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Sample records for peptidyl-prolyl isomerase prsa2

  1. Development of a mariner-based transposon and identification of Listeria monocytogenes determinants, including the peptidyl-prolyl isomerase PrsA2, that contribute to its hemolytic phenotype.

    PubMed

    Zemansky, Jason; Kline, Benjamin C; Woodward, Joshua J; Leber, Jess H; Marquis, Hélène; Portnoy, Daniel A

    2009-06-01

    Listeriolysin O (LLO) is a pore-forming toxin that mediates phagosomal escape and cell-to-cell spread of the intracellular pathogen Listeria monocytogenes. In order to identify factors that control the production, activity, or secretion of this essential virulence factor, we constructed a Himar1 mariner transposon delivery system and screened 50,000 mutants for a hypohemolytic phenotype on blood agar plates. Approximately 200 hypohemolytic mutants were identified, and the 51 most prominent mutants were screened ex vivo for intracellular growth defects. Eight mutants with a phenotype were identified, and they contained insertions in the following genes: lmo0964 (similar to yjbH), lmo1268 (clpX), lmo1401 (similar to ymdB), lmo1575 (similar to ytqI), lmo1695 (mprF), lmo1821 (similar to prpC), lmo2219 (prsA2), and lmo2460 (similar to cggR). Some of these genes are involved in previously unexplored areas of research with L. monocytogenes: the genes yjbH and clpX regulate the disulfide stress response in Bacillus subtilis, and the prpC phosphatase has been implicated in virulence in other gram-positive pathogens. Here we demonstrate that prsA2, an extracytoplasmic peptidyl-prolyl cis/trans isomerase, is critical for virulence and contributes to the folding of LLO and to the activity of another virulence factor, the broad-range phospholipase C (PC-PLC). Furthermore, although it has been shown that prsA2 expression is linked to PrfA, the master virulence transcription factor in L. monocytogenes pathogenesis, we demonstrate that prsA2 is not directly controlled by PrfA. Finally, we show that PrsA2 is involved in flagellum-based motility, indicating that this factor likely serves a broad physiological role.

  2. Development of a mariner-Based Transposon and Identification of Listeria monocytogenes Determinants, Including the Peptidyl-Prolyl Isomerase PrsA2, That Contribute to Its Hemolytic Phenotype▿

    PubMed Central

    Zemansky, Jason; Kline, Benjamin C.; Woodward, Joshua J.; Leber, Jess H.; Marquis, Hélène; Portnoy, Daniel A.

    2009-01-01

    Listeriolysin O (LLO) is a pore-forming toxin that mediates phagosomal escape and cell-to-cell spread of the intracellular pathogen Listeria monocytogenes. In order to identify factors that control the production, activity, or secretion of this essential virulence factor, we constructed a Himar1 mariner transposon delivery system and screened 50,000 mutants for a hypohemolytic phenotype on blood agar plates. Approximately 200 hypohemolytic mutants were identified, and the 51 most prominent mutants were screened ex vivo for intracellular growth defects. Eight mutants with a phenotype were identified, and they contained insertions in the following genes: lmo0964 (similar to yjbH), lmo1268 (clpX), lmo1401 (similar to ymdB), lmo1575 (similar to ytqI), lmo1695 (mprF), lmo1821 (similar to prpC), lmo2219 (prsA2), and lmo2460 (similar to cggR). Some of these genes are involved in previously unexplored areas of research with L. monocytogenes: the genes yjbH and clpX regulate the disulfide stress response in Bacillus subtilis, and the prpC phosphatase has been implicated in virulence in other gram-positive pathogens. Here we demonstrate that prsA2, an extracytoplasmic peptidyl-prolyl cis/trans isomerase, is critical for virulence and contributes to the folding of LLO and to the activity of another virulence factor, the broad-range phospholipase C (PC-PLC). Furthermore, although it has been shown that prsA2 expression is linked to PrfA, the master virulence transcription factor in L. monocytogenes pathogenesis, we demonstrate that prsA2 is not directly controlled by PrfA. Finally, we show that PrsA2 is involved in flagellum-based motility, indicating that this factor likely serves a broad physiological role. PMID:19376879

  3. Peptidyl-prolyl cis-trans isomerases: structure and functions.

    PubMed

    Pliyev, B K; Gurvits, B Y

    1999-07-01

    Peptidyl-prolyl cis-trans isomerases (PPI) catalyze cis-trans isomerization of imide bonds in peptides and proteins. This review summarizes the literature on the structure and functions of PPIs, their involvement in protein folding, and organization of PPI-containing receptors and membrane channels. A possible role of several PPIs in distant interactions between cells is discussed.

