal, Thomas; Perez, Kathryn A.; Einsle, Oliver; Howard, James B.; Rees, Douglas C.

    2014-01-01

    The mechanism of nitrogenase remains enigmatic, with a major unresolved issue concerning how inhibitors and substrates bind to the active site. We report a crystal structure of carbon monoxide (CO) inhibited nitrogenase MoFe-protein at 1.50 Å resolution, revealing a CO molecule bridging Fe2 and Fe6 of the FeMo-cofactor. The μ2 binding geometry is achieved by replacing a belt-sulfur atom (S2B) and highlights the generation of a reactive iron species uncovered by the displacement of sulfur. The CO inhibition is fully reversible as established by regain of enzyme activity and reappearance of S2B in the 1.43 Å resolution structure of the reactivated enzyme. The substantial and reversible reorganization of the FeMo-cofactor accompanying CO binding was unanticipated and provides insights into a catalytically competent state of nitrogenase. PMID:25258081

  5. Ca cofactor of the water-oxidation complex: Evidence for a Mn/Ca heteronuclear cluster

    SciTech Connect

    Cinco, Roehl M.; Robblee, John H.; Messinger, Johannes; Fernandez, Carmen; McFarlane, Karen L.; Pizarro, Shelly A.; Sauer, Ken; Yachandra, Vittal K.

    2001-07-25

    Calcium and chloride are necessary cofactors for the proper function of the oxygen-evolving complex (OEC) of Photosystem II (PS II). Located in the thylakoid membranes of green plants, cyanobacteria and algae, PS II and the OEC catalyze the light-driven oxidation of water into dioxygen (released into the biosphere), protons and electrons for carbon fixation. The actual chemistry of water oxidation is performed by a cluster of four manganese atoms, along with the requisite cofactors Ca{sup 2+} and Cl{sup -}. While the Mn complex has been extensively studied by X-ray absorption techniques, comparatively less is known about the Ca{sup 2+} cofactor. The fewer number of studies on the Ca{sup 2+} cofactor have sometimes relied on substituting the native cofactor with strontium or other metals, and have stirred some debate about the structure of the binding site. past efforts using Mn EXAFS on Sr-substituted PSII are suggestive of a close link between the Mn cluster and Sr, within 3.5 {angstrom}. The most recent published study using Sr EXAFS on similar samples confirms this finding of a 3.5 {angstrom} distance between Mn and Sr. This finding was base3d on a second Fourier peak (R {approx} 3 {angstrom}) in the Sr EXAFS from functional samples, but is absent from inactive, hydroxylamine-treated PS II. This Fourier peak II was found to fit best to two Mn at 3.5 {angstrom} rather than lighter atoms (carbon). Nevertheless, other experiments have given contrary results. They wanted to extend the technique by using polarized Sr EXAFS on layered Sr-substituted samples, to provide important angle information. Polarized EXAFS involves collecting spectra for different incident angles ({theta}) between the membrane normal of the layered sample and the X-ray electric field vector. Dichroism in the EXAFS can occur, depending on how the particular absorber-backscatterer (A-B) vector is aligned with the electric field. Through analysis of the dichroism, they extract the average number

  6. Structure of a bacterial microcompartment shell protein bound to a cobalamin cofactor

    PubMed Central

    Thompson, Michael C.; Crowley, Christopher S.; Kopstein, Jeffrey; Bobik, Thomas A.; Yeates, Todd O.

    2014-01-01

    The EutL shell protein is a key component of the ethanolamine-utilization microcompartment, which serves to compartmentalize ethanolamine degradation in diverse bacteria. The apparent function of this shell protein is to facilitate the selective diffusion of large cofactor molecules between the cytoplasm and the lumen of the microcompartment. While EutL is implicated in molecular-transport phenomena, the details of its function, including the identity of its transport substrate, remain unknown. Here, the 2.1 Å resolution X-ray crystal structure of a EutL shell protein bound to cobalamin (vitamin B12) is presented and the potential relevance of the observed protein–ligand interaction is briefly discussed. This work represents the first structure of a bacterial microcompartment shell protein bound to a potentially relevant cofactor molecule. PMID:25484204

  7. Menaquinone-7 Is Specific Cofactor in Tetraheme Quinol Dehydrogenase CymA

    PubMed Central

    McMillan, Duncan G. G.; Marritt, Sophie J.; Butt, Julea N.; Jeuken, Lars J. C.

    2012-01-01

    Little is known about enzymatic quinone-quinol interconversions in the lipid membrane when compared with our knowledge of substrate transformations by globular enzymes. Here, the smallest example of a quinol dehydrogenase in nature, CymA, has been studied. CymA is a monotopic membrane tetraheme c-type cytochrome belonging to the NapC/NirT family and central to anaerobic respiration in Shewanella sp. Using protein-film electrochemistry, it is shown that vesicle-bound menaquinone-7 is not only a substrate for this enzyme but is also required as a cofactor when converting other quinones. Here, we propose that the high concentration of quinones in the membrane negates the evolutionary pressure to create a high affinity active site. However, the instability and reactivity of reaction intermediate, semiquinone, might require a cofactor that functions to minimize damaging side reactions. PMID:22393052

  8. The History of the Discovery of the Molybdenum Cofactor and Novel Aspects of its Biosynthesis in Bacteria.

    PubMed

    Leimkühler, Silke; Wuebbens, Margot M; Rajagopalan, K V

    2011-05-01

    Biosynthesis of the molybdenum cofactor in bacteria is described with a detailed analysis of each individual reaction leading to the formation of stable intermediates during the synthesis of molybdopterin from GTP. As a starting point, the discovery of molybdopterin and the elucidation of its structure through the study of stable degradation products are described. Subsequent to molybdopterin synthesis, the molybdenum atom is added to the molybdopterin dithiolene group to form the molybdenum cofactor. This cofactor is either inserted directly into specific molybdoenzymes or is further modified by the addition of nucleotides to the molybdopterin phosphate group or the replacement of ligands at the molybdenum center.

  9. The History of the Discovery of the Molybdenum Cofactor and Novel Aspects of its Biosynthesis in Bacteria

    PubMed Central

    Leimkühler, Silke; Wuebbens, Margot M.; Rajagopalan, K.V.

    2010-01-01

    Biosynthesis of the molybdenum cofactor in bacteria is described with a detailed analysis of each individual reaction leading to the formation of stable intermediates during the synthesis of molybdopterin from GTP. As a starting point, the discovery of molybdopterin and the elucidation of its structure through the study of stable degradation products are described. Subsequent to molybdopterin synthesis, the molybdenum atom is added to the molybdopterin dithiolene group to form the molybdenum cofactor. This cofactor is either inserted directly into specific molybdoenzymes or is further modified by the addition of nucleotides to the molybdopterin phosphate group or the replacement of ligands at the molybdenum center. PMID:21528011

  10. Potential role of Arabidopsis PHP as an accessory subunit of the PAF1 transcriptional cofactor.

    PubMed

    Park, Sunchung; Ek-Ramos, Maria Julissa; Oh, Sookyung; van Nocker, Steven

    2011-08-01

    Paf1C is a transcriptional cofactor that has been implicated in various transcription-associated mechanisms spanning initiation, elongation and RNA processing, and is important for multiple aspects of development in Arabidopsis. Our recent studies suggest Arabidopsis Paf1C is crucial for proper regulation of genes within H3K27me3-enriched chromatin, and that a protein named PHP may act as an accessory subunit of Paf1C that promotes this function.

  11. Co-Factor Binding Confers Substrate Specificity to Xylose Reductase from Debaryomyces hansenii

    PubMed Central

    Singh, Appu Kumar; Mondal, Alok K.; Kumaran, S.

    2012-01-01

    Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from Debaryomyces hansenii (DhXR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. DhXR reduces D-xylose with increased specificity and shows no activity towards “non-substrate” sugars like L-rhamnose. Interestingly, apo-DhXR binds to D-xylose and L-rhamnose with similar affinity (Kd∼5.0–10.0 mM). Crystal structure of apo-DhXR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-DhXR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4–5 fold molar excess. Comparison of Kd values with Km values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism. PMID:23049810

  12. Co-factor binding confers substrate specificity to xylose reductase from Debaryomyces hansenii.

    PubMed

    Biswas, Dipanwita; Pandya, Vaibhav; Singh, Appu Kumar; Mondal, Alok K; Kumaran, S

    2012-01-01

    Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from Debaryomyces hansenii (DhXR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. DhXR reduces D-xylose with increased specificity and shows no activity towards "non-substrate" sugars like L-rhamnose. Interestingly, apo-DhXR binds to D-xylose and L-rhamnose with similar affinity (K(d)∼5.0-10.0 mM). Crystal structure of apo-DhXR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-DhXR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4-5 fold molar excess. Comparison of K(d) values with K(m) values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism.

  13. Regulation of Prp43-mediated disassembly of spliceosomes by its cofactors Ntr1 and Ntr2

    PubMed Central

    Fourmann, Jean-Baptiste; Tauchert, Marcel J.; Ficner, Ralf

    2017-01-01

    Abstract The DEAH-box NTPase Prp43 disassembles spliceosomes in co-operation with the cofactors Ntr1/Spp382 and Ntr2, forming the NTR complex. How Prp43 is regulated by its cofactors to discard selectively only intron-lariat spliceosomes (ILS) and defective spliceosomes and to prevent disassembly of earlier and properly assembled/wild-type spliceosomes remains unclear. First, we show that Ntr1΄s G-patch motif (Ntr1GP) can be replaced by the GP motif of Pfa1/Sqs1, a Prp43΄s cofactor in ribosome biogenesis, demonstrating that the specific function of Ntr1GP is to activate Prp43 for spliceosome disassembly and not to guide Prp43 to its binding site in the spliceosome. Furthermore, we show that Ntr1΄s C-terminal domain (CTD) plays a safeguarding role by preventing Prp43 from disrupting wild-type spliceosomes other than the ILS. Ntr1 and Ntr2 can also discriminate between wild-type and defective spliceosomes. In both type of spliceosomes, Ntr1-CTD impedes Prp43-mediated disassembly while the Ntr1GP promotes disassembly. Intriguingly, Ntr2 plays a specific role in defective spliceosomes, likely by stabilizing Ntr1 and allowing Prp43 to enter a productive interaction with the GP motif of Ntr1. Our data indicate that Ntr1 and Ntr2 act as ‘doorkeepers’ and suggest that both cofactors inspect the RNP structure of spliceosomal complexes thereby targeting suboptimal spliceosomes for Prp43-mediated disassembly. PMID:27923990

  14. Thermodynamic analysis of interactions between cofactor and neuronal nitric oxide synthase.

    PubMed

    Sanae, Ryuhei; Kurokawa, Fumiaki; Oda, Masayuki; Ishijima, Sumio; Sagami, Ikuko

    2011-03-15

    The thermodynamics of cofactor binding to the isolated reductase domain (Red) of nNOS and its mutants have been studied by isothermal titration calorimetry. The NADP(+) and 2',5'-ADP binding stoichiometry to Red were both 1:1, consistent with a one-site kinetic model instead of a two-site model. The binding constant (K(D) = 71 nM) and the large heat capacity change (ΔC(p) = -440 cal mol(-1) K(-1)) for 2',5'-ADP were remarkably different from those for NADP(+) (1.7 μM and -140 cal mol(-1) K(-1), respectively). These results indicate that the nicotinamide moiety as well as the adenosine moiety has an important role in binding to nNOS. They also suggest that the thermodynamics of the conformational change in Red caused by cofactor binding are significantly different from the conformational changes that occur in cytochrome c reductase, in which the nicotinamide moiety of the cofactor is not essential for binding. Analysis of the deletion mutant of the autoinhibitory helix (RedΔ40) revealed that the deletion resulted in a decrease in the binding affinity of 2',5'-ADP with more unfavorable enthalpy gain. In the case of RedCaM, which contains a calmodulin (CaM) binding site, the presence of Ca(2+)/CaM caused a 6.7-fold increase in the binding affinity for 2',5'-ADP that was mostly due to the favorable entropy change. These results are consistent with a model in which Ca(2+)/CaM induces a conformational change in NOS to a flexible "open" form from a "closed" form that locked by cofactor binding, and this change facilitates the electron transfer required for catalysis.

  15. Potential role of Arabidopsis PHP as an accessory subunit of the PAF1 transcriptional cofactor

    PubMed Central

    Park, Sunchung; Ek-Ramos, Maria Julissa; Oh, Sookyung

    2011-01-01

    Paf1C is a transcriptional cofactor that has been implicated in various transcription-associated mechanisms spanning initiation, elongation and RNA processing, and is important for multiple aspects of development in Arabidopsis. Our recent studies suggest Arabidopsis Paf1C is crucial for proper regulation of genes within H3K27me3-enriched chromatin, and that a protein named PHP may act as an accessory subunit of Paf1C that promotes this function. PMID:21720211

  16. Carbon dioxide fixation as a central redox cofactor recycling mechanism in bacteria.

    PubMed

    McKinlay, James B; Harwood, Caroline S

    2010-06-29

    The Calvin-Benson-Bassham cycle (Calvin cycle) catalyzes virtually all primary productivity on Earth and is the major sink for atmospheric CO(2). A less appreciated function of CO(2) fixation is as an electron-accepting process. It is known that anoxygenic phototrophic bacteria require the Calvin cycle to accept electrons when growing with light as their sole energy source and organic substrates as their sole carbon source. However, it was unclear why and to what extent CO(2) fixation is required when the organic substrates are more oxidized than biomass. To address these questions we measured metabolic fluxes in the photosynthetic bacterium Rhodopseudomonas palustris grown with (13)C-labeled acetate. R. palustris metabolized 22% of acetate provided to CO(2) and then fixed 68% of this CO(2) into cell material using the Calvin cycle. This Calvin cycle flux enabled R. palustris to reoxidize nearly half of the reduced cofactors generated during conversion of acetate to biomass, revealing that CO(2) fixation plays a major role in cofactor recycling. When H(2) production via nitrogenase was used as an alternative cofactor recycling mechanism, a similar amount of CO(2) was released from acetate, but only 12% of it was reassimilated by the Calvin cycle. These results underscore that N(2) fixation and CO(2) fixation have electron-accepting roles separate from their better-known roles in ammonia production and biomass generation. Some nonphotosynthetic heterotrophic bacteria have Calvin cycle genes, and their potential to use CO(2) fixation to recycle reduced cofactors deserves closer scrutiny.

  17. Carbon dioxide fixation as a central redox cofactor recycling mechanism in bacteria

    PubMed Central

    McKinlay, James B.; Harwood, Caroline S.

    2010-01-01

    The Calvin-Benson-Bassham cycle (Calvin cycle) catalyzes virtually all primary productivity on Earth and is the major sink for atmospheric CO2. A less appreciated function of CO2 fixation is as an electron-accepting process. It is known that anoxygenic phototrophic bacteria require the Calvin cycle to accept electrons when growing with light as their sole energy source and organic substrates as their sole carbon source. However, it was unclear why and to what extent CO2 fixation is required when the organic substrates are more oxidized than biomass. To address these questions we measured metabolic fluxes in the photosynthetic bacterium Rhodopseudomonas palustris grown with 13C-labeled acetate. R. palustris metabolized 22% of acetate provided to CO2 and then fixed 68% of this CO2 into cell material using the Calvin cycle. This Calvin cycle flux enabled R. palustris to reoxidize nearly half of the reduced cofactors generated during conversion of acetate to biomass, revealing that CO2 fixation plays a major role in cofactor recycling. When H2 production via nitrogenase was used as an alternative cofactor recycling mechanism, a similar amount of CO2 was released from acetate, but only 12% of it was reassimilated by the Calvin cycle. These results underscore that N2 fixation and CO2 fixation have electron-accepting roles separate from their better-known roles in ammonia production and biomass generation. Some nonphotosynthetic heterotrophic bacteria have Calvin cycle genes, and their potential to use CO2 fixation to recycle reduced cofactors deserves closer scrutiny. PMID:20558750

  18. Stress and Activity of Molybdenum-Containing Complex (Molybdenum Cofactor) in Winter Wheat Seeds

    PubMed Central

    Vunkova-Radeva, Reneta; Schiemann, Johan; Mendel, Ralf-Reiner; Salcheva, Galitona; Georgieva, Damyana

    1988-01-01

    Molybdenum, applied in vivo, restored the damage from low temperature with winter wheat (Triticum aestivum, var “Sadovo 1”) grown on acid soil and, in addition, sharply increased productivity (G Salcheva, D Georgieva, 1982; G Salcheva et al., 1977, 1979). Two fractions with molybdenum-cofactor activity in seeds were detected. One of them has a molecular weight of about 230 kilodaltons corresponding to xanthine oxidase activity and leaf nitrate reductase activity. The other has a molecular weight of about 60 kilodaltons. The ratio between the molybdenum-cofactor activity of these fractions was different in `mother' seeds used in the experiment, in seeds obtained from the damaged plants, and in seeds obtained from the damaged plants restored by in vivo molybdenum addition. Every one of these fractions consisted of several components in which molybdenum-cofactor activity and stability in vitro was different. We suggest that plants store molybdenum as molybdenum carriers in these low molecular weight fractions. Images Fig. 2 PMID:16666178

  19. Substrate Recognition and Catalysis by the Cofactor-Independent Dioxygenase DpgC+

    SciTech Connect

    Fielding,E.; Widboom, P.; Bruner, S.