  4. Structural and Biochemical Characterization of the Human Cyclophilin Family of Peptidyl-Prolyl Isomerases

    SciTech Connect

    Davis, Tara L.; Walker, John R.; Campagna-Slater, Valérie; Finerty, Jr., Patrick J.; Paramanathan, Ragika; Bernstein, Galina; MacKenzie, Farrell; Tempel, Wolfram; Ouyang, Hui; Lee, Wen Hwa; Eisenmesser, Elan Z.; Dhe-Paganon, Sirano

    2011-12-14

    Peptidyl-prolyl isomerases catalyze the conversion between cis and trans isomers of proline. The cyclophilin family of peptidyl-prolyl isomerases is well known for being the target of the immunosuppressive drug cyclosporin, used to combat organ transplant rejection. There is great interest in both the substrate specificity of these enzymes and the design of isoform-selective ligands for them. However, the dearth of available data for individual family members inhibits attempts to design drug specificity; additionally, in order to define physiological functions for the cyclophilins, definitive isoform characterization is required. In the current study, enzymatic activity was assayed for 15 of the 17 human cyclophilin isomerase domains, and binding to the cyclosporin scaffold was tested. In order to rationalize the observed isoform diversity, the high-resolution crystallographic structures of seven cyclophilin domains were determined. These models, combined with seven previously solved cyclophilin isoforms, provide the basis for a family-wide structure:function analysis. Detailed structural analysis of the human cyclophilin isomerase explains why cyclophilin activity against short peptides is correlated with an ability to ligate cyclosporin and why certain isoforms are not competent for either activity. In addition, we find that regions of the isomerase domain outside the proline-binding surface impart isoform specificity for both in vivo substrates and drug design. We hypothesize that there is a well-defined molecular surface corresponding to the substrate-binding S2 position that is a site of diversity in the cyclophilin family. Computational simulations of substrate binding in this region support our observations. Our data indicate that unique isoform determinants exist that may be exploited for development of selective ligands and suggest that the currently available small-molecule and peptide-based ligands for this class of enzyme are insufficient for isoform

  5. Drosophila peptidyl-prolyl isomerase Pin1 modulates circadian rhythms via regulating levels of PERIOD.

    PubMed

    Kang, So Who; Lee, Euna; Cho, Eunjoo; Seo, Ji Hye; Ko, Hyuk Wan; Kim, Eun Young

    2015-07-31

    In animal circadian clock machinery, the phosphorylation program of PERIOD (PER) leads to the spatio-temporal regulation of diverse PER functions, which are crucial for the maintenance of ~24-hr circadian rhythmicity. The peptidyl-prolyl isomerase PIN1 modulates the diverse functions of its substrates by inducing conformational changes upon recognizing specific phosphorylated residues. Here, we show that overexpression of Drosophila pin1, dodo (dod), lengthens the locomotor behavioral period. Using Drosophila S2 cells, we demonstrate that Dod associates preferentially with phosphorylated species of PER, which delays the phosphorylation-dependent degradation of PER. Consistent with this, PER protein levels are higher in flies overexpressing dod. Taken together, we suggest that Dod plays a role in the maintenance of circadian period by regulating PER metabolism.

  6. Trypanosomatid pin1-type peptidyl-prolyl isomerase is cytosolic and not essential for cell proliferation.

    PubMed

    Erben, Esteban D; Nardelli, Sheila C; de Jesus, Teresa C L; Schenkman, Sergio; Tellez-Iñon, Maria T

    2013-01-01

    Pin1-type peptidyl-prolyl cis/trans isomerases (PPIases) isomerise the peptide bond of specific phosphorylated (Ser/Thr)-Pro residues, regulating various cellular events. Previously, we reported a Pin1-type PPIase in Trypanosoma cruzi, but little is known about its function and subcellular localization. Immunofluorescence analysis revealed that in contrast with Pin1-like proteins from diverse organisms, TcPin1 mainly localized in the cytoplasm and was excluded from the nuclei. In addition, RNAi-mediated downregulation of TbPin1 in Trypanosoma brucei did not abolish cell proliferation. Using yeast two-hybrid assay, we identified a MORN domain-containing protein as putative Pin1-binding partners. These data suggest that Pin1-mediated signaling mechanism plays a different role in protozoan parasites. © 2012 The Author(s) Journal of Eukaryotic Microbiology © 2012 International Society of Protistologists.

  7. Non-catalytic participation of the Pin1 peptidyl-prolyl isomerase domain in target binding

    PubMed Central

    Innes, Brendan T.; Bailey, Melanie L.; Brandl, Christopher J.; Shilton, Brian H.; Litchfield, David W.

    2012-01-01

    Pin1 is a phosphorylation-dependent peptidyl-prolyl isomerase (PPIase) that has the potential to add an additional level of regulation within protein kinase mediated signaling pathways. Furthermore, there is a mounting body of evidence implicating Pin1 in the emergence of pathological phenotypes in neurodegeneration and cancer through the isomerization of a wide variety of substrates at peptidyl-prolyl bonds where the residue preceding proline is a phosphorylated serine or threonine residue (i.e., pS/T-P motifs). A key step in this regulatory process is the interaction of Pin-1 with its substrates. This is a complex process since Pin1 is composed of two domains, the catalytic PPIase domain, and a type IV WW domain, both of which recognize pS/T-P motifs. The observation that the WW domain exhibits considerably higher binding affinity for pS/T-P motifs has led to predictions that the two domains may have distinct roles in mediating the actions of Pin1 on its substrates. To evaluate the participation of its individual domains in target binding, we performed GST pulldowns to monitor interactions between various forms of Pin1 and mitotic phospho-proteins that revealed two classes of Pin-1 interacting proteins, differing in their requirement for residues within the PPIase domain. From these observations, we consider models for Pin1-substrate interactions and the potential functions of the different classes of Pin1 interacting proteins. We also compare sequences that are recognized by Pin1 within its individual interaction partners to investigate the underlying basis for its different types of interactions. PMID:23407864

  8. Structural and Biochemical Characterization of the Human Cyclophilin Family of Peptidyl-Prolyl Isomerases

    PubMed Central

    Davis, Tara L.; Walker, John R.; Campagna-Slater, Valérie; Finerty, Patrick J.; Paramanathan, Ragika; Bernstein, Galina; MacKenzie, Farrell; Tempel, Wolfram; Ouyang, Hui; Lee, Wen Hwa; Eisenmesser, Elan Z.; Dhe-Paganon, Sirano

    2010-01-01

    Peptidyl-prolyl isomerases catalyze the conversion between cis and trans isomers of proline. The cyclophilin family of peptidyl-prolyl isomerases is well known for being the target of the immunosuppressive drug cyclosporin, used to combat organ transplant rejection. There is great interest in both the substrate specificity of these enzymes and the design of isoform-selective ligands for them. However, the dearth of available data for individual family members inhibits attempts to design drug specificity; additionally, in order to define physiological functions for the cyclophilins, definitive isoform characterization is required. In the current study, enzymatic activity was assayed for 15 of the 17 human cyclophilin isomerase domains, and binding to the cyclosporin scaffold was tested. In order to rationalize the observed isoform diversity, the high-resolution crystallographic structures of seven cyclophilin domains were determined. These models, combined with seven previously solved cyclophilin isoforms, provide the basis for a family-wide structure∶function analysis. Detailed structural analysis of the human cyclophilin isomerase explains why cyclophilin activity against short peptides is correlated with an ability to ligate cyclosporin and why certain isoforms are not competent for either activity. In addition, we find that regions of the isomerase domain outside the proline-binding surface impart isoform specificity for both in vivo substrates and drug design. We hypothesize that there is a well-defined molecular surface corresponding to the substrate-binding S2 position that is a site of diversity in the cyclophilin family. Computational simulations of substrate binding in this region support our observations. Our data indicate that unique isoform determinants exist that may be exploited for development of selective ligands and suggest that the currently available small-molecule and peptide-based ligands for this class of enzyme are insufficient for isoform