    2007-01-01

    The enzyme DpgC belongs to a small class of oxygenases not dependent on accessory cofactors for activity. DpgC is in the biosynthetic pathway for the nonproteinogenic amino acid 3, 5-dihydroxyphenylglycine in actinomycetes bacteria responsible for the production of the vancomycin/teicoplanin family of antibiotic natural products. The X-ray structure of DpgC confirmed the absence of cofactors and defined a novel hydrophobic dioxygen binding pocket adjacent to a bound substrate analogue. In this paper, the role specific amino acids play in substrate recognition and catalysis is examined through biochemical and structural characterization of site-specific enzyme mutations and alternate substrates. The results establish the importance of three amino acids, Arg254, Glu299, and Glu189, in the chemistry of DpgC. Arg254 and Glu189 join to form a specific contact with one of the phenolic hydroxyls of the substrate, and this interaction plays a key role in both substrate recognition and catalysis. The X-ray crystal structure of Arg254Lys was determined to address the role this residue plays in the chemistry. In addition, characterization of alternate substrate analogues demonstrates the presence and position of phenol groups are necessary for both enzyme recognition and downstream oxidation chemistry. Overall, this work defines the mechanism of substrate recognition and specificity by the cofactor-independent dioxygenase DpgC.

  20. A network analysis of cofactor-protein interactions for analyzing associations between human nutrition and diseases

    PubMed Central

    Scott-Boyer, Marie Pier; Lacroix, Sébastien; Scotti, Marco; Morine, Melissa J.; Kaput, Jim; Priami, Corrado

    2016-01-01

    The involvement of vitamins and other micronutrients in intermediary metabolism was elucidated in the mid 1900’s at the level of individual biochemical reactions. Biochemical pathways remain the foundational knowledgebase for understanding how micronutrient adequacy modulates health in all life stages. Current daily recommended intakes were usually established on the basis of the association of a single nutrient to a single, most sensitive adverse effect and thus neglect interdependent and pleiotropic effects of micronutrients on biological systems. Hence, the understanding of the impact of overt or sub-clinical nutrient deficiencies on biological processes remains incomplete. Developing a more complete view of the role of micronutrients and their metabolic products in protein-mediated reactions is of importance. We thus integrated and represented cofactor-protein interaction data from multiple and diverse sources into a multi-layer network representation that links cofactors, cofactor-interacting proteins, biological processes, and diseases. Network representation of this information is a key feature of the present analysis and enables the integration of data from individual biochemical reactions and protein-protein interactions into a systems view, which may guide strategies for targeted nutritional interventions aimed at improving health and preventing diseases. PMID:26777674

  1. The nitrogenase FeMo-cofactor and P-cluster pair: 2. 2 [Angstrom] resolution structures

    SciTech Connect

    Chan, M.K.; Jongsun Kim; Rees, D.C. )

    1993-05-07

    Structures recently proposed for the FeMo-cofactor and P-cluster pair of the nitrogenase molybdenum-iron (MoFe)-protein from Azotobacter vinelandii have been crystallographically verified at 2.2 angstrom resolution. Significantly, no hexacoordinate sulfur atoms are observed in either type of metal center. Consequently, the six bridged iron atoms in the FeMo-cofactor are trigonally coordinated by nonprotein ligands, although there may be some iron-iron bonding interactions that could provide a fourth coordination interaction for these sites. Two of the cluster sulfurs in the P-cluster pair are very close together ([approximately]2.1 angstroms), indicating that they form a disulfide bond. These findings indicate that a cavity exists in the interior of the FeMo-cofactor that could be involved in substrate binding and suggest that redox reactions at the P-cluster pair may be linked to transitions of two cluster-bound sulfurs between disulfide and sulfide oxidation states. 12 refs., 3 figs.

  2. Substrate recognition and catalysis by the cofactor-independent dioxygenase DpgC.

    PubMed

    Fielding, Elisha N; Widboom, Paul F; Bruner, Steven D

    2007-12-11

    The enzyme DpgC belongs to a small class of oxygenases not dependent on accessory cofactors for activity. DpgC is in the biosynthetic pathway for the nonproteinogenic amino acid 3,5-dihydroxyphenylglycine in actinomycetes bacteria responsible for the production of the vancomycin/teicoplanin family of antibiotic natural products. The X-ray structure of DpgC [Widboom, P. W., Fielding, E. N., Liu, Y., and Bruner, S. D. (2007) Nature 447, 342-345] confirmed the absence of cofactors and defined a novel hydrophobic dioxygen binding pocket adjacent to a bound substrate analogue. In this paper, the role specific amino acids play in substrate recognition and catalysis is examined through biochemical and structural characterization of site-specific enzyme mutations and alternate substrates. The results establish the importance of three amino acids, Arg254, Glu299, and Glu189, in the chemistry of DpgC. Arg254 and Glu189 join to form a specific contact with one of the phenolic hydroxyls of the substrate, and this interaction plays a key role in both substrate recognition and catalysis. The X-ray crystal structure of Arg254Lys was determined to address the role this residue plays in the chemistry. In addition, characterization of alternate substrate analogues demonstrates the presence and position of phenol groups are necessary for both enzyme recognition and downstream oxidation chemistry. Overall, this work defines the mechanism of substrate recognition and specificity by the cofactor-independent dioxygenase DpgC.

  3. A Live Zebrafish-Based Screening System for Human Nuclear Receptor Ligand and Cofactor Discovery

    PubMed Central

    Tiefenbach, Jens; Moll, Pamela R.; Nelson, Meryl R.; Hu, Chun; Baev, Lilia; Kislinger, Thomas; Krause, Henry M.

    2010-01-01

    Nuclear receptors (NRs) belong to a superfamily of transcription factors that regulate numerous homeostatic, metabolic and reproductive processes. Taken together with their modulation by small lipophilic molecules, they also represent an important and successful class of drug targets. Although many NRs have been targeted successfully, the majority have not, and one third are still orphans. Here we report the development of an in vivo GFP-based reporter system suitable for monitoring NR activities in all cells and tissues using live zebrafish (Danio rerio). The human NR fusion proteins used also contain a new affinity tag cassette allowing the purification of receptors with bound molecules from responsive tissues. We show that these constructs 1) respond as expected to endogenous zebrafish hormones and cofactors, 2) facilitate efficient receptor and cofactor purification, 3) respond robustly to NR hormones and drugs and 4) yield readily quantifiable signals. Transgenic lines representing the majority of human NRs have been established and are available for the investigation of tissue- and isoform-specific ligands and cofactors. PMID:20339547

  4. Rapid X-ray Photoreduction of Dimetal-Oxygen Cofactors in Ribonucleotide Reductase

    PubMed Central

    Sigfridsson, Kajsa G. V.; Chernev, Petko; Leidel, Nils; Popović-Bijelić, Ana; Gräslund, Astrid; Haumann, Michael

    2013-01-01

    Prototypic dinuclear metal cofactors with varying metallation constitute a class of O2-activating catalysts in numerous enzymes such as ribonucleotide reductase. Reliable structures are required to unravel the reaction mechanisms. However, protein crystallography data may be compromised by x-ray photoreduction (XRP). We studied XPR of Fe(III)Fe(III) and Mn(III)Fe(III) sites in the R2 subunit of Chlamydia trachomatis ribonucleotide reductase using x-ray absorption spectroscopy. Rapid and biphasic x-ray photoreduction kinetics at 20 and 80 K for both cofactor types suggested sequential formation of (III,II) and (II,II) species and similar redox potentials of iron and manganese sites. Comparing with typical x-ray doses in crystallography implies that (II,II) states are reached in <1 s in such studies. First-sphere metal coordination and metal-metal distances differed after chemical reduction at room temperature and after XPR at cryogenic temperatures, as corroborated by model structures from density functional theory calculations. The inter-metal distances in the XPR-induced (II,II) states, however, are similar to R2 crystal structures. Therefore, crystal data of initially oxidized R2-type proteins mostly contain photoreduced (II,II) cofactors, which deviate from the native structures functional in O2 activation, explaining observed variable metal ligation motifs. This situation may be remedied by novel femtosecond free electron-laser protein crystallography techniques. PMID:23400774

  5. A network analysis of cofactor-protein interactions for analyzing associations between human nutrition and diseases.

    PubMed

    Scott-Boyer, Marie Pier; Lacroix, Sébastien; Scotti, Marco; Morine, Melissa J; Kaput, Jim; Priami, Corrado

    2016-01-18

    The involvement of vitamins and other micronutrients in intermediary metabolism was elucidated in the mid 1900's at the level of individual biochemical reactions. Biochemical pathways remain the foundational knowledgebase for understanding how micronutrient adequacy modulates health in all life stages. Current daily recommended intakes were usually established on the basis of the association of a single nutrient to a single, most sensitive adverse effect and thus neglect interdependent and pleiotropic effects of micronutrients on biological systems. Hence, the understanding of the impact of overt or sub-clinical nutrient deficiencies on biological processes remains incomplete. Developing a more complete view of the role of micronutrients and their metabolic products in protein-mediated reactions is of importance. We thus integrated and represented cofactor-protein interaction data from multiple and diverse sources into a multi-layer network representation that links cofactors, cofactor-interacting proteins, biological processes, and diseases. Network representation of this information is a key feature of the present analysis and enables the integration of data from individual biochemical reactions and protein-protein interactions into a systems view, which may guide strategies for targeted nutritional interventions aimed at improving health and preventing diseases.

  6. Studies on Escherichia coli RNase P RNA with Zn2+ as the catalytic cofactor

    PubMed Central

    Cuzic, Simona; Hartmann, Roland K.

    2005-01-01

    We demonstrate, for the first time, catalysis by Escherichia coli ribonuclease P (RNase P) RNA with Zn2+ as the sole divalent metal ion cofactor in the presence of ammonium, but not sodium or potassium salts. Hill analysis suggests a role for two or more Zn2+ ions in catalysis. Whereas Zn2+ destabilizes substrate ground state binding to an extent that precludes reliable Kd determination, Co(NH3)63+ and Sr2+ in particular, both unable to support catalysis by themselves, promote high-substrate affinity. Zn2+ and Co(NH3)63+ substantially reduce the fraction of precursor tRNA molecules capable of binding to RNase P RNA. Stimulating and inhibitory effects of Sr2+ on the ribozyme reaction with Zn2+ as cofactor could be rationalized by a model involving two Sr2+ ions (or two classes of Sr2+ ions). Both ions improve substrate affinity in a cooperative manner, but one of the two inhibits substrate conversion in a non-competitive mode with respect to the substrate and the Zn2+. A single 2′-fluoro modification at nt −1 of the substrate substantially weakened the inhibitory effect of Sr2+. Our results demonstrate that the studies on RNase P RNA with metal cofactors other than Mg2+ entail complex effects on structural equilibria of ribozyme and substrate RNAs as well as E·S formation apart from the catalytic performance. PMID:15867194

  7. Biochemical Characterization of Molybdenum Cofactor-free Nitrate Reductase from Neurospora crassa*

    PubMed Central

    Ringel, Phillip; Krausze, Joern; van den Heuvel, Joop; Curth, Ute; Pierik, Antonio J.; Herzog, Stephanie; Mendel, Ralf R.; Kruse, Tobias

    2013-01-01

    Nitrate reductase (NR) is a complex molybdenum cofactor (Moco)-dependent homodimeric metalloenzyme that is vitally important for autotrophic organism as it catalyzes the first and rate-limiting step of nitrate assimilation. Beside Moco, eukaryotic NR also binds FAD and heme as additional redox active cofactors, and these are involved in electron transfer from NAD(P)H to the enzyme molybdenum center where reduction of nitrate to nitrite takes place. We report the first biochemical characterization of a Moco-free eukaryotic NR from the fungus Neurospora crassa, documenting that Moco is necessary and sufficient to induce dimer formation. The molybdenum center of NR reconstituted in vitro from apo-NR and Moco showed an EPR spectrum identical to holo-NR. Analysis of mutants unable to bind heme or FAD revealed that insertion of Moco into NR occurs independent from the insertion of any other NR redox cofactor. Furthermore, we showed that at least in vitro the active site formation of NR is an autonomous process. PMID:23539622

  8. Pyridoxine-dependent epilepsy with elevated urinary α-amino adipic semialdehyde in molybdenum cofactor deficiency.

    PubMed

    Struys, Eduard Alexander; Nota, Benjamin; Bakkali, Abdellatif; Al Shahwan, Saad; Salomons, Gajja Sophi; Tabarki, Brahim

    2012-12-01

    α-Amino adipic semialdehyde (α-AASA) accumulates in body fluids from patients with pyridoxine-dependent epilepsy because of mutations in antiquitin (ALDH7A1) and serves as the biomarker for this condition. We have recently found that the urinary excretion of α-AASA was also increased in molybdenum cofactor and sulfite oxidase deficiencies. The seizures in pyridoxine-dependent epilepsy are caused by lowered cerebral levels of pyridoxal-5-phosphate (PLP), the bioactive form of pyridoxine (vitamin B(6)), which can be corrected by the supplementation of pyridoxine. The nonenzymatic trapping of PLP by the cyclic form of α-AASA is causative for the lowered cerebral PLP levels. We describe 2 siblings with clinically evident pyridoxine-responsive seizures associated with increased urinary excretion of α-AASA. Subsequent metabolic investigations revealed several metabolic abnormities, all indicative for molybdenum cofactor deficiency. Molecular investigations indeed revealed a known homozygous mutation in the MOCS2 gene. Based upon the clinically evident pyridoxine-responsive seizures in these 2 siblings, we recommend considering pyridoxine supplementation to patients affected with molybdenum cofactor or sulfite oxidase deficiencies.

  9. Directing electron transfer within Photosystem I by breaking H-bonds in the cofactor branches

    PubMed Central

    Li, Yajing; van der Est, Art; Lucas, Marie Gabrielle; Ramesh, V. M.; Gu, Feifei; Petrenko, Alexander; Lin, Su; Webber, Andrew N.; Rappaport, Fabrice; Redding, Kevin

    2006-01-01

    Photosystem I has two branches of cofactors down which light-driven electron transfer (ET) could potentially proceed, each consisting of a pair of chlorophylls (Chls) and a phylloquinone (PhQ). Forward ET from PhQ to the next ET cofactor (FX) is described by two kinetic components with decay times of ≈20 and ≈200 ns, which have been proposed to represent ET from PhQB and PhQA, respectively. Immediately preceding each quinone is a Chl (ec3), which receives a H-bond from a nearby tyrosine. To decrease the reduction potential of each of these Chls, and thus modify the relative yield of ET within the targeted branch, this H-bond was removed by conversion of each Tyr to Phe in the green alga Chlamydomonas reinhardtii. Together, transient optical absorption spectroscopy performed in vivo and transient electron paramagnetic resonance data from thylakoid membranes showed that the mutations affect the relative amplitudes, but not the lifetimes, of the two kinetic components representing ET from PhQ to FX. The mutation near ec3A increases the fraction of the faster component at the expense of the slower component, with the opposite effect seen in the ec3B mutant. We interpret this result as a decrease in the relative use of the targeted branch. This finding suggests that in Photosystem I, unlike type II reaction centers, the relative efficiency of the two branches is extremely sensitive to the energetics of the embedded redox cofactors. PMID:16467143

  10. Stepwise isotope editing of [FeFe]-hydrogenases exposes cofactor dynamics

    PubMed Central

    Senger, Moritz; Mebs, Stefan; Duan, Jifu; Wittkamp, Florian; Apfel, Ulf-Peter; Heberle, Joachim; Haumann, Michael; Stripp, Sven Timo

    2016-01-01

    The six-iron cofactor of [FeFe]-hydrogenases (H-cluster) is the most efficient H2-forming catalyst in nature. It comprises a diiron active site with three carbon monoxide (CO) and two cyanide (CN−) ligands in the active oxidized state (Hox) and one additional CO ligand in the inhibited state (Hox-CO). The diatomic ligands are sensitive reporter groups for structural changes of the cofactor. Their vibrational dynamics were monitored by real-time attenuated total reflection Fourier-transform infrared spectroscopy. Combination of 13CO gas exposure, blue or red light irradiation, and controlled hydration of three different [FeFe]-hydrogenase proteins produced 8 Hox and 16 Hox-CO species with all possible isotopic exchange patterns. Extensive density functional theory calculations revealed the vibrational mode couplings of the carbonyl ligands and uniquely assigned each infrared spectrum to a specific labeling pattern. For Hox-CO, agreement between experimental and calculated infrared frequencies improved by up to one order of magnitude for an apical CN− at the distal iron ion of the cofactor as opposed to an apical CO. For Hox, two equally probable isomers with partially rotated ligands were suggested. Interconversion between these structures implies dynamic ligand reorientation at the H-cluster. Our experimental protocol for site-selective 13CO isotope editing combined with computational species assignment opens new perspectives for characterization of functional intermediates in the catalytic cycle. PMID:27432985

  11. Cofactor specificity motifs and the induced fit mechanism in class I ketol-acid reductoisomerases.

    PubMed

    Cahn, Jackson K B; Brinkmann-Chen, Sabine; Spatzal, Thomas; Wiig, Jared A; Buller, Andrew R; Einsle, Oliver; Hu, Yilin; Ribbe, Markus W; Arnold, Frances H

    2015-06-15

    Although most sequenced members of the industrially important ketol-acid reductoisomerase (KARI) family are class I enzymes, structural studies to date have focused primarily on the class II KARIs, which arose through domain duplication. In the present study, we present five new crystal structures of class I KARIs. These include the first structure of a KARI with a six-residue β2αB (cofactor specificity determining) loop and an NADPH phosphate-binding geometry distinct from that of the seven- and 12-residue loops. We also present the first structures of naturally occurring KARIs that utilize NADH as cofactor. These results show insertions in the specificity loops that confounded previous attempts to classify them according to loop length. Lastly, we explore the conformational changes that occur in class I KARIs upon binding of cofactor and metal ions. The class I KARI structures indicate that the active sites close upon binding NAD(P)H, similar to what is observed in the class II KARIs of rice and spinach and different from the opening of the active site observed in the class II KARI of Escherichia coli. This conformational change involves a decrease in the bending of the helix that runs between the domains and a rearrangement of the nicotinamide-binding site. © The Authors Journal Compilation © 2015 Biochemical Society.