  9. Identification and functional analysis of a novel parvulin-type peptidyl-prolyl isomerase from Gossypium hirsutum.

    PubMed

    Wang, Ping; Li, Xin-Zheng; Cui, Hao-Ran; Feng, Yue-guang; Wang, Xiao-Yun

    2014-03-01

    Plants have developed a variety of adaptive mechanisms to cope with stresses. A novel salt-induced gene was isolated during the screening of a NaCl-induced cDNA library of cotton seedlings. The gene was registered as accession number AY972810 in GenBank. Phylogenetic analysis suggested that the protein encoded by the gene belongs to the parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases, EC 5.2.1.8). Northern blot analysis indicated that the mRNA accumulation of GhPPI was induced by salt stress. Subcellular localization revealed that GhPPI (Gossypium hirsutum peptidyl-prolyl isomerase) was localized in the nucleus. The purified recombinant GhPPI could accelerate the initial velocity of the cis-trans conversion of peptidyl-prolyl bonds of a tetrapeptide in a GhPPI concentration-dependent manner. Recombinant GhPPI also suppressed protein aggregation under denaturing conditions using Gdn-HCl (guanidine hydrochloride), suggesting an additional chaperone activity. Several amino acid residues in GhPPI were speculated to be involved in substrate binding or catalysis based on molecular modeling and docking results. The activity of the peptidyl-prolyl isomerase was affected when the relevant amino acids were mutated. Among the 11 mutants, five amino acids mutations led to the enzyme activities decreased to 30% as that of wild type, and two reduced to approximately 60%. To the best of our knowledge, this is the first report of a plant parvulin PPIase involved in the salt stress response. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  10. Cellular peptidyl-prolyl cis/trans isomerase Pin1 facilitates replication of feline coronavirus.

    PubMed

    Tanaka, Yoshikazu; Amano, Arisa; Morisaki, Masateru; Sato, Yuka; Sasaki, Takashi

    2016-02-01

    Although feline coronavirus (FCoV) causes feline infectious peritonitis (FIP), which is a fatal infectious disease, there are no effective therapeutic medicines or vaccines. Previously, in vitro studies have shown that cyclosporin (CsA) and FK506 inhibit virus replication in diverse coronaviruses. CsA and FK506 are targets of clinically relevant immunosuppressive drugs and bind to cellular cyclophilins (Cyps) or FK506 binding proteins (FKBPs), respectively. Both Cyp and FKBP have peptidyl-prolyl cis-trans isomerase (PPIase) activity. However, protein interacting with NIMA (Pin1), a member of the parvulin subfamily of PPIases that differs from Cyps and FKBPs, is essential for various signaling pathways. Here we demonstrated that genetic silencing or knockout of Pin1 resulted in decreased FCoV replication in vitro. Dipentamethylene thiuram monosulfide, a specific inhibitor of Pin1, inhibited FCoV replication. These data indicate that Pin1 modulates FCoV propagation. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Mycobacterium tuberculosis Peptidyl-Prolyl Isomerases Are Immunogenic, Alter Cytokine Profile and Aid in Intracellular Survival.