  12. Cofactor-free light-driven whole-cell cytochrome P450 catalysis.

    PubMed

    Park, Jong Hyun; Lee, Sahng Ha; Cha, Gun Su; Choi, Da Som; Nam, Dong Heon; Lee, Jae Hyung; Lee, Jung-Kul; Yun, Chul-Ho; Jeong, Ki Jun; Park, Chan Beum

    2015-01-12

    Cytochromes P450 can catalyze various regioselective and stereospecific oxidation reactions of non-functionalized hydrocarbons. Here, we have designed a novel light-driven platform for cofactor-free, whole-cell P450 photo-biocatalysis using eosin Y (EY) as a photosensitizer. EY can easily enter into the cytoplasm of Escherichia coli and bind specifically to the heme domain of P450. The catalytic turnover of P450 was mediated through the direct transfer of photoinduced electrons from the photosensitized EY to the P450 heme domain under visible light illumination. The photoactivation of the P450 catalytic cycle in the absence of cofactors and redox partners is successfully conducted using many bacterial P450s (variants of P450 BM3) and human P450s (CYPs 1A1, 1A2, 1B1, 2A6, 2E1, and 3A4) for the bioconversion of different substrates, including marketed drugs (simvastatin, lovastatin, and omeprazole) and a steroid (17β-estradiol), to demonstrate the general applicability of the light-driven, cofactor-free system. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Directing electron transfer within Photosystem I by breaking H-bonds in the cofactor branches.

    PubMed

    Li, Yajing; van der Est, Art; Lucas, Marie Gabrielle; Ramesh, V M; Gu, Feifei; Petrenko, Alexander; Lin, Su; Webber, Andrew N; Rappaport, Fabrice; Redding, Kevin

    2006-02-14

    Photosystem I has two branches of cofactors down which light-driven electron transfer (ET) could potentially proceed, each consisting of a pair of chlorophylls (Chls) and a phylloquinone (PhQ). Forward ET from PhQ to the next ET cofactor (FX) is described by two kinetic components with decay times of approximately 20 and approximately 200 ns, which have been proposed to represent ET from PhQB and PhQA, respectively. Immediately preceding each quinone is a Chl (ec3), which receives a H-bond from a nearby tyrosine. To decrease the reduction potential of each of these Chls, and thus modify the relative yield of ET within the targeted branch, this H-bond was removed by conversion of each Tyr to Phe in the green alga Chlamydomonas reinhardtii. Together, transient optical absorption spectroscopy performed in vivo and transient electron paramagnetic resonance data from thylakoid membranes showed that the mutations affect the relative amplitudes, but not the lifetimes, of the two kinetic components representing ET from PhQ to F(X). The mutation near ec3A increases the fraction of the faster component at the expense of the slower component, with the opposite effect seen in the ec3B mutant. We interpret this result as a decrease in the relative use of the targeted branch. This finding suggests that in Photosystem I, unlike type II reaction centers, the relative efficiency of the two branches is extremely sensitive to the energetics of the embedded redox cofactors.

  14. Biochemical characterization of molybdenum cofactor-free nitrate reductase from Neurospora crassa.

    PubMed

    Ringel, Phillip; Krausze, Joern; van den Heuvel, Joop; Curth, Ute; Pierik, Antonio J; Herzog, Stephanie; Mendel, Ralf R; Kruse, Tobias

    2013-05-17

    Nitrate reductase (NR) is a complex molybdenum cofactor (Moco)-dependent homodimeric metalloenzyme that is vitally important for autotrophic organism as it catalyzes the first and rate-limiting step of nitrate assimilation. Beside Moco, eukaryotic NR also binds FAD and heme as additional redox active cofactors, and these are involved in electron transfer from NAD(P)H to the enzyme molybdenum center where reduction of nitrate to nitrite takes place. We report the first biochemical characterization of a Moco-free eukaryotic NR from the fungus Neurospora crassa, documenting that Moco is necessary and sufficient to induce dimer formation. The molybdenum center of NR reconstituted in vitro from apo-NR and Moco showed an EPR spectrum identical to holo-NR. Analysis of mutants unable to bind heme or FAD revealed that insertion of Moco into NR occurs independent from the insertion of any other NR redox cofactor. Furthermore, we showed that at least in vitro the active site formation of NR is an autonomous process.

  15. Stepwise isotope editing of [FeFe]-hydrogenases exposes cofactor dynamics.

    PubMed

    Senger, Moritz; Mebs, Stefan; Duan, Jifu; Wittkamp, Florian; Apfel, Ulf-Peter; Heberle, Joachim; Haumann, Michael; Stripp, Sven Timo

    2016-07-26

    The six-iron cofactor of [FeFe]-hydrogenases (H-cluster) is the most efficient H2-forming catalyst in nature. It comprises a diiron active site with three carbon monoxide (CO) and two cyanide (CN(-)) ligands in the active oxidized state (Hox) and one additional CO ligand in the inhibited state (Hox-CO). The diatomic ligands are sensitive reporter groups for structural changes of the cofactor. Their vibrational dynamics were monitored by real-time attenuated total reflection Fourier-transform infrared spectroscopy. Combination of (13)CO gas exposure, blue or red light irradiation, and controlled hydration of three different [FeFe]-hydrogenase proteins produced 8 Hox and 16 Hox-CO species with all possible isotopic exchange patterns. Extensive density functional theory calculations revealed the vibrational mode couplings of the carbonyl ligands and uniquely assigned each infrared spectrum to a specific labeling pattern. For Hox-CO, agreement between experimental and calculated infrared frequencies improved by up to one order of magnitude for an apical CN(-) at the distal iron ion of the cofactor as opposed to an apical CO. For Hox, two equally probable isomers with partially rotated ligands were suggested. Interconversion between these structures implies dynamic ligand reorientation at the H-cluster. Our experimental protocol for site-selective (13)CO isotope editing combined with computational species assignment opens new perspectives for characterization of functional intermediates in the catalytic cycle.

  16. Proline dehydrogenase from Thermus thermophilus does not discriminate between FAD and FMN as cofactor

    PubMed Central

    Huijbers, Mieke M. E.; Martínez-Júlvez, Marta; Westphal, Adrie H.; Delgado-Arciniega, Estela; Medina, Milagros; van Berkel, Willem J. H.

    2017-01-01

    Flavoenzymes are versatile biocatalysts containing either FAD or FMN as cofactor. FAD often binds to a Rossmann fold, while FMN prefers a TIM-barrel or flavodoxin-like fold. Proline dehydrogenase is denoted as an exception: it possesses a TIM barrel-like fold while binding FAD. Using a riboflavin auxotrophic Escherichia coli strain and maltose-binding protein as solubility tag, we produced the apoprotein of Thermus thermophilus ProDH (MBP-TtProDH). Remarkably, reconstitution with FAD or FMN revealed that MBP-TtProDH has no preference for either of the two prosthetic groups. Kinetic parameters of both holo forms are similar, as are the dissociation constants for FAD and FMN release. Furthermore, we show that the holo form of MBP-TtProDH, as produced in E. coli TOP10 cells, contains about three times more FMN than FAD. In line with this flavin content, the crystal structure of TtProDH variant ΔABC, which lacks helices αA, αB and αC, shows no electron density for an AMP moiety of the cofactor. To the best of our knowledge, this is the first example of a flavoenzyme that does not discriminate between FAD and FMN as cofactor. Therefore, classification of TtProDH as an FAD-binding enzyme should be reconsidered. PMID:28256579

  17. Activity of the Molybdopterin-Containing Xanthine Dehydrogenase of Rhodobacter capsulatus Can Be Restored by High Molybdenum Concentrations in a moeA Mutant Defective in Molybdenum Cofactor Biosynthesis

    PubMed Central

    Leimkühler, Silke; Angermüller, Sieglinde; Schwarz, Günter; Mendel, Ralf R.; Klipp, Werner

    1999-01-01

    During the screening for Rhodobacter capsulatus mutants defective in xanthine degradation, one Tn5 mutant which was able to grow with xanthine as a sole nitrogen source only in the presence of high molybdate concentrations (1 mM), a phenotype resembling Escherichia coli mogA mutants, was identified. Unexpectedly, the corresponding Tn5 insertion was located within the moeA gene. Partial DNA sequence analysis and interposon mutagenesis of regions flanking R. capsulatus moeA revealed that no further genes essential for molybdopterin biosynthesis are located in the vicinity of moeA and revealed that moeA forms a monocistronic transcriptional unit in R. capsulatus. Amino acid sequence alignments of R. capsulatus MoeA (414 amino acids [aa]) with E. coli MogA (195 aa) showed that MoeA contains an internal domain homologous to MogA, suggesting similar functions of these proteins in the biosynthesis of the molybdenum cofactor. Interposon mutants defective in moeA did not exhibit dimethyl sulfoxide reductase or nitrate reductase activity, which both require the molybdopterin guanine dinucleotide (MGD) cofactor, even after addition of 1 mM molybdate to the medium. In contrast, the activity of xanthine dehydrogenase, which binds the molybdopterin (MPT) cofactor, was restored to wild-type levels after the addition of 1 mM molybdate to the growth medium. Analysis of fluorescent derivatives of the molybdenum cofactor of purified xanthine dehydrogenase isolated from moeA and modA mutant strains, respectively, revealed that MPT is inserted into the enzyme only after molybdenum chelation, and both metal chelation and Mo-MPT insertion can occur only under high molybdate concentrations in the absence of MoeA. These data support a model for the biosynthesis of the molybdenum cofactor in which the biosynthesis of MPT and MGD are split at a stage when the molybdenum atom is added to MPT. PMID:10498704

  18. Cellular Phone Towers

    MedlinePlus

    ... the call. How are people exposed to the energy from cellular phone towers? As people use cell ... where people can be exposed to them. The energy from a cellular phone tower antenna, like that ...

  19. Redox-dependent substrate-cofactor interactions in the Michaelis-complex of a flavin-dependent oxidoreductase

    NASA Astrophysics Data System (ADS)

    Werther, Tobias; Wahlefeld, Stefan; Salewski, Johannes; Kuhlmann, Uwe; Zebger, Ingo; Hildebrandt, Peter; Dobbek, Holger

    2017-07-01

    How an enzyme activates its substrate for turnover is fundamental for catalysis but incompletely understood on a structural level. With redox enzymes one typically analyses structures of enzyme-substrate complexes in the unreactive oxidation state of the cofactor, assuming that the interaction between enzyme and substrate is independent of the cofactors oxidation state. Here, we investigate the Michaelis complex of the flavoenzyme xenobiotic reductase A with the reactive reduced cofactor bound to its substrates by X-ray crystallography and resonance Raman spectroscopy and compare it to the non-reactive oxidized Michaelis complex mimics. We find that substrates bind in different orientations to the oxidized and reduced flavin, in both cases flattening its structure. But only authentic Michaelis complexes display an unexpected rich vibrational band pattern uncovering a strong donor-acceptor complex between reduced flavin and substrate. This interaction likely activates the catalytic ground state of the reduced flavin, accelerating the reaction within a compressed cofactor-substrate complex.

  20. Systems biology analysis merging phenotype, metabolomic and genomic data identifies Non-SMC Condensin I Complex, Subunit G (NCAPG) and cellular maintenance processes as major contributors to genetic variability in bovine feed efficiency.

    PubMed

    Widmann, Philipp; Reverter, Antonio; Weikard, Rosemarie; Suhre, Karsten; Hammon, Harald M; Albrecht, Elke; Kuehn, Christa

    2015-01-01

    Feed efficiency is a paramount factor for livestock economy. Previous studies had indicated a substantial heritability of several feed efficiency traits. In our study, we investigated the genetic background of residual feed intake, a commonly used parameter of feed efficiency, in a cattle resource population generated from crossing dairy and beef cattle. Starting from a whole genome association analysis, we subsequently performed combined phenotype-metabolome-genome analysis taking a systems biology approach by inferring gene networks based on partial correlation and information theory approaches. Our data about biological processes enriched with genes from the feed efficiency network suggest that genetic variation in feed efficiency is driven by genetic modulation of basic processes relevant to general cellular functions. When looking at the predicted upstream regulators from the feed efficiency network, the Tumor Protein P53 (TP53) and Transforming Growth Factor beta 1 (TGFB1) genes stood out regarding significance of overlap and number of target molecules in the data set. These results further support the hypothesis that TP53 is a major upstream regulator for genetic variation of feed efficiency. Furthermore, our data revealed a significant effect of both, the Non-SMC Condensin I Complex, Subunit G (NCAPG) I442M (rs109570900) and the Growth /differentiation factor 8 (GDF8) Q204X (rs110344317) loci, on residual feed intake and feed conversion. For both loci, the growth promoting allele at the onset of puberty was associated with a negative, but favorable effect on residual feed intake. The elevated energy demand for increased growth triggered by the NCAPG 442M allele is obviously not fully compensated for by an increased efficiency in converting feed into body tissue. As a consequence, the individuals carrying the NCAPG 442M allele had an additional demand for energy uptake that is reflected by the association of the allele with increased daily energy intake as

  1. Systems Biology Analysis Merging Phenotype, Metabolomic and Genomic Data Identifies Non-SMC Condensin I Complex, Subunit G (NCAPG) and Cellular Maintenance Processes as Major Contributors to Genetic Variability in Bovine Feed Efficiency

    PubMed Central

    Widmann, Philipp; Reverter, Antonio; Weikard, Rosemarie; Suhre, Karsten; Hammon, Harald M.; Albrecht, Elke; Kuehn, Christa

    2015-01-01

    Feed efficiency is a paramount factor for livestock economy. Previous studies had indicated a substantial heritability of several feed efficiency traits. In our study, we investigated the genetic background of residual feed intake, a commonly used parameter of feed efficiency, in a cattle resource population generated from crossing dairy and beef cattle. Starting from a whole genome association analysis, we subsequently performed combined phenotype-metabolome-genome analysis taking a systems biology approach by inferring gene networks based on partial correlation and information theory approaches. Our data about biological processes enriched with genes from the feed efficiency network suggest that genetic variation in feed efficiency is driven by genetic modulation of basic processes relevant to general cellular functions. When looking at the predicted upstream regulators from the feed efficiency network, the Tumor Protein P53 (TP53) and Transforming Growth Factor beta 1 (TGFB1) genes stood out regarding significance of overlap and number of target molecules in the data set. These results further support the hypothesis that TP53 is a major upstream regulator for genetic variation of feed efficiency. Furthermore, our data revealed a significant effect of both, the Non-SMC Condensin I Complex, Subunit G (NCAPG) I442M (rs109570900) and the Growth /differentiation factor 8 (GDF8) Q204X (rs110344317) loci, on residual feed intake and feed conversion. For both loci, the growth promoting allele at the onset of puberty was associated with a negative, but favorable effect on residual feed intake. The elevated energy demand for increased growth triggered by the NCAPG 442M allele is obviously not fully compensated for by an increased efficiency in converting feed into body tissue. As a consequence, the individuals carrying the NCAPG 442M allele had an additional demand for energy uptake that is reflected by the association of the allele with increased daily energy intake as

  2. Hierarchical cellular materials

    SciTech Connect

    Gibson, L.J.

    1991-12-31

    In this paper a method for estimating the contributions of both the composite and the cellular microstructures to the overall material properties and the mechanical efficiency of natural cellular solids will be described. The method will be demonstrated by focusing on the Young`s modulus; similar techniques can be used for other material properties. The results suggest efficient microstructures for engineered cellular materials.

  3. Hierarchical cellular materials

    SciTech Connect

    Gibson, L.J.

    1991-01-01

    In this paper a method for estimating the contributions of both the composite and the cellular microstructures to the overall material properties and the mechanical efficiency of natural cellular solids will be described. The method will be demonstrated by focusing on the Young's modulus; similar techniques can be used for other material properties. The results suggest efficient microstructures for engineered cellular materials.

  4. Sub-cellular mRNA localization modulates the regulation of gene expression by small RNAs in bacteria

    NASA Astrophysics Data System (ADS)

    Teimouri, Hamid; Korkmazhan, Elgin; Stavans, Joel; Levine, Erel

    2017-10-01

    Small non-coding RNAs can exert significant regulatory activity on gene expression in bacteria. In recent years, substantial progress has been made in understanding bacterial gene expression by sRNAs. However, recent findings that demonstrate that families of mRNAs show non-trivial sub-cellular distributions raise the question of how localization may affect the regulatory activity of sRNAs. Here we address this question within a simple mathematical model. We show that the non-uniform spatial distributions of mRNA can alter the threshold-linear response that characterizes sRNAs that act stoichiometrically, and modulate the hierarchy among targets co-regulated by the same sRNA. We also identify conditions where the sub-cellular organization of cofactors in the sRNA pathway can induce spatial heterogeneity on sRNA targets. Our results suggest that under certain conditions, interpretation and modeling of natural and synthetic gene regulatory circuits need to take into account the spatial organization of the transcripts of participating genes.

  5. Feline leukemia virus T entry is dependent on both expression levels and specific interactions between cofactor and receptor.

    PubMed

    Cheng, Heather H; Anderson, Maria M; Overbaugh, Julie

    2007-03-01

    Feline leukemia virus (FeLV) subgroup T uses both a multiple membrane-spanning receptor, FePit1, and a soluble cofactor, FeLIX, to enter feline cells. FeLIX is expressed from endogenous FeLV-related sequence and resembles the receptor binding domain (RBD) of the viral envelope protein. It remains unclear whether FeLV-T receptor activity requires specific residues within FePit1 and FeLIX and/or a threshold level of receptor/cofactor expression. To address this, we examined FeLV-T infection of cells expressing variable levels of FePit1 and other gammaretroviral receptors in the presence of variable amounts of soluble cofactor, either RBD or the envelope surface subunit (SU). Cofactor-receptor pairs fall into three groups with regard to mediating FeLV-T infection: those that are efficient at all concentrations tested, such as FePit1 and FeLIX; those requiring high expression of both cofactor and receptor; and those that are non-functional as receptors even at high expression. This suggests that both expression levels and specific interactions with receptor and cofactor are critical for mediating entry of FeLV-T.

  6. A water-forming NADH oxidase from Lactobacillus pentosus suitable for the regeneration of synthetic biomimetic cofactors.

    PubMed

    Nowak, Claudia; Beer, Barbara; Pick, André; Roth, Teresa; Lommes, Petra; Sieber, Volker

    2015-01-01

    The cell-free biocatalytic production of fine chemicals by oxidoreductases has continuously grown over the past years. Since especially dehydrogenases depend on the stoichiometric use of nicotinamide pyridine cofactors, an integrated efficient recycling system is crucial to allow process operation under economic conditions. Lately, the variety of cofactors for biocatalysis was broadened by the utilization of totally synthetic and cheap biomimetics. Though, to date the regeneration has been limited to chemical or electrochemical methods. Here, we report an enzymatic recycling by the flavoprotein NADH-oxidase from Lactobacillus pentosus (LpNox). Since this enzyme has not been described before, we first characterized it in regard to its optimal reaction parameters. We found that the heterologously overexpressed enzyme only contained 13% FAD. In vitro loading of the enzyme with FAD, resulted in a higher specific activity towards its natural cofactor NADH as well as different nicotinamide derived biomimetics. Apart from the enzymatic recycling, which gives water as a by-product by transferring four electrons onto oxygen, unbound FAD can also catalyze the oxidation of biomimetic cofactors. Here a two electron process takes place yielding H2O2 instead. The enzymatic and chemical recycling was compared in regard to reaction kinetics for the natural and biomimetic cofactors. With LpNox and FAD, two recycling strategies for biomimetic cofactors are described with either water or hydrogen peroxide as by-product.

  7. The GlcN6P cofactor plays multiple catalytic roles in the glmS ribozyme.

    PubMed

    Bingaman, Jamie L; Zhang, Sixue; Stevens, David R; Yennawar, Neela H; Hammes-Schiffer, Sharon; Bevilacqua, Philip C

    2017-04-01

    RNA enzymes (ribozymes) have remarkably diverse biological roles despite having limited chemical diversity. Protein enzymes enhance their reactivity through recruitment of cofactors; likewise, the naturally occurring glmS ribozyme uses the glucosamine-6-phosphate (GlcN6P) organic cofactor for phosphodiester bond cleavage. Prior structural and biochemical studies have implicated GlcN6P as the general acid. Here we describe new catalytic roles of GlcN6P through experiments and calculations. Large stereospecific normal thio effects and a lack of metal-ion rescue in the holoribozyme indicate that nucleobases and the cofactor play direct chemical roles and align the active site for self-cleavage. Large stereospecific inverse thio effects in the aporibozyme suggest that the GlcN6P cofactor disrupts an inhibitory interaction of the nucleophile. Strong metal-ion rescue in the aporibozyme reveals that this cofactor also provides electrostatic stabilization. Ribozyme organic cofactors thus perform myriad catalytic roles, thereby allowing RNA to compensate for its limited functional diversity.

  8. A water-forming NADH oxidase from Lactobacillus pentosus suitable for the regeneration of synthetic biomimetic cofactors

    PubMed Central

    Nowak, Claudia; Beer, Barbara; Pick, André; Roth, Teresa; Lommes, Petra; Sieber, Volker

    2015-01-01

    The cell-free biocatalytic production of fine chemicals by oxidoreductases has continuously grown over the past years. Since especially dehydrogenases depend on the stoichiometric use of nicotinamide pyridine cofactors, an integrated efficient recycling system is crucial to allow process operation under economic conditions. Lately, the variety of cofactors for biocatalysis was broadened by the utilization of totally synthetic and cheap biomimetics. Though, to date the regeneration has been limited to chemical or electrochemical methods. Here, we report an enzymatic recycling by the flavoprotein NADH-oxidase from Lactobacillus pentosus (LpNox). Since this enzyme has not been described before, we first characterized it in regard to its optimal reaction parameters. We found that the heterologously overexpressed enzyme only contained 13% FAD. In vitro loading of the enzyme with FAD, resulted in a higher specific activity towards its natural cofactor NADH as well as different nicotinamide derived biomimetics. Apart from the enzymatic recycling, which gives water as a by-product by transferring four electrons onto oxygen, unbound FAD can also catalyze the oxidation of biomimetic cofactors. Here a two electron process takes place yielding H2O2 instead. The enzymatic and chemical recycling was compared in regard to reaction kinetics for the natural and biomimetic cofactors. With LpNox and FAD, two recycling strategies for biomimetic cofactors are described with either water or hydrogen peroxide as by-product. PMID:26441891

  9. Attenuation of the suppressive activity of cellular splicing factor SRSF3 by Kaposi sarcoma–associated herpesvirus ORF57 protein is required for RNA splicing

    PubMed Central

    Majerciak, Vladimir; Lu, Mathew; Li, Xiaofan

    2014-01-01

    Kaposi sarcoma–associated herpesvirus (KSHV) ORF57 is a multifunctional post-transcriptional regulator essential for viral gene expression during KSHV lytic infection. ORF57 requires interactions with various cellular proteins for its function. Here, we identified serine/arginine-rich splicing factor 3 (SRSF3, formerly known as SRp20) as a cellular cofactor involved in ORF57-mediated splicing of KSHV K8β RNA. In the absence of ORF57, SRSF3 binds to a suboptimal K8β intron and inhibits K8β splicing. Knockdown of SRSF3 promotes K8β splicing, mimicking the effect of ORF57. The N-terminal half of ORF57 binds to the RNA recognition motif of SRSF3, which prevents SRSF3 from associating with the K8β intron RNA and therefore attenuates the suppressive effect of SRSF3 on K8β splicing. ORF57 also promotes splicing of heterologous non-KSHV transcripts that are negatively regulated by SRSF3, indicating that the effect of ORF57 on SRSF3 activity is independent of RNA target. SPEN proteins, previously identified as ORF57-interacting partners, suppress ORF57 splicing activity by displacing ORF57 from SRSF3–RNA complexes. In summary, we have identified modulation of SRSF3 activity as the molecular mechanism by which ORF57 promotes RNA splicing. PMID:25234929

  10. Attenuation of the suppressive activity of cellular splicing factor SRSF3 by Kaposi sarcoma-associated herpesvirus ORF57 protein is required for RNA splicing.

    PubMed

    Majerciak, Vladimir; Lu, Mathew; Li, Xiaofan; Zheng, Zhi-Ming

    2014-11-01

    Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 is a multifunctional post-transcriptional regulator essential for viral gene expression during KSHV lytic infection. ORF57 requires interactions with various cellular proteins for its function. Here, we identified serine/arginine-rich splicing factor 3 (SRSF3, formerly known as SRp20) as a cellular cofactor involved in ORF57-mediated splicing of KSHV K8β RNA. In the absence of ORF57, SRSF3 binds to a suboptimal K8β intron and inhibits K8β splicing. Knockdown of SRSF3 promotes K8β splicing, mimicking the effect of ORF57. The N-terminal half of ORF57 binds to the RNA recognition motif of SRSF3, which prevents SRSF3 from associating with the K8β intron RNA and therefore attenuates the suppressive effect of SRSF3 on K8β splicing. ORF57 also promotes splicing of heterologous non-KSHV transcripts that are negatively regulated by SRSF3, indicating that the effect of ORF57 on SRSF3 activity is independent of RNA target. SPEN proteins, previously identified as ORF57-interacting partners, suppress ORF57 splicing activity by displacing ORF57 from SRSF3-RNA complexes. In summary, we have identified modulation of SRSF3 activity as the molecular mechanism by which ORF57 promotes RNA splicing.

  11. Viral Activation of Cellular Metabolism

    PubMed Central

    Sanchez, Erica L.; Lagunoff, Michael

    2015-01-01

    To ensure optimal environments for their replication and spread, viruses have evolved to alter many host cell pathways. In the last decade, metabolomic studies have shown that eukaryotic viruses induce large-scale alterations in host cellular metabolism. Most viruses examined to date induce aerobic glycolysis also known as the Warburg effect. Many viruses tested also induce fatty acid synthesis as well as glutaminolysis. These modifications of carbon source utilization by infected cells can increase available energy for virus replication and virion production, provide specific cellular substrates for virus particles and create viral replication niches while increasing infected cell survival. Each virus species also likely requires unique metabolic changes for successful spread and recent research has identified additional virus-specific metabolic changes induced by many virus species. A better understanding of the metabolic alterations required for each virus may lead to novel therapeutic approaches through targeted inhibition of specific cellular metabolic pathways. PMID:25812764

  12. Investigation of the cofactor-binding site of Zymomonas mobilis pyruvate decarboxylase by site-directed mutagenesis.

    PubMed Central

    Candy, J M; Duggleby, R G

    1994-01-01

    Several enzymes require thiamin diphosphate (ThDP) as an essential cofactor, and we have used one of these, pyruvate decarboxylase (PDC; EC 4.1.1.1) from Zymomonas mobilis, as a model for this group of enzymes. It is well suited for this purpose because of its stability, ease of purification and its simple kinetic properties. A sequence motif of approx. 30 residues, beginning with a glycine-aspartate-glycine (-GDG-) triplet and ending with a double asparagine (-NN-) sequence, has been identified in many of these enzymes [Hawkins, Borges and Perham (1989) FEBS Lett. 255, 77-82]. Other residues within this putative ThDP-binding motif are conserved, but to a lesser extent, including a glutamate and a proline residue. The role of the elements of this motif has been clarified by the determination of the three-dimensional structure of three of these enzymes [Muller, Lindqvist, Furey, Schulz, Jordan and Schneider (1993) Structure 1, 95-103]. Four of the residues within this motif were modified by site-directed mutagenesis of the cloned PDC gene to evaluate their role in cofactor binding. The mutant proteins were expressed in Escherichia coli and found to purify normally, indicating that the tertiary structure of these enzymes had not been grossly perturbed by the amino acid substitutions. We have shown previously [Diefenbach, Candy, Mattick and Duggleby (1992) FEBS Lett. 296, 95-98] that changing the aspartate in the -GDG- sequence to glycine, threonine or asparagine yields an inactive enzyme that is unable to bind ThDP, therefore verifying the role of the ThDP-binding motif. Here we demonstrate that substitution with glutamate yields an active enzyme with a greatly reduced affinity for both ThDP and Mg2+, but with normal kinetics for pyruvate. Unlike the wild-type tetrameric enzyme, this mutant protein usually exists as a dimer. Replacement of the second asparagine of the -NN- sequence by glutamine also yields an inactive enzyme which is unable to bind ThDP, whereas

  13. Chemoselective, Enzymatic C-H Bond Amination Catalyzed by a Cytochrome P450 Containing an Ir(Me)-PIX Cofactor.

    PubMed

    Dydio, Paweł; Key, Hanna M; Hayashi, Hiroki; Clark, Douglas S; Hartwig, John F

    2017-02-08

    Cytochrome P450 enzymes have been engineered to catalyze abiological C-H bond amination reactions, but the yields of these reactions have been limited by low chemoselectivity for the amination of C-H bonds over competing reduction of the azide substrate to a sulfonamide. Here we report that P450s derived from a thermophilic organism and containing an iridium porphyrin cofactor (Ir(Me)-PIX) in place of the heme catalyze enantioselective intramolecular C-H bond amination reactions of sulfonyl azides. These reactions occur with chemoselectivity for insertion of the nitrene units into C-H bonds over reduction of the azides to the sulfonamides that is higher and with substrate scope that is broader than those of enzymes containing iron porphyrins. The products from C-H amination are formed in up to 98% yield and ∼300 TON. In one case, the enantiomeric excess reaches 95:5 er, and the reactions can occur with divergent site selectivity. The chemoselectivity for C-H bond amination is greater than 20:1 in all cases. Variants of the Ir(Me)-PIX CYP119 displaying these properties were identified rapidly by evaluating CYP119 mutants containing Ir(Me)-PIX in cell lysates, rather than as purified enzymes. This study sets the stage to discover suitable enzymes to catalyze challenging C-H amination reactions.

  14. An Arabidopsis F-box protein acts as a transcriptional co-factor to regulate floral development.

    PubMed

    Chae, Eunyoung; Tan, Queenie K-G; Hill, Theresa A; Irish, Vivian F

    2008-04-01

    Plants flower in response to both environmental and endogenous signals. The Arabidopsis LEAFY (LFY) transcription factor is crucial in integrating these signals, and acts in part by activating the expression of multiple floral homeotic genes. LFY-dependent activation of the homeotic APETALA3 (AP3) gene requires the activity of UNUSUAL FLORAL ORGANS (UFO), an F-box component of an SCF ubiquitin ligase, yet how this regulation is effected has remained unclear. Here, we show that UFO physically interacts with LFY both in vitro and in vivo, and this interaction is necessary to recruit UFO to the AP3 promoter. Furthermore, a transcriptional repressor domain fused to UFO reduces endogenous LFY activity in plants, supporting the idea that UFO acts as part of a transcriptional complex at the AP3 promoter. Moreover, chemical or genetic disruption of proteasome activity compromises LFY-dependent AP3 activation, indicating that protein degradation is required to promote LFY activity. These results define an unexpected role for an F-box protein in functioning as a DNA-associated transcriptional co-factor in regulating floral homeotic gene expression. These results suggest a novel mechanism for promoting flower development via protein degradation and concomitant activation of the LFY transcription factor. This mechanism may be widely conserved, as homologs of UFO and LFY have been identified in a wide array of plant species.

  15. Cofactors-loaded quaternary structure of lysine-specific demethylase 5C (KDM5C) protein: Computational model.

    PubMed

    Peng, Yunhui; Alexov, Emil

    2016-12-01

    The KDM5C gene (also known as JARID1C and SMCX) is located on the X chromosome and encodes a ubiquitously expressed 1560-aa protein, which plays an important role in lysine methylation (specifically reverses tri- and di-methylation of Lys4 of histone H3). Currently, 13 missense mutations in KDM5C have been linked to X-linked mental retardation. However, the molecular mechanism of disease is currently unknown due to the experimental difficulties in expressing such large protein and the lack of experimental 3D structure. In this work, we utilize homology modeling, docking, and experimental data to predict 3D structures of KDM5C domains and their mutual arrangement. The resulting quaternary structure includes KDM5C JmjN, ARID, PHD1, JmjC, ZF domains, substrate histone peptide, enzymatic cofactors, and DNA. The predicted quaternary structure was investigated with molecular dynamic simulation for its stability, and further analysis was carried out to identify features measured experimentally. The predicted structure of KDM5C was used to investigate the effects of disease-causing mutations and it was shown that the mutations alter domain stability and inter-domain interactions. The structural model reported in this work could prompt experimental investigations of KDM5C domain-domain interaction and exploration of undiscovered functionalities. Proteins 2016; 84:1797-1809. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  16. A Bicarbonate Cofactor Modulates 1,4-Dihydroxy-2-naphthoyl-Coenzyme A Synthase in Menaquinone Biosynthesis of Escherichia coli*

    PubMed Central

    Jiang, Ming; Chen, Minjiao; Guo, Zu-Feng; Guo, Zhihong

    2010-01-01

    1,4-Dihydroxy-2-naphthoyl coenzyme A (DHNA-CoA) synthase is a typical crotonase-fold protein catalyzing an intramolecular Claisen condensation in the menaquinone biosynthetic pathway. We have characterized this enzyme from Escherichia coli and found that it is activated by bicarbonate in a concentration-dependent manner. The bicarbonate binding site has been identified in the crystal structure of a virtually identical ortholog (96.8% sequence identity) from Salmonella typhimurium through comparison with a bicarbonate-insensitive orthologue. Kinetic properties of the enzyme and its site-directed mutants of the bicarbonate binding site indicate that the exogenous bicarbonate anion is essential to the enzyme activity. With this essential catalytic role, the simple bicarbonate anion is an enzyme cofactor, which is usually a small organic molecule derived from vitamins, a metal ion, or a metal-containing polyatomic anionic complex. This finding leads to classification of the DHNA-CoA synthases into two evolutionarily conserved subfamilies: type I enzymes that are bicarbonate-dependent and contain a conserved glycine at the bicarbonate binding site; and type II enzymes that are bicarbonate-independent and contain a conserved aspartate at the position similar to the enzyme-bound bicarbonate. In addition, the unique location of the enzyme-bound bicarbonate allows it to be proposed as a catalytic base responsible for abstraction of the α-proton of the thioester substrate in the enzymatic reaction, suggesting a unified catalytic mechanism for all DHNA-CoA synthases. PMID:20643650

  17. A bicarbonate cofactor modulates 1,4-dihydroxy-2-naphthoyl-coenzyme a synthase in menaquinone biosynthesis of Escherichia coli.

    PubMed

    Jiang, Ming; Chen, Minjiao; Guo, Zu-Feng; Guo, Zhihong

    2010-09-24

    1,4-Dihydroxy-2-naphthoyl coenzyme A (DHNA-CoA) synthase is a typical crotonase-fold protein catalyzing an intramolecular Claisen condensation in the menaquinone biosynthetic pathway. We have characterized this enzyme from Escherichia coli and found that it is activated by bicarbonate in a concentration-dependent manner. The bicarbonate binding site has been identified in the crystal structure of a virtually identical ortholog (96.8% sequence identity) from Salmonella typhimurium through comparison with a bicarbonate-insensitive orthologue. Kinetic properties of the enzyme and its site-directed mutants of the bicarbonate binding site indicate that the exogenous bicarbonate anion is essential to the enzyme activity. With this essential catalytic role, the simple bicarbonate anion is an enzyme cofactor, which is usually a small organic molecule derived from vitamins, a metal ion, or a metal-containing polyatomic anionic complex. This finding leads to classification of the DHNA-CoA synthases into two evolutionarily conserved subfamilies: type I enzymes that are bicarbonate-dependent and contain a conserved glycine at the bicarbonate binding site; and type II enzymes that are bicarbonate-independent and contain a conserved aspartate at the position similar to the enzyme-bound bicarbonate. In addition, the unique location of the enzyme-bound bicarbonate allows it to be proposed as a catalytic base responsible for abstraction of the α-proton of the thioester substrate in the enzymatic reaction, suggesting a unified catalytic mechanism for all DHNA-CoA synthases.

  18. WT1 and its transcriptional cofactor BASP1 redirect the differentiation pathway of an established blood cell line.

    PubMed

    Goodfellow, Sarah J; Rebello, Michelle R; Toska, Eneda; Zeef, Leo A H; Rudd, Sean G; Medler, Kathryn F; Roberts, Stefan G E

    2011-04-01

    The Wilms' tumour suppressor WT1 (Wilms' tumour 1) is a transcriptional regulator that plays a central role in organogenesis, and is mutated or aberrantly expressed in several childhood and adult malignancies. We previously identified BASP1 (brain acid-soluble protein 1) as a WT1 cofactor that suppresses the transcriptional activation function of WT1. In the present study we have analysed the dynamic between WT1 and BASP1 in the regulation of gene expression in myelogenous leukaemia K562 cells. Our findings reveal that BASP1 is a significant regulator of WT1 that is recruited to WT1-binding sites and suppresses WT1-mediated transcriptional activation at several WT1 target genes. We find that WT1 and BASP1 can divert the differentiation programme of K562 cells to a non-blood cell type following induction by the phorbol ester PMA. WT1 and BASP1 co-operate to induce the differentiation of K562 cells to a neuronal-like morphology that exhibits extensive arborization, and the expression of several genes involved in neurite outgrowth and synapse formation. Functional analysis revealed the relevance of the transcriptional reprogramming and morphological changes, in that the cells elicited a response to the neurotransmitter ATP. Taken together, the results of the present study reveal that WT1 and BASP1 can divert the lineage potential of an established blood cell line towards a cell with neuronal characteristics.

  19. Regulation of Carotenoid Biosynthesis by Shade Relies on Specific Subsets of Antagonistic Transcription Factors and Cofactors.

    PubMed

    Bou-Torrent, Jordi; Toledo-Ortiz, Gabriela; Ortiz-Alcaide, Miriam; Cifuentes-Esquivel, Nicolas; Halliday, Karen J; Martinez-García, Jaime F; Rodriguez-Concepcion, Manuel

    2015-11-01

    Carotenoids are photosynthetic pigments essential for the protection against excess light. During deetiolation, their production is regulated by a dynamic repression-activation module formed by PHYTOCHROME-INTERACTING FACTOR1 (PIF1) and LONG HYPOCOTYL5 (HY5). These transcription factors directly and oppositely control the expression of the gene encoding PHYTOENE SYNTHASE (PSY), the first and main rate-determining enzyme of the carotenoid pathway. Antagonistic modules also regulate the responses of deetiolated plants to vegetation proximity and shade (i.e. to the perception of far-red light-enriched light filtered through or reflected from neighboring plants). These responses, aimed to adapt to eventual shading from plant competitors, include a reduced accumulation of carotenoids. Here, we show that PIF1 and related photolabile PIFs (but not photostable PIF7) promote the shade-triggered decrease in carotenoid accumulation. While HY5 does not appear to be required for this process, other known PIF antagonists were found to modulate the expression of the Arabidopsis (Arabidopsis thaliana) PSY gene and the biosynthesis of carotenoids early after exposure to shade. In particular, PHYTOCHROME-RAPIDLY REGULATED1, a transcriptional cofactor that prevents the binding of true transcription factors to their target promoters, was found to interact with PIF1 and hence directly induce PSY expression. By contrast, a change in the levels of the transcriptional cofactor LONG HYPOCOTYL IN FAR RED1, which also binds to PIF1 and other PIFs to regulate shade-related elongation responses, did not impact PSY expression or carotenoid accumulation. Our data suggest that the fine-regulation of carotenoid biosynthesis in response to shade relies on specific modules of antagonistic transcriptional factors and cofactors.

  20. Crystal structure of Trypanosoma cruzi tyrosine aminotransferase: substrate specificity is influenced by cofactor binding mode.

    PubMed Central

    Blankenfeldt, W.; Nowicki, C.; Montemartini-Kalisz, M.; Kalisz, H. M.; Hecht, H. J.

    1999-01-01

    The crystal structure of tyrosine aminotransferase (TAT) from the parasitic protozoan Trypanosoma cruzi, which belongs to the aminotransferase subfamily Igamma, has been determined at 2.5 A resolution with the R-value R = 15.1%. T. cruzi TAT shares less than 15% sequence identity with aminotransferases of subfamily Ialpha but shows only two larger topological differences to the aspartate aminotransferases (AspATs). First, TAT contains a loop protruding from the enzyme surface in the larger cofactor-binding domain, where the AspATs have a kinked alpha-helix. Second, in the smaller substrate-binding domain, TAT has a four-stranded antiparallel beta-sheet instead of the two-stranded beta-sheet in the AspATs. The position of the aromatic ring of the pyridoxal-5'-phosphate cofactor is very similar to the AspATs but the phosphate group, in contrast, is closer to the substrate-binding site with one of its oxygen atoms pointing toward the substrate. Differences in substrate specificities of T. cruzi TAT and subfamily Ialpha aminotransferases can be attributed by modeling of substrate complexes mainly to this different position of the cofactor-phosphate group. Absence of the arginine, which in the AspATs fixes the substrate side-chain carboxylate group by a salt bridge, contributes to the inability of T. cruzi TAT to transaminate acidic amino acids. The preference of TAT for tyrosine is probably related to the ability of Asn17 in TAT to form a hydrogen bond to the tyrosine side-chain hydroxyl group. PMID:10595543

  1. Nonracemic Antifolates Stereoselectively Recruit Alternate Cofactors and Overcome Resistance in S. aureus.

    PubMed

    Keshipeddy, Santosh; Reeve, Stephanie M; Anderson, Amy C; Wright, Dennis L

    2015-07-22

    While antifolates such as Bactrim (trimethoprim-sulfamethoxazole; TMP-SMX) continue to play an important role in treating community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA), resistance-conferring mutations, specifically F98Y of dihydrofolate reductase (DHFR), have arisen and compromise continued use. In an attempt to extend the lifetime of this important class, we have developed a class of propargyl-linked antifolates (PLAs) that exhibit potent inhibition of the enzyme and bacterial strains. Probing the role of the configuration at the single propargylic stereocenter in these inhibitors required us to develop a new approach to nonracemic 3-aryl-1-butyne building blocks by the pairwise use of asymmetric conjugate addition and aldehyde dehydration protocols. Using this new route, a series of nonracemic PLA inhibitors was prepared and shown to possess potent enzyme inhibition (IC50 values <50 nM), antibacterial effects (several with MIC values <1 μg/mL) and to form stable ternary complexes with both wild-type and resistant mutants. Unexpectedly, crystal structures of a pair of individual enantiomers in the wild-type DHFR revealed that the single change in configuration of the stereocenter drove the selection of an alternative NADPH cofactor, with the minor α-anomer appearing with R-27. Remarkably, this cofactor switching becomes much more prevalent when the F98Y mutation is present. The observation of cofactor site plasticity leads to a postulate for the structural basis of TMP resistance in DHFR and also suggests design strategies that can be used to target these resistant enzymes.

  2. Dimeric human sulfotransferase 1B1 displays cofactor-dependent subunit communication

    PubMed Central

    Tibbs, Zachary E; Falany, Charles N

    2015-01-01

    The cytosolic sulfotransferases (SULTs) are dimeric enzymes that catalyze the transformation of hydrophobic drugs and hormones into hydrophilic sulfate esters thereby providing the body with an important pathway for regulating small molecule activity and excretion. While SULT dimerization is highly conserved, the necessity for the interaction has not been established. To perform its function, a SULT must efficiently bind the universal sulfate donor, 3′-phosphoadenosine-5′-phosphosulfate (PAPS), and release the byproduct, 3′, 5′-diphosphoadenosine (PAP), following catalysis. We hypothesize this efficient binding and release of PAPS/PAP may be connected to SULT dimerization. To allow for the visualization of dynamic protein interactions critical for addressing this hypothesis and to generate kinetically testable hypotheses, molecular dynamic simulations (MDS) of hSULT1B1 were performed with PAPS and PAP bound to each dimer subunit in various combinations. The results suggest the dimer subunits may possess the capability of communicating with one another in a manner dependent on the presence of the cofactor. PAP or PAPS binding to a single side of the dimer results in decreased backbone flexibility of both the bound and unbound subunits, implying the dimer subunits may not act independently. Further, binding of PAP to one subunit of the dimer and PAPS to the other caused increased flexibility in the subunit bound to the inactive cofactor (PAP). These results suggest SULT dimerization may be important in maintaining cofactor binding/release properties of SULTs and provide hypothetical explanations for SULT half-site reactivity and substrate inhibition, which can be analyzed in vitro. PMID:26236487

  3. Cofactor-dependent conformational heterogeneity of GAD65 and its role in autoimmunity and neurotransmitter homeostasis

    PubMed Central

    Kass, Itamar; Hoke, David E.; Costa, Mauricio G. S.; Reboul, Cyril F.; Porebski, Benjamin T.; Cowieson, Nathan P.; Leh, Hervé; Pennacchietti, Eugenia; McCoey, Julia; Kleifeld, Oded; Borri Voltattorni, Carla; Langley, David; Roome, Brendan; Mackay, Ian R.; Christ, Daniel; Perahia, David; Buckle, Malcolm; Paiardini, Alessandro; De Biase, Daniela; Buckle, Ashley M.

    2014-01-01

    The human neuroendocrine enzyme glutamate decarboxylase (GAD) catalyses the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) using pyridoxal 5′-phosphate as a cofactor. GAD exists as two isoforms named according to their respective molecular weights: GAD65 and GAD67. Although cytosolic GAD67 is typically saturated with the cofactor (holoGAD67) and constitutively active to produce basal levels of GABA, the membrane-associated GAD65 exists mainly as the inactive apo form. GAD65, but not GAD67, is a prevalent autoantigen, with autoantibodies to GAD65 being detected at high frequency in patients with autoimmune (type 1) diabetes and certain other autoimmune disorders. The significance of GAD65 autoinactivation into the apo form for regulation of neurotransmitter levels and autoantibody reactivity is not understood. We have used computational and experimental approaches to decipher the nature of the holo → apo conversion in GAD65 and thus, its mechanism of autoinactivation. Molecular dynamics simulations of GAD65 reveal coupling between the C-terminal domain, catalytic loop, and pyridoxal 5′-phosphate–binding domain that drives structural rearrangement, dimer opening, and autoinactivation, consistent with limited proteolysis fragmentation patterns. Together with small-angle X-ray scattering and fluorescence spectroscopy data, our findings are consistent with apoGAD65 existing as an ensemble of conformations. Antibody-binding kinetics suggest a mechanism of mutually induced conformational changes, implicating the flexibility of apoGAD65 in its autoantigenicity. Although conformational diversity may provide a mechanism for cofactor-controlled regulation of neurotransmitter biosynthesis, it may also come at a cost of insufficient development of immune self-tolerance that favors the production of GAD65 autoantibodies. PMID:24927554

  4. Cofactor-dependent conformational heterogeneity of GAD65 and its role in autoimmunity and neurotransmitter homeostasis.

    PubMed

    Kass, Itamar; Hoke, David E; Costa, Mauricio G S; Reboul, Cyril F; Porebski, Benjamin T; Cowieson, Nathan P; Leh, Hervé; Pennacchietti, Eugenia; McCoey, Julia; Kleifeld, Oded; Borri Voltattorni, Carla; Langley, David; Roome, Brendan; Mackay, Ian R; Christ, Daniel; Perahia, David; Buckle, Malcolm; Paiardini, Alessandro; De Biase, Daniela; Buckle, Ashley M

    2014-06-24

    The human neuroendocrine enzyme glutamate decarboxylase (GAD) catalyses the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) using pyridoxal 5'-phosphate as a cofactor. GAD exists as two isoforms named according to their respective molecular weights: GAD65 and GAD67. Although cytosolic GAD67 is typically saturated with the cofactor (holoGAD67) and constitutively active to produce basal levels of GABA, the membrane-associated GAD65 exists mainly as the inactive apo form. GAD65, but not GAD67, is a prevalent autoantigen, with autoantibodies to GAD65 being detected at high frequency in patients with autoimmune (type 1) diabetes and certain other autoimmune disorders. The significance of GAD65 autoinactivation into the apo form for regulation of neurotransmitter levels and autoantibody reactivity is not understood. We have used computational and experimental approaches to decipher the nature of the holo → apo conversion in GAD65 and thus, its mechanism of autoinactivation. Molecular dynamics simulations of GAD65 reveal coupling between the C-terminal domain, catalytic loop, and pyridoxal 5'-phosphate-binding domain that drives structural rearrangement, dimer opening, and autoinactivation, consistent with limited proteolysis fragmentation patterns. Together with small-angle X-ray scattering and fluorescence spectroscopy data, our findings are consistent with apoGAD65 existing as an ensemble of conformations. Antibody-binding kinetics suggest a mechanism of mutually induced conformational changes, implicating the flexibility of apoGAD65 in its autoantigenicity. Although conformational diversity may provide a mechanism for cofactor-controlled regulation of neurotransmitter biosynthesis, it may also come at a cost of insufficient development of immune self-tolerance that favors the production of GAD65 autoantibodies.

  5. Communication between Thiamin Cofactors in the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Active Centers

    PubMed Central

    Nemeria, Natalia S.; Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Mossad, Madouna; Tittmann, Kai; Furey, William; Jordan, Frank

    2010-01-01

    Kinetic, spectroscopic, and structural analysis tested the hypothesis that a chain of residues connecting the 4′-aminopyrimidine N1′ atoms of thiamin diphosphates (ThDPs) in the two active centers of the Escherichia coli pyruvate dehydrogenase complex E1 component provides a signal transduction pathway. Substitution of the three acidic residues (Glu571, Glu235, and Glu237) and Arg606 resulted in impaired binding of the second ThDP, once the first active center was filled, suggesting a pathway for communication between the two ThDPs. 1) Steady-state kinetic and fluorescence quenching studies revealed that upon E571A, E235A, E237A, and R606A substitutions, ThDP binding in the second active center was affected. 2) Analysis of the kinetics of thiazolium C2 hydrogen/deuterium exchange of enzyme-bound ThDP suggests half-of-the-sites reactivity for the E1 component, with fast (activated site) and slow exchanging sites (dormant site). The E235A and E571A variants gave no evidence for the slow exchanging site, indicating that only one of two active sites is filled with ThDP. 3) Titration of the E235A and E237A variants with methyl acetylphosphonate monitored by circular dichroism suggested that only half of the active sites were filled with a covalent predecarboxylation intermediate analog. 4) Crystal structures of E235A and E571A in complex with ThDP revealed the structural basis for the spectroscopic and kinetic observations and showed that either substitution affects cofactor binding, despite the fact that Glu235 makes no direct contact with the cofactor. The role of the conserved Glu571 residue in both catalysis and cofactor orientation is revealed by the combined results for the first time. PMID:20106967

  6. Engineering of alanine dehydrogenase from Bacillus subtilis for novel cofactor specificity.

    PubMed

    Lerchner, Alexandra; Jarasch, Alexander; Skerra, Arne

    2016-09-01

    The l-alanine dehydrogenase of Bacillus subtilis (BasAlaDH), which is strictly dependent on NADH as redox cofactor, efficiently catalyzes the reductive amination of pyruvate to l-alanine using ammonia as amino group donor. To enable application of BasAlaDH as regenerating enzyme in coupled reactions with NADPH-dependent alcohol dehydrogenases, we alterated its cofactor specificity from NADH to NADPH via protein engineering. By introducing two amino acid exchanges, D196A and L197R, high catalytic efficiency for NADPH was achieved, with kcat /KM  = 54.1 µM(-1)  Min(-1) (KM  = 32 ± 3 µM; kcat  = 1,730 ± 39 Min(-1) ), almost the same as the wild-type enzyme for NADH (kcat /KM  = 59.9 µM(-1)  Min(-1) ; KM  = 14 ± 2 µM; kcat  = 838 ± 21 Min(-1) ). Conversely, recognition of NADH was much diminished in the mutated enzyme (kcat /KM  = 3 µM(-1)  Min(-1) ). BasAlaDH(D196A/L197R) was applied in a coupled oxidation/transamination reaction of the chiral dicyclic dialcohol isosorbide to its diamines, catalyzed by Ralstonia sp. alcohol dehydrogenase and Paracoccus denitrificans ω-aminotransferase, thus allowing recycling of the two cosubstrates NADP(+) and l-Ala. An excellent cofactor regeneration with recycling factors of 33 for NADP(+) and 13 for l-Ala was observed with the engineered BasAlaDH in a small-scale biocatalysis experiment. This opens a biocatalytic route to novel building blocks for industrial high-performance polymers.

  7. Mining the human complexome database identifies RBM14 as an XPO1-associated protein involved in HIV-1 Rev function.

    PubMed

    Budhiraja, Sona; Liu, Hongbing; Couturier, Jacob; Malovannaya, Anna; Qin, Jun; Lewis, Dorothy E; Rice, Andrew P

    2015-04-01

    By recruiting the host protein XPO1 (CRM1), the HIV-1 Rev protein mediates the nuclear export of incompletely spliced viral transcripts. We mined data from the recently described human nuclear complexome to identify a host protein, RBM14, which associates with XPO1 and Rev and is involved in Rev function. Using a Rev-dependent p24 reporter plasmid, we found that RBM14 depletion decreased Rev activity and Rev-mediated enhancement of the cytoplasmic levels of unspliced viral transcripts. RBM14 depletion also reduced p24 expression during viral infection, indicating that RBM14 is limiting for Rev function. RBM14 has previously been shown to localize to nuclear paraspeckles, a structure implicated in retaining unspliced HIV-1 transcripts for either Rev-mediated nuclear export or degradation. We found that depletion of NEAT1 RNA, a long noncoding RNA required for paraspeckle integrity, abolished the ability of overexpressed RBM14 to enhance Rev function, indicating the dependence of RBM14 function on paraspeckle integrity. Our study extends the known host cell interactome of Rev and XPO1 and further substantiates a critical role for paraspeckles in the mechanism of action of Rev. Our study also validates the nuclear complexome as a database from which viral cofactors can be mined. This study mined a database of nuclear protein complexes to identify a cellular protein named RBM14 that is associated with XPO1 (CRM1), a nuclear protein that binds to the HIV-1 Rev protein and mediates nuclear export of incompletely spliced viral RNAs. Functional assays demonstrated that RBM14, a protein found in paraspeckle structures in the nucleus, is involved in HIV-1 Rev function. This study validates the nuclear complexome database as a reference that can be mined to identify viral cofactors. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  8. Substrate, Product, and Cofactor: the Extraordinarily Flexible Relationship between the CDE Superfamily and Heme

    PubMed Central

    Celis, Arianna I.; DuBois, Jennifer L.

    2015-01-01

    PFam Clan 0032, also known as the CDE superfamily, is a diverse group of at least 20 protein families sharing a common α, β-barrel domain. Of these, six different groups bind heme inside the barrel’s interior, using it alternately as a cofactor, substrate, or product. Focusing on these six, an integrated picture of structure, sequence, taxonomy, and mechanism is presented here, detailing how a single structural motif might be able to mediate such an array of functions with one of nature’s most important small molecules. PMID:25778630

  9. FrsA functions as a cofactor-independent decarboxylase to control metabolic flux.

    PubMed

    Lee, Kyung-Jo; Jeong, Chang-Sook; An, Young Jun; Lee, Hyun-Jung; Park, Soon-Jung; Seok, Yeong-Jae; Kim, Pil; Lee, Jung-Hyun; Lee, Kyu-Ho; Cha, Sun-Shin

    2011-05-29

    The interaction between fermentation-respiration switch (FrsA) protein and glucose-specific enzyme IIA(Glc) increases glucose fermentation under oxygen-limited conditions. We show that FrsA converts pyruvate to acetaldehyde and carbon dioxide in a cofactor-independent manner and that its pyruvate decarboxylation activity is enhanced by the dephosphorylated form of IIA(Glc) (d-IIA(Glc)). Crystal structures of FrsA and its complex with d-IIA(Glc) revealed residues required for catalysis as well as the structural basis for the activation by d-IIA(Glc).

  10. Biochemical and genetic characterization of three molybdenum cofactor hydroxylases in Arabidopsis thaliana.

    PubMed

    Hoff, T; Frandsen, G I; Rocher, A; Mundy, J

    1998-07-09

    Aldehyde oxidases and xanthine dehydrogenases/oxidases belong to the molybdenum cofactor dependent hydroxylase class of enzymes. Zymograms show that Arabidopsis thaliana has at least three different aldehyde oxidases and one xanthine oxidase. Three different cDNA clones encoding putative aldehyde oxidases (AtAO1, 2, 3) were isolated. An aldehyde oxidase is the last step in abscisic acid (ABA) biosynthesis. AtAO1 is mainly expressed in seeds and roots which might reflect that it is involved in ABA biosynthesis.

  11. Substituted quinoline quinones as surrogates for the PQQ cofactor: an electrochemical and computational study.

    PubMed

    Dorfner, Walter L; Carroll, Patrick J; Schelter, Eric J

    2015-04-17

    Pyrroloquinoline quinones (PQQ) are important cofactors that shuttle redox equivalents in diverse metalloproteins. Quinoline 7,8-quinones have been synthesized and characterized as surrogates for PQQ to elucidate redox energetics within metalloenzyme active sites. The quinoline 7,8-quinones were accessed using polymer-supported iodoxybenzoic acid and the compounds evaluated using solution electrochemistry. Together with a family of quinones, the products were evaluated computationally and used to generate a predictive correlation between a computed ΔG and the experimental reduction potentials.

  12. Evidence for a reduced heparin cofactor II biological activity in diabetes.

    PubMed

    Ceriello, A; Quatraro, A; Dello Russo, P; Marchi, E; Milani, M R; Giugliano, D

    1990-01-01

    A reduction of heparin cofactor II (HCII) biological activity, despite its normal plasma concentration, is reported in insulin-dependent diabetic patients. A good linear correlation between HCII activity and concentration is present in normal controls but not in diabetics. In these subjects HCII activity correlates inversely with fasting blood glucose and glycated proteins but not with Hb A1. These data demonstrate the presence of a depressed HCII activity in the presence of its normal plasma concentration in insulin-dependent diabetics and suggest a role for short-term metabolic control in conditioning this phenomenon.

  13. Systems-based Analysis of RIG-I-dependent Signaling Identifies KHSRP as an Inhibitor of RIG-I Receptor Activation

    PubMed Central

    Soonthornvacharin, Stephen; Rodriguez-Frandsen, Ariel; Zhou, Yingyao; Galvez, Felipe; Huffmaster, Nicholas J.; Tripathi, Shashank; Balasubramaniam, Vinod RMT; Inoue, Atsushi; de Castro, Elisa; Moulton, Hong; Stein, David A.; Sánchez-Aparicio, María Teresa; De Jesus, Paul D.; Nguyen, Quy; König, Renate; Krogan, Nevan J.; García-Sastre, Adolfo; Yoh, Sunnie M.; Chanda, Sumit K.

    2017-01-01

    Retinoic acid inducible gene-I (RIG-I) receptor recognizes 5′-triphosphorylated RNA and triggers a signaling cascade that results in the induction of type-I IFN-dependent responses. Its precise regulation represents a pivotal balance between antiviral defenses and autoimmunity. To elucidate cellular cofactors that regulate RIG-I signaling, we performed two global RNAi analyses to identify both positive and negative regulatory nodes operating on the signaling pathway during virus infection. These factors were integrated with experimentally and computationally derived interactome data to build a RIG-I protein interaction network. Our analysis revealed diverse cellular processes, including the unfolded protein response, WNT signaling, and RNA metabolism, as critical cellular components governing innate responses to non-self RNA species. Importantly, we identified K-Homology Splicing Regulatory Protein (KHSRP) as a negative regulator of this pathway. We find that KHSRP associates with the regulatory domain of RIG-I to maintain the receptor in an inactive state and attenuate it’s sensing of viral RNA (vRNA). Consistent with increased RIG-I antiviral signaling in the absence of KHSRP, viral replication is reduced when KHSRP expression is knocked down both in vitro and in vivo. Taken together, these data indicate that KHSRP functions as a checkpoint regulator of the innate immune response to pathogen challenge. PMID:28248290

  14. Systems-based analysis of RIG-I-dependent signalling identifies KHSRP as an inhibitor of RIG-I receptor activation.

    PubMed

    Soonthornvacharin, Stephen; Rodriguez-Frandsen, Ariel; Zhou, Yingyao; Galvez, Felipe; Huffmaster, Nicholas J; Tripathi, Shashank; Balasubramaniam, Vinod R M T; Inoue, Atsushi; de Castro, Elisa; Moulton, Hong; Stein, David A; Sánchez-Aparicio, María Teresa; De Jesus, Paul D; Nguyen, Quy; König, Renate; Krogan, Nevan J; García-Sastre, Adolfo; Yoh, Sunnie M; Chanda, Sumit K

    2017-03-01

    Retinoic acid-inducible gene I (RIG-I) receptor recognizes 5'-triphosphorylated RNA and triggers a signalling cascade that results in the induction of type-I interferon (IFN)-dependent responses. Its precise regulation represents a pivotal balance between antiviral defences and autoimmunity. To elucidate the cellular cofactors that regulate RIG-I signalling, we performed two global RNA interference analyses to identify both positive and negative regulatory nodes operating on the signalling pathway during virus infection. These factors were integrated with experimentally and computationally derived interactome data to build a RIG-I protein interaction network. Our analysis revealed diverse cellular processes, including the unfolded protein response, Wnt signalling and RNA metabolism, as critical cellular components governing innate responses to non-self RNA species. Importantly, we identified K-Homology Splicing Regulatory Protein (KHSRP) as a negative regulator of this pathway. We find that KHSRP associates with the regulatory domain of RIG-I to maintain the receptor in an inactive state and attenuate its sensing of viral RNA (vRNA). Consistent with increased RIG-I antiviral signalling in the absence of KHSRP, viral replication is reduced when KHSRP expression is knocked down both in vitro and in vivo. Taken together, these data indicate that KHSRP functions as a checkpoint regulator of the innate immune response to pathogen challenge.

  15. Cofactor-binding sites in proteins of deviating sequence: comparative analysis and clustering in torsion angle, cavity, and fold space.

    PubMed

    Stegemann, Björn; Klebe, Gerhard

    2012-02-01

    Small molecules are recognized in protein-binding pockets through surface-exposed physicochemical properties. To optimize binding, they have to adopt a conformation corresponding to a local energy minimum within the formed protein-ligand complex. However, their conformational flexibility makes them competent to bind not only to homologous proteins of the same family but also to proteins of remote similarity with respect to the shape of the binding pockets and folding pattern. Considering drug action, such observations can give rise to unexpected and undesired cross reactivity. In this study, datasets of six different cofactors (ADP, ATP, NAD(P)(H), FAD, and acetyl CoA, sharing an adenosine diphosphate moiety as common substructure), observed in multiple crystal structures of protein-cofactor complexes exhibiting sequence identity below 25%, have been analyzed for the conformational properties of the bound ligands, the distribution of physicochemical properties in the accommodating protein-binding pockets, and the local folding patterns next to the cofactor-binding site. State-of-the-art clustering techniques have been applied to group the different protein-cofactor complexes in the different spaces. Interestingly, clustering in cavity (Cavbase) and fold space (DALI) reveals virtually the same data structuring. Remarkable relationships can be found among the different spaces. They provide information on how conformations are conserved across the host proteins and which distinct local cavity and fold motifs recognize the different portions of the cofactors. In those cases, where different cofactors are found to be accommodated in a similar fashion to the same fold motifs, only a commonly shared substructure of the cofactors is used for the recognition process. Copyright © 2011 Wiley Periodicals, Inc.

  16. A Cellular Biophysics Textbook

    NASA Astrophysics Data System (ADS)

    Wilder, Alan Joseph

    2011-12-01

    In the past two decades, great advances have been made in understanding of the biophysical mechanisms of the protein machines that carry out the fundamental processes of the cell. It is now known that all major eukaryotic cellular processes require a complicated assemblage of proteins acting via a series of concerted motions. In order to grasp current understanding of cellular mechanisms, the new generation of cell biologists needs to be trained in the general characteristics of these cellular properties and the methods with which to study them. This cellular biophysics textbook, to be used in conjunction with the cellular biophysics course (MCB143) at UC-Davis, provides a great tool in the instruction of the new generation of cellular biologists. It provides a hierarchical view of the cell, from atoms to protein machines and explains in depth the mechanisms of cytoskeletal force generators as an example of these principles.

  17. Correlating two-photon excited fluorescence imaging of breast cancer cellular redox state with seahorse flux analysis of normalized cellular oxygen consumption

    NASA Astrophysics Data System (ADS)

    Hou, Jue; Wright, Heather J.; Chan, Nicole; Tran, Richard; Razorenova, Olga V.; Potma, Eric O.; Tromberg, Bruce J.

    2016-06-01

    Two-photon excited fluorescence (TPEF) imaging of the cellular cofactors nicotinamide adenine dinucleotide and oxidized flavin adenine dinucleotide is widely used to measure cellular metabolism, both in normal and pathological cells and tissues. When dual-wavelength excitation is used, ratiometric TPEF imaging of the intrinsic cofactor fluorescence provides a metabolic index of cells-the "optical redox ratio" (ORR). With increased interest in understanding and controlling cellular metabolism in cancer, there is a need to evaluate the performance of ORR in malignant cells. We compare TPEF metabolic imaging with seahorse flux analysis of cellular oxygen consumption in two different breast cancer cell lines (MCF-7 and MDA-MB-231). We monitor metabolic index in living cells under both normal culture conditions and, for MCF-7, in response to cell respiration inhibitors and uncouplers. We observe a significant correlation between the TPEF-derived ORR and the flux analyzer measurements (R=0.7901, p<0.001). Our results confirm that the ORR is a valid dynamic index of cell metabolism under a range of oxygen consumption conditions relevant for cancer imaging.

  18. Identity of cofactor bound to mycothiol conjugate amidase (Mca) influenced by expression and purification conditions.

    PubMed

    Kocabas, Evren; Liu, Hualan; Hernick, Marcy

    2015-08-01

    Mycothiol serves as the primary reducing agent in Mycobacterium species, and is also a cofactor for the detoxification of xenobiotics. Mycothiol conjugate amidase (Mca) is a metalloamidase that catalyzes the cleavage of MS-conjugates to form a mercapturic acid, which is excreted from the mycobacterium, and 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside. Herein we report on the metal cofactor preferences of Mca from Mycobacterium smegmatis and Mycobacterium tuberculosis. Importantly, results from homology models of Mca from M. smegmatis and M. tuberculosis suggest that the metal binding site of Mca is identical to that of the closely related protein N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside deacetylase (MshB). This finding is supported by results from zinc ion affinity measurements that indicate Mca and MshB have comparable K(D)(ZnII) values (~10-20 pM). Furthermore, results from pull-down experiments using Halo-Mca indicate that Mca purifies with (stoichiometric) Fe(2+) when purified under anaerobic conditions, and Zn(2+) when purified under aerobic conditions. Consequently, Mca is likely a Fe(2+)-dependent enzyme under physiological conditions; with Zn(2+)-Mca an experimental artifact that could become biologically relevant under oxidatively stressed conditions. Importantly, these findings suggest that efforts towards the design of Mca inhibitors should include targeting the Fe(2+) form of the enzyme.

  19. Structural basis for a molecular allosteric control mechanism of cofactor binding to nuclear receptors.

    PubMed

    Osz, Judit; Brélivet, Yann; Peluso-Iltis, Carole; Cura, Vincent; Eiler, Sylvia; Ruff, Marc; Bourguet, William; Rochel, Natacha; Moras, Dino

    2012-03-06

    Transcription regulation by steroid hormones, vitamin derivatives, and metabolites is mediated by nuclear receptors (NRs), which play an important role in ligand-dependent gene expression and human health. NRs function as homodimers or heterodimers and are involved in a combinatorial, coordinated and sequentially orchestrated exchange between coregulators (corepressors, coactivators). The architecture of DNA-bound functional dimers positions the coregulators proteins. We previously demonstrated that retinoic acid (RAR-RXR) and vitamin D3 receptors (VDR-RXR) heterodimers recruit only one coactivator molecule asymmetrically without steric hindrance for the binding of a second cofactor. We now address the problem of homodimers for which the presence of two identical targets enhances the functional importance of the mode of binding. Using structural and biophysical methods and RAR as a model, we could dissect the molecular mechanism of coactivator recruitment to homodimers. Our study reveals an allosteric mechanism whereby binding of a coactivator promotes formation of nonsymmetrical RAR homodimers with a 21 stoichiometry. Ligand conformation and the cofactor binding site of the unbound receptor are affected through the dimer interface. A similar control mechanism is observed with estrogen receptor (ER) thus validating the negative cooperativity model for an established functional homodimer. Correlation with published data on other NRs confirms the general character of this regulatory pathway.

  20. A simple method to engineer a protein-derived redox cofactor for catalysis

    PubMed Central

    Shin, Sooim; Choi, Moonsung; Williamson, Heather R.; Davidson, Victor L.

    2014-01-01

    The 6x-Histidine tag which is commonly used for purification of recombinant proteins was converted to a catalytic redox-active center by incorporation of Co2+. Two examples of the biological activity of this engineered protein-derived cofactor are presented. After inactivation of the natural diheme cofactor of MauG, it was shown that the Co2+-loaded 6xHis-tag could substitute for the hemes in the H2O2-driven catalysis of tryptophan tryptophylquinone biosynthesis. To further demonstrate that the Co2+-loaded 6xHis-tag could mediate long range electron transfer, it was shown that addition of H2O2 to the Co2+-loaded 6xHis-tagged Cu1+ amicyanin oxidizes the copper site which is 20 Å away. These results provide proof of principle for this simple method by which to introduce a catalytic redox-active site into proteins for potential applications in research and biotechnology. PMID:24858537

  1. Immobilized cofactor derivatives for kinetic-based enzyme capture strategies: direct coupling of NAD(P)+.

    PubMed

    Oakey, Laura; Mulcahy, Patricia

    2004-12-15

    This study reevaluates the potential for direct coupling of NAD(P)(+) to a carboxylate-terminating spacer arm using carbodiimide-promoted coupling in an attempt to develop a greatly simplified synthetic method for cofactor immobilization that would support the more widespread adoption of kinetic-based enzyme capture (KBEC) strategies for protein purification applications and protein-detecting arrays/proteomic studies. Direct coupling of NAD(+) to epoxy (1,4-butanediol diglycidyl ether)-activated Sepharose is also described. Depending on the synthetic method used, the position of attachment of cofactor is concluded to be primarily through the pyrophosphate or ribosyl hydroxyl groups. Total substitution levels varied from 0.5 to 2 micromol/g wet weight with 28-67% accessibility. Model bioaffinity chromatographic studies employing KBEC strategies are reported for bovine heart L-lactate dehydrogenase, yeast alcohol dehydrogenase, l-phenylalanine dehydrogenase from Sporosarcina, glutamate dehydrogenase (GDH) from Candida utilis, and GDH from bovine liver. The NAD(+) derivative prepared using epoxy-activated Sepharose shows most potential for further development based on total substitution levels, the apparent absence of nonbiospecific interference, reversible biospecific adsorption of some of the test enzymes using soluble KBEC/stripping ligand tactics, and the relative simplicity of the synthetic method.

  2. A simple method to engineer a protein-derived redox cofactor for catalysis.

    PubMed

    Shin, Sooim; Choi, Moonsung; Williamson, Heather R; Davidson, Victor L

    2014-10-01

    The 6×-Histidine tag which is commonly used for purification of recombinant proteins was converted to a catalytic redox-active center by incorporation of Co(2+). Two examples of the biological activity of this engineered protein-derived cofactor are presented. After inactivation of the natural diheme cofactor of MauG, it was shown that the Co(2+)-loaded 6×His-tag could substitute for the hemes in the H2O2-driven catalysis of tryptophan tryptophylquinone biosynthesis. To further demonstrate that the Co(2+)-loaded 6×His-tag could mediate long range electron transfer, it was shown that addition of H2O2 to the Co(2+)-loaded 6×His-tagged Cu(1+) amicyanin oxidizes the copper site which is 20Å away. These results provide proof of principle for this simple method by which to introduce a catalytic redox-active site into proteins for potential applications in research and biotechnology.

  3. Early features in neuroimaging of two siblings with molybdenum cofactor deficiency.

    PubMed

    Higuchi, Ryuzo; Sugimoto, Takuya; Tamura, Akira; Kioka, Naomi; Tsuno, Yoshinobu; Higa, Asumi; Yoshikawa, Norishige

    2014-01-01

    We report the features of neuroimaging within 24 hours after birth in 2 siblings with molybdenum cofactor deficiency. The first sibling was delivered by emergency cesarean section because of fetal distress and showed pedaling and crawling seizures soon after birth. Brain ultrasound revealed subcortical multicystic lesions in the frontal white matter, and brain MRI at 4 hours after birth showed restricted diffusion in the entire cortex, except for the area adjacent to the subcortical cysts. The second sibling was delivered by elective cesarean section. Cystic lesions were seen in the frontal white matter on ultrasound, and brain MRI showed low signal intensity on T1-weighted image and high signal intensity on T2-weighted image in bifrontal white matter within 24 hours after birth, at which time the infant sucked sluggishly. Clonic spasm appeared at 29 hours after birth. The corpus callosum could not be seen clearly on ultrasound or MRI in both infants. Cortical atrophy and white matter cystic lesions spread to the entire hemisphere and resulted in severe brain atrophy within ~1 month in both infants. Subcortical multicystic lesions on ultrasound and a cortex with nonuniform, widespread, restricted diffusion on diffusion-weighted images are early features of neuroimaging in patients with molybdenum cofactor deficiency type A.

  4. Crystallization and preliminary crystallographic analysis of molybdenum-cofactor biosynthesis protein C from Thermus thermophilus

    SciTech Connect

    Kanaujia, Shankar Prasad; Ranjani, Chellamuthu Vasuki; Jeyakanthan, Jeyaraman; Baba, Seiki; Chen, Lirong; Liu, Zhi-Jie; Wang, Bi-Cheng; Nishida, Masami; Ebihara, Akio; Shinkai, Akeo; Kuramitsu, Seiki; Shiro, Yoshitsugu; Sekar, Kanagaraj; Yokoyama, Shigeyuki

    2010-12-03

    The Gram-negative aerobic eubacterium Thermus thermophilus is an extremely important thermophilic microorganism that was originally isolated from a thermal vent environment in Japan. The molybdenum cofactor in this organism is considered to be an essential component required by enzymes that catalyze diverse key reactions in the global metabolism of carbon, nitrogen and sulfur. The molybdenum-cofactor biosynthesis protein C derived from T. thermophilus was crystallized in two different space groups. Crystals obtained using the first crystallization condition belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 64.81, b = 109.84, c = 115.19 {angstrom}, {beta} = 104.9{sup o}; the crystal diffracted to a resolution of 1.9 {angstrom}. The other crystal form belonged to space group R32, with unit-cell parameters a = b = 106.57, c = 59.25 {angstrom}, and diffracted to 1.75 {angstrom} resolution. Preliminary calculations reveal that the asymmetric unit contains 12 monomers and one monomer for the crystals belonging to space group P2{sub 1} and R32, respectively.

  5. Effect of mitochondrial cofactors and antioxidants supplementation on cognition in the aged canine.

    PubMed

    Snigdha, Shikha; de Rivera, Christina; Milgram, Norton W; Cotman, Carl W

    2016-01-01

    A growing body of research has focused on modifiable risk factors for prevention and attenuation of cognitive decline in aging. This has led to an unprecedented interest in the relationship between diet and cognitive function. Several preclinical and epidemiologic studies suggest that dietary intervention can be used to improve cognitive function but randomized controlled trials are increasingly failing to replicate these findings. Here, we use a canine model of aging to evaluate the effects of specific components of diet supplementation which contain both antioxidants and a combination of mitochondrial cofactors (lipoic acid [LA] and acetyl-l-carnitine) on a battery of cognitive functions. Our data suggest that supplementation with mitochondrial cofactors, but not LA or antioxidant alone, selectively improve long-term recall in aged canines. Furthermore, we found evidence that LA alone could have cognitive impairing effects. These results contrast to those of a previous longitudinal study in aged canine. Our data demonstrate that one reason for this difference may be the nutritional status of animals at baseline for the 2 studies. Overall, this study suggests that social, cognitive, and physical activity together with optimal dietary intake (rather than diet alone) promotes successful brain aging.

  6. [Pathogenesis of AIDS: possible role of co-factors in HIV reactivation].

    PubMed

    Veronesi, R; Focaccia, R; Mazza, C C

    1989-01-01

    One of the most intriguing aspects concerning the pathogenesis of AIDS is the long period of latency of the HIV in human cells, not causing any cytopatic effect in some and, on the other hand, causing cell destruction, at short periods, in others. The various agents and the mechanisms they adopt to reactivate the latente HIV, were described. Also the frequent epidemiological observation on the presence of both such agents and the HIV in AIDS patients allowed the authors to speculate on the probable important role of a cohort of co-factors which determine the destiny of such individuals. Special considerations were made in respect to the hepatitis B virus, cytomegalovirus, herpesviruses (HHV-1, e and 6), EB virus, HTLV-1 and 2 retroviruses, group B arbovirus Maguary, malaria and other endemic infectious diseases which victimize millions of Brazilians. Accepting the importance of such co-factors acting on the viral gens that regulate the HIV expression in the host cell, it was speculated on the possible role of vaccines, such as the hepatitis B vaccine, and some antiviral drugs which could be useful in the indirect prevention of AIDS-disease in both HIV-carriers and those practising AIDS-high-risk-activities.

  7. Convenient synthesis of deazaflavin cofactor FO and its activity in F(420)-dependent NADP reductase.

    PubMed

    Hossain, Mohammad S; Le, Cuong Q; Joseph, Ebenezer; Nguyen, Toan Q; Johnson-Winters, Kayunta; Foss, Frank W

    2015-05-14

    F420 and FO are phenolic 5-deazaflavin cofactors that complement nicotinamide and flavin redox coenzymes in biochemical oxidoreductases and photocatalytic systems. Specifically, these 5-deazaflavins lack the single electron reactivity with O2 of riboflavin-derived coenzymes (FMN and FAD), and, in general, have a more negative redox potential than NAD(P)(+). For example, F420-dependent NADP(+) oxidoreductase (Fno) is critical to the conversion of CO2 to CH4 by methanogenic archaea, while FO functions as a light-harvesting agent in DNA repair. The preparation of these cofactors is an obstacle to their use in biochemical studies and biotechnology. Here, a convenient synthesis of FO was achieved by improving the redox stability of synthetic intermediates containing a polar, electron-rich aminophenol fragment. Improved yields and simplified purification techniques for FO are described. Additionally, Fno activity was restored with FO in the absence of F420. Investigating the FO-dependent NADP(+)/NADPH redox process by stopped-flow spectrophotometry, steady state kinetics were defined as having a Km of 4.00 ± 0.39 μM and a kcat of 5.27 ± 0.14 s(-1). The preparation of FO should enable future biochemical studies and novel uses of F420 mimics.

  8. Epitope mapping of 10 monoclonal antibodies against the pig analogue of human membrane cofactor protein (MCP)

    PubMed Central

    PéRez De La Lastra, J M; Van Den Berg, C W; Bullido, R; Almazán, F; Domínguez, J; Llanes, D; Morgan, B P

    1999-01-01

    Pig membrane cofactor protein (MCP; CD46) is a 50 000–60 000 MW glycoprotein that is expressed on a wide variety of cells, including erythrocytes. Pig MCP has cofactor activity for factor I-mediated cleavage of C3b and is an efficient regulator of the classical and alternative pathway of human and pig complement. A panel of 10 monoclonal antibodies (mAbs) was collected from two different laboratories; all of these mAbs were raised against pig leucocytes and all recognized the same complex banding pattern on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) of erythrocyte membranes. All were shown to be reactive with pig MCP and were divided into four groups of mutually competitive antibodies based on competition studies for membrane-bound MCP and for soluble MCP, the latter by surface plasmon resonance (SPR) analysis. The antigenic properties of membrane-bound and soluble MCP were similar, although some interesting differences were revealed. None of the 10 mAbs were cross-reactive with human MCP and only one showed cross-reactivity with leucocytes from a panel of large mammals – a weak cross-reactivity with a subset of dog leucocytes. All antibodies in one of the epitope groups and some in a second epitope group were able to block the functional activity of pig MCP, as measured by inhibition of MCP-catalysed C3 degradation by factor I. PMID:10233756

  9. Distribution and Properties of the Genes Encoding the Biosynthesis of the Bacterial Cofactor, Pyrroloquinoline Quinone†

    PubMed Central

    Shen, Yao-Qing; Bonnot, Florence; Imsand, Erin M.; RoseFigura, Jordan M.; Sjölander, Kimmen; Klinman, Judith P.

    2012-01-01

    Pyrroloquinoline quinone (PQQ) is a small, redox-active molecule that serves as a cofactor for several bacterial dehydrogenases, introducing pathways for carbon utilization that confer a growth advantage. Early studies had implicated a ribosomally translated peptide as the substrate for PQQ production. This study presents a sequence and structure based analysis of the components of the pqq operon. We find the necessary components for PQQ production are present in 126 prokaryotes, most of which are Gram- negative and a number of which are pathogens. A total of five gene products, PqqA, PqqB, PqqC, PqqD and PqqE, are concluded to be obligatory for PQQ production. Three of the gene products in the pqq operon, PqqB, PqqC and PqqE, are members of large protein superfamilies. By combining evolutionary conservation patterns with information from three-dimensional structures, we are able to differentiate the gene products involved in PQQ biosynthesis from those with divergent functions. The observed persistence of a conserved gene order within analyzed operons strongly suggests a role for protein/protein interactions in the course of cofactor biosynthesis. These studies propose previously unidentified roles for several of the gene products as well as possible new targets for antibiotic design and application. PMID:22324760

  10. Discovery and validation of information theory-based transcription factor and cofactor binding site motifs.

    PubMed

    Lu, Ruipeng; Mucaki, Eliseos J; Rogan, Peter K

    2016-11-28

    Data from ChIP-seq experiments can derive the genome-wide binding specificities of transcription factors (TFs) and other regulatory proteins. We analyzed 765 ENCODE ChIP-seq peak datasets of 207 human TFs with a novel motif discovery pipeline based on recursive, thresholded entropy minimization. This approach, while obviating the need to compensate for skewed nucleotide composition, distinguishes true binding motifs from noise, quantifies the strengths of individual binding sites based on computed affinity and detects adjacent cofactor binding sites that coordinate with the targets of primary, immunoprecipitated TFs. We obtained contiguous and bipartite information theory-based position weight matrices (iPWMs) for 93 sequence-specific TFs, discovered 23 cofactor motifs for 127 TFs and revealed six high-confidence novel motifs. The reliability and accuracy of these iPWMs were determined via four independent validation methods, including the detection of experimentally proven binding sites, explanation of effects of characterized SNPs, comparison with previously published motifs and statistical analyses. We also predict previously unreported TF coregulatory interactions (e.g. TF complexes). These iPWMs constitute a powerful tool for predicting the effects of sequence variants in known binding sites, performing mutation analysis on regulatory SNPs and predicting previously unrecognized binding sites and target genes.

  11. FAD Synthesis and Degradation in the Nucleus Create a Local Flavin Cofactor Pool*

    PubMed Central

    Giancaspero, Teresa Anna; Busco, Giovanni; Panebianco, Concetta; Carmone, Claudia; Miccolis, Angelica; Liuzzi, Grazia Maria; Colella, Matilde; Barile, Maria

    2013-01-01

    FAD is a redox cofactor ensuring the activity of many flavoenzymes mainly located in mitochondria but also relevant for nuclear redox activities. The last enzyme in the metabolic pathway producing FAD is FAD synthase (EC 2.7.7.2), a protein known to be localized both in cytosol and in mitochondria. FAD degradation to riboflavin occurs via still poorly characterized enzymes, possibly belonging to the NUDIX hydrolase family. By confocal microscopy and immunoblotting experiments, we demonstrate here the existence of FAD synthase in the nucleus of different experimental rat models. HPLC experiments demonstrated that isolated rat liver nuclei contain ∼300 pmol of FAD·mg−1 protein, which was mainly protein-bound FAD. A mean FAD synthesis rate of 18.1 pmol·min−1·mg−1 protein was estimated by both HPLC and continuous coupled enzymatic spectrophotometric assays. Rat liver nuclei were also shown to be endowed with a FAD pyrophosphatase that hydrolyzes FAD with an optimum at alkaline pH and is significantly inhibited by adenylate-containing nucleotides. The coordinate activity of these FAD forming and degrading enzymes provides a potential mechanism by which a dynamic pool of flavin cofactor is created in the nucleus. These data, which significantly add to the biochemical comprehension of flavin metabolism and its subcellular compartmentation, may also provide the basis for a more detailed comprehension of the role of flavin homeostasis in biologically and clinically relevant epigenetic events. PMID:23946482

  12. FAD synthesis and degradation in the nucleus create a local flavin cofactor pool.

    PubMed

    Giancaspero, Teresa Anna; Busco, Giovanni; Panebianco, Concetta; Carmone, Claudia; Miccolis, Angelica; Liuzzi, Grazia Maria; Colella, Matilde; Barile, Maria

    2013-10-04

    FAD is a redox cofactor ensuring the activity of many flavoenzymes mainly located in mitochondria but also relevant for nuclear redox activities. The last enzyme in the metabolic pathway producing FAD is FAD synthase (EC 2.7.7.2), a protein known to be localized both in cytosol and in mitochondria. FAD degradation to riboflavin occurs via still poorly characterized enzymes, possibly belonging to the NUDIX hydrolase family. By confocal microscopy and immunoblotting experiments, we demonstrate here the existence of FAD synthase in the nucleus of different experimental rat models. HPLC experiments demonstrated that isolated rat liver nuclei contain ∼300 pmol of FAD·mg(-1) protein, which was mainly protein-bound FAD. A mean FAD synthesis rate of 18.1 pmol·min(-1)·mg(-1) protein was estimated by both HPLC and continuous coupled enzymatic spectrophotometric assays. Rat liver nuclei were also shown to be endowed with a FAD pyrophosphatase that hydrolyzes FAD with an optimum at alkaline pH and is significantly inhibited by adenylate-containing nucleotides. The coordinate activity of these FAD forming and degrading enzymes provides a potential mechanism by which a dynamic pool of flavin cofactor is created in the nucleus. These data, which significantly add to the biochemical comprehension of flavin metabolism and its subcellular compartmentation, may also provide the basis for a more detailed comprehension of the role of flavin homeostasis in biologically and clinically relevant epigenetic events.

  13. Neutrino mass matrices with two vanishing cofactors and Fritzsch texture for charged lepton mass matrix

    NASA Astrophysics Data System (ADS)

    Wang, Weijian; Guo, Shu-Yuan; Wang, Zhi-Gang

    2016-04-01

    In this paper, we study the cofactor 2 zero neutrino mass matrices with the Fritzsch-type structure in charged lepton mass matrix (CLMM). In the numerical analysis, we perform a scan over the parameter space of all the 15 possible patterns to get a large sample of viable scattering points. Among the 15 possible patterns, three of them can accommodate the latest lepton mixing and neutrino mass data. We compare the predictions of the allowed patterns with their counterparts with diagonal CLMM. In this case, the severe cosmology bound on the neutrino mass set a strong constraint on the parameter space, rendering two patterns only marginally allowed. The Fritzsch-type CLMM will have impact on the viable parameter space and give rise to different phenomenological predictions. Each allowed pattern predicts the strong correlations between physical variables, which is essential for model selection and can be probed in future experiments. It is found that under the no-diagonal CLMM, the cofactor zeros structure in neutrino mass matrix is unstable as the running of renormalization group (RG) from seesaw scale to the electroweak scale. A way out of the problem is to propose the flavor symmetry under the models with a TeV seesaw scale. The inverse seesaw model and a loop-induced model are given as two examples.

  14. Cofactor Editing by the G-protein Metallochaperone Domain Regulates the Radical B12 Enzyme IcmF*♦

    PubMed Central

    Li, Zhu; Kitanishi, Kenichi; Twahir, Umar T.; Cracan, Valentin; Chapman, Derrell; Warncke, Kurt; Banerjee, Ruma

    2017-01-01

    IcmF is a 5′-deoxyadenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the carbon skeleton rearrangement of isobutyryl-CoA to butyryl-CoA. It is a bifunctional protein resulting from the fusion of a G-protein chaperone with GTPase activity and the cofactor- and substrate-binding mutase domains with isomerase activity. IcmF is prone to inactivation during catalytic turnover, thus setting up its dependence on a cofactor repair system. Herein, we demonstrate that the GTPase activity of IcmF powers the ejection of the inactive cob(II)alamin cofactor and requires the presence of an acceptor protein, adenosyltransferase, for receiving it. Adenosyltransferase in turn converts cob(II)alamin to AdoCbl in the presence of ATP and a reductant. The repaired cofactor is then reloaded onto IcmF in a GTPase-gated step. The mechanistic details of cofactor loading and offloading from the AdoCbl-dependent IcmF are distinct from those of the better characterized and homologous methylmalonyl-CoA mutase/G-protein chaperone system. PMID:28130442

  15. Flavin Adenine Dinucleotide and N(5) ,N(10) -Methenyltetrahydrofolate are the in planta Cofactors of Arabidopsis thaliana Cryptochrome 3.

    PubMed

    Göbel, Tanja; Reisbacher, Stefan; Batschauer, Alfred; Pokorny, Richard

    2017-01-01

    Members of the cryptochrome/photolyase family (CPF) of proteins utilize noncovalently bound light-absorbing cofactors for their biological function. Usually, the identity of these cofactors is determined after expression in heterologous systems leaving the question unanswered whether these cofactors are identical to the indigenous ones. Here, cryptochrome 3 from Arabidopsis thaliana was expressed as a fusion with the green fluorescent protein in Arabidopsis plants. Besides the confirmation of the earlier report of its localization in chloroplasts, our data indicate that fractions of the fusion protein are present in the stroma and associated with thylakoids, respectively. Furthermore, it is shown that the fusion protein expressed in planta contains the same cofactors as the His6 -tagged protein expressed in Escherichia coli, that is, flavin adenine dinucleotide and N(5) ,N(10) -methenyltetrahydrofolate. This demonstrates that the heterologously expressed cryptochrome 3, characterized in a number of previous studies, is a valid surrogate of the corresponding protein expressed in plants. To our knowledge, this is also a first conclusive analysis of cofactors bound to an Arabidopsis protein belonging to the CPF and purified from plant tissue. © 2016 The Authors. Photochemistry and Photobiology published by Wiley Periodicals, Inc. on behalf of American Society for Photobiology.

  16. Crystal structures, dynamics and functional implications of molybdenum-cofactor biosynthesis protein MogA from two thermophilic organisms

    PubMed Central

    Kanaujia, Shankar Prasad; Jeyakanthan, Jeyaraman; Shinkai, Akeo; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Sekar, Kanagaraj

    2011-01-01

    Molybdenum-cofactor (Moco) biosynthesis is an evolutionarily conserved pathway in almost all kingdoms of life, including humans. Two proteins, MogA and MoeA, catalyze the last step of this pathway in bacteria, whereas a single two-domain protein carries out catalysis in eukaryotes. Here, three crystal structures of the Moco-biosynthesis protein MogA from the two thermophilic organisms Thermus thermophilus (TtMogA; 1.64 Å resolution, space group P21) and Aquifex aeolicus (AaMogA; 1.70 Å resolution, space group P21 and 1.90 Å resolution, space group P1) have been determined. The functional roles and the residues involved in oligomerization of the protein molecules have been identified based on a comparative analysis of these structures with those of homologous proteins. Furthermore, functional roles have been proposed for the N- and C-terminal residues. In addition, a possible protein–protein complex of MogA and MoeA has been proposed and the residues involved in protein–protein interactions are discussed. Several invariant water molecules and those present at the subunit interfaces have been identified and their possible structural and/or functional roles are described in brief. In addition, molecular-dynamics and docking studies with several small molecules (including the substrate and the product) have been carried out in order to estimate their binding affinities towards AaMogA and TtMogA. The results obtained are further compared with those obtained for homologous eukaryotic proteins. PMID:21206014

  17. Genome-Wide Tissue-Specific Occupancy of the Hox Protein Ultrabithorax and Hox Cofactor Homothorax in Drosophila

    PubMed Central

    Slattery, Matthew; Ma, Lijia; Négre, Nicolas; White, Kevin P.; Mann, Richard S.

    2011-01-01

    The Hox genes are responsible for generating morphological diversity along the anterior-posterior axis during animal development. The Drosophila Hox gene Ultrabithorax (Ubx), for example, is required for specifying the identity of the third thoracic (T3) segment of the adult, which includes the dorsal haltere, an appendage required for flight, and the ventral T3 leg. Ubx mutants show homeotic transformations of the T3 leg towards the identity of the T2 leg and the haltere towards the wing. All Hox genes, including Ubx, encode homeodomain containing transcription factors, raising the question of what target genes Ubx regulates to generate these adult structures. To address this question, we carried out whole genome ChIP-chip studies to identify all of the Ubx bound regions in the haltere and T3 leg imaginal discs, which are the precursors to these adult structures. In addition, we used ChIP-chip to identify the sites bound by the Hox cofactor, Homothorax (Hth). In contrast to previous ChIP-chip studies carried out in Drosophila embryos, these binding studies reveal that there is a remarkable amount of tissue- and transcription factor-specific binding. Analyses of the putative target genes bound and regulated by these factors suggest that Ubx regulates many downstream transcription factors and developmental pathways in the haltere and T3 leg. Finally, we discovered additional DNA sequence motifs that in some cases are specific for individual data sets, arguing that Ubx and/or Hth work together with many regionally expressed transcription factors to execute their functions. Together, these data provide the first whole-genome analysis of the binding sites and target genes regulated by Ubx to specify the morphologies of the adult T3 segment of the fly. PMID:21483663

  18. Crystal structures, dynamics and functional implications of molybdenum-cofactor biosynthesis protein MogA from two thermophilic organisms.

    PubMed

    Kanaujia, Shankar Prasad; Jeyakanthan, Jeyaraman; Shinkai, Akeo; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Sekar, Kanagaraj

    2011-01-01

    Molybdenum-cofactor (Moco) biosynthesis is an evolutionarily conserved pathway in almost all kingdoms of life, including humans. Two proteins, MogA and MoeA, catalyze the last step of this pathway in bacteria, whereas a single two-domain protein carries out catalysis in eukaryotes. Here, three crystal structures of the Moco-biosynthesis protein MogA from the two thermophilic organisms Thermus thermophilus (TtMogA; 1.64 Å resolution, space group P2(1)) and Aquifex aeolicus (AaMogA; 1.70 Å resolution, space group P2(1) and 1.90 Å resolution, space group P1) have been determined. The functional roles and the residues involved in oligomerization of the protein molecules have been identified based on a comparative analysis of these structures with those of homologous proteins. Furthermore, functional roles have been proposed for the N- and C-terminal residues. In addition, a possible protein-protein complex of MogA and MoeA has been proposed and the residues involved in protein-protein interactions are discussed. Several invariant water molecules and those present at the subunit interfaces have been identified and their possible structural and/or functional roles are described in brief. In addition, molecular-dynamics and docking studies with several small molecules (including the substrate and the product) have been carried out in order to estimate their binding affinities towards AaMogA and TtMogA. The results obtained are further compared with those obtained for homologous eukaryotic proteins.

  19. Modelling cellular behaviour

    NASA Astrophysics Data System (ADS)

    Endy, Drew; Brent, Roger

    2001-01-01

    Representations of cellular processes that can be used to compute their future behaviour would be of general scientific and practical value. But past attempts to construct such representations have been disappointing. This is now changing. Increases in biological understanding combined with advances in computational methods and in computer power make it possible to foresee construction of useful and predictive simulations of cellular processes.

  20. New functional assays to selectively quantify the activated protein C- and tissue factor pathway inhibitor-cofactor activities of protein S in plasma.

    PubMed

    Alshaikh, N A; Rosing, J; Thomassen, M C L G D; Castoldi, E; Simioni, P; Hackeng, T M

    2017-02-17

    Essentials Protein S is a cofactor of activated protein C (APC) and tissue factor pathway inhibitor (TFPI). There are no assays to quantify separate APC and TFPI cofactor activities of protein S in plasma. We developed assays to measure the APC- and TFPI-cofactor activities of protein S in plasma. The assays were sensitive to protein S deficiency, and not affected by the Factor V Leiden mutation.

  1. Metabolic autofluorescence imaging of head and neck cancer organoids quantifies cellular heterogeneity and treatment response (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Shah, Amy T.; Heaster, Tiffany M.; Skala, Melissa C.

    2017-02-01

    Treatment options for head and neck cancer are limited, and can cause an impaired ability to eat, talk, and breathe. Therefore, optimized and personalized therapies could reduce unnecessary toxicities from ineffective treatments. Organoids are generated from primary tumor tissue and provide a physiologically-relevant in vitro model to measure drug response. Additionally, multiphoton fluorescence lifetime imaging (FLIM) of the metabolic cofactors NAD(P)H and FAD can resolve dynamic cellular response to anti-cancer treatment. This study applies FLIM of NAD(P)H and FAD to head and neck cancer organoids. Head and neck cancer tissue was digested and grown in culture as three-dimensional organoids. Gold standard measures of therapeutic response in vivo indicate stable disease after treatment with cetuximab (antibody therapy) or cisplatin (chemotherapy), and treatment response after combination treatment. In parallel, organoids were treated with cetuximab, cisplatin, or combination therapy for 24 hours. Treated organoids exhibit decreased NAD(P)H lifetime (p<0.05) and increased FAD lifetime (p<0.05) compared with control organoids. Additionally, analysis of cellular heterogeneity identifies distinct subpopulations of cells in response to treatment. A quantitative heterogeneity index predicts in vivo treatment response and demonstrates increased cellular heterogeneity in organoids treated with cetuximab or cisplatin compared with combination treatment. Mapping of cell subpopulations enables characterization of spatial relationships between cell subpopulations. Ultimately, an organoid model combined with metabolic fluorescence imaging could provide a high-throughput platform for drug discovery. Organoids grown from patient tissue could enable individualized treatment planning. These achievements could optimize quality of life and treatment outcomes for head and neck cancer patients.

  2. Activation and inhibition of rubber transferases by metal cofactors and pyrophosphate substrates.

    PubMed

    Scott, Deborah J; da Costa, Bernardo M T; Espy, Stephanie C; Keasling, Jay D; Cornish, Katrina

    2003-09-01

    Metal cofactors are necessary for the activity of alkylation by prenyl transfer in enzyme-catalyzed reactions. Rubber transferase (RuT, a cis-prenyl transferase) associated with purified rubber particles from Hevea brasiliensis, Parthenium argentatum and Ficus elastica can use magnesium and manganese interchangably to achieve maximum velocity. We define the concentration of activator required for maximum velocity as [A](max). The [A](max)(Mg2+) in F. elastica (100 mM) is 10 times the [A](max)(Mg2+) for either H. brasiliensis (10 mM) or P. argentatum (8 mM). The [A](max)(Mn2+) in F. elastica (11 mM), H. brasiliensis (3.8 mM) and P. argentatum (6.8 mM) and the [A](max)(Mg2+) in H. brasiliensis (10 mM) and P. argentatum (8 mM) are similar. The differences in [A](max)(Mg2+) correlate with the actual endogenous Mg(2+) concentrations in the latex of living plants. Extremely low Mn(2+) levels in vivo indicate that Mg(2+) is the RuT cofactor in living H. brasiliensis and F. elastica trees. Kinetic analyses demonstrate that FPP-Mg(2+) and FPP-Mn(2+) are active substrates for rubber molecule initiation, although free FPP and metal cations, Mg(2+) and Mn(2+), can interact independently at the active site with the following relative dissociation constants K(d)(FPP) cofactor requirements, and are membrane-bound enzymes.

  3. Crystal structure of archaeal photolyase from Sulfolobus tokodaii with two FAD molecules: implication of a novel light-harvesting cofactor.

    PubMed

    Fujihashi, Masahiro; Numoto, Nobutaka; Kobayashi, Yukiko; Mizushima, Akira; Tsujimura, Masanari; Nakamura, Akira; Kawarabayasi, Yutaka; Miki, Kunio

    2007-01-26

    UV exposure of DNA molecules induces serious DNA lesions. The cyclobutane pyrimidine dimer (CPD) photolyase repairs CPD-type - lesions by using the energy of visible light. Two chromophores for different roles have been found in this enzyme family; one catalyzes the CPD repair reaction and the other works as an antenna pigment that harvests photon energy. The catalytic cofactor of all known photolyases is FAD, whereas several light-harvesting cofactors are found. Currently, 5,10-methenyltetrahydrofolate (MTHF), 8-hydroxy-5-deaza-riboflavin (8-HDF) and FMN are the known light-harvesting cofactors, and some photolyases lack the chromophore. Three crystal structures of photolyases from Escherichia coli (Ec-photolyase), Anacystis nidulans (An-photolyase), and Thermus thermophilus (Tt-photolyase) have been determined; however, no archaeal photolyase structure is available. A similarity search of archaeal genomic data indicated the presence of a homologous gene, ST0889, on Sulfolobus tokodaii strain7. An enzymatic assay reveals that ST0889 encodes photolyase from S. tokodaii (St-photolyase). We have determined the crystal structure of the St-photolyase protein to confirm its structural features and to investigate the mechanism of the archaeal DNA repair system with light energy. The crystal structure of the St-photolyase is superimposed very well on the three known photolyases including the catalytic cofactor FAD. Surprisingly, another FAD molecule is found at the position of the light-harvesting cofactor. This second FAD molecule is well accommodated in the crystal structure, suggesting that FAD works as a novel light-harvesting cofactor of photolyase. In addition, two of the four CPD recognition residues in the crystal structure of An-photolyase are not found in St-photolyase, which might utilize a different mechanism to recognize the CPD from that of An-photolyase.

  4. Plasmonic Nanostructured Cellular Automata

    NASA Astrophysics Data System (ADS)

    Alkhazraji, Emad; Ghalib, A.; Manzoor, K.; Alsunaidi, M. A.

    2017-03-01

    In this work, we have investigated the scattering plasmonic resonance characteristics of silver nanospheres with a geometrical distribution that is modelled by Cellular Automata using time-domain numerical analysis. Cellular Automata are discrete mathematical structures that model different natural phenomena. Two binary one-dimensional Cellular Automata rules are considered to model the nanostructure, namely rule 30 and rule 33. The analysis produces three-dimensional scattering profiles of the entire plasmonic nanostructure. For the Cellular Automaton rule 33, the introduction of more Cellular Automata generations resulted only in slight red and blue shifts in the plasmonic modes with respect to the first generation. On the other hand, while rule 30 introduced significant red shifts in the resonance peaks at early generations, at later generations however, a peculiar effect is witnessed in the scattering profile as new peaks emerge as a feature of the overall Cellular Automata structure rather than the sum of the smaller parts that compose it. We strongly believe that these features that emerge as a result adopting the different 256 Cellular Automata rules as configuration models of nanostructures in different applications and systems might possess a great potential in enhancing their capability, sensitivity, efficiency, and power utilization.

  5. Active Site Metal Ion in UDP-3-O-((R)-3-Hydroxymyristoyl)-N-acetylglucosamine Deacetylase (LpxC) Switches between Fe(II) and Zn(II) Depending on Cellular Conditions*

    PubMed Central

    Gattis, Samuel G.; Hernick, Marcy; Fierke, Carol A.

    2010-01-01

    UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the deacetylation of UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine to form UDP-3-O-myristoylglucosamine and acetate in Gram-negative bacteria. This second, and committed, step in lipid A biosynthesis is a target for antibiotic development. LpxC was previously identified as a mononuclear Zn(II) metalloenzyme; however, LpxC is 6–8-fold more active with the oxygen-sensitive Fe(II) cofactor (Hernick, M., Gat