    PubMed

    Pandey, Saurabh; Tripathi, Deeksha; Khubaib, Mohd; Kumar, Ashutosh; Sheikh, Javaid A; Sumanlatha, Gaddam; Ehtesham, Nasreen Z; Hasnain, Seyed E

    2017-01-01

    Mycobacterium tuberculosis (M. tb) has two peptidyl-prolyl isomerases (Ppiases) PpiA and PpiB, popularly known as cyclophilin A and cyclophilin B. The role of cyclophilins in processes such as signaling, cell surface recognition, chaperoning, and heat shock response has been well-documented. We present evidence that M. tb Ppiases modulate the host immune response. ELISA results revealed significant presence of antibodies to M. tb Ppiases in patient sera as compared to sera from healthy individuals. Treatment of THP-1 cells with increasing concentrations of rPpiA, induced secretion of pro-inflammatory cytokines TNF-α and IL-6. Alternatively, treatment with rPpiB inhibited secretion of TNF-α and induced secretion of IL-10. Furthermore, heterologous expression of M. tb PpiA and PpiB in Mycobacterium smegmatis increased bacterial survival in THP-1 cells as compared to those transformed with the vector control. Our results demonstrate that M. tb Ppiases are immunogenic proteins that can possibly modulate host immune response and enhance persistence of the pathogen within the host by subverting host cell generated stresses.

  12. Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α

    PubMed Central

    Han, Hyeong-jun; Kwon, Nayoung; Choi, Min-A; Jung, Kyung Oh; Piao, Juan-Yu; Ngo, Hoang Kieu Chi; Kim, Su-Jung; Kim, Do-Hee; Chung, June-Key; Cha, Young-Nam; Youn, Hyewon; Choi, Bu Young; Min, Sang-Hyun; Surh, Young-Joon

    2016-01-01

    Peptidyl prolyl isomerase (PIN1) regulates the functional activity of a subset of phosphoproteins through binding to phosphorylated Ser/Thr-Pro motifs and subsequently isomerization of the phosphorylated bonds. Interestingly, PIN1 is overexpressed in many types of malignancies including breast, prostate, lung and colon cancers. However, its oncogenic functions have not been fully elucidated. Here, we report that PIN1 directly interacts with hypoxia-inducible factor (HIF)-1α in human colon cancer (HCT116) cells. PIN1 binding to HIF-1α occurred in a phosphorylation-dependent manner. We also found that PIN1 interacted with HIF-1α at both exogenous and endogenous levels. Notably, PIN1 binding stabilized the HIF-1α protein, given that their levels were significantly increased under hypoxic conditions. The stabilization of HIF-1α resulted in increased transcriptional activity, consequently upregulating expression of vascular endothelial growth factor, a major contributor to angiogenesis. Silencing of PIN1 or pharmacologic inhibition of its activity abrogated the angiogenesis. By utilizing a bioluminescence imaging technique, we were able to demonstrate that PIN1 inhibition dramatically reduced the tumor volume in a subcutaneous mouse xenograft model and angiogenesis as well as hypoxia-induced transcriptional activity of HIF-1α. These results suggest that PIN1 interacting with HIF-1α is a potential cancer chemopreventive and therapeutic target. PMID:26784107

  13. Cyclic Peptidyl Inhibitors against Human Peptidyl-Prolyl Isomerase Pin1

    PubMed Central

    Liu, Tao; Liu, Yu; Kao, Hung-Ying; Pei, Dehua

    2010-01-01

    Peptidyl-prolyl isomerase Pin1 regulates the function and/or stability of phosphoproteins by altering the conformation of specific pSer/pThr-Pro peptide bonds. In this work, a cyclic peptide library was synthesized and screened against the catalytic domain of human Pin1. The selected inhibitors contained a consensus motif of D-pThr-Pip-Nal (where Pip is L-piperidine-2-carboxylic acid and Nal is L-2-naphthylalanine). Representative compounds were tested for binding to Pin1 by isothermal titration calorimetry and inhibition of Pin1 activity and the most potent inhibitors had KD (and KI) values in the low nanomolar range. Treatment of breast cancer cells with the inhibitors, which were rendered membrane permeable by attachment of an octaarginine sequence, inhibited cell proliferation and increased the protein levels of two previously established Pin1 substrates, PML and SMRT. Finally, a second generation of cell permeable Pin1 inhibitors was designed by replacing the noncritical residues within the cyclic peptide ring with arginine residues and shown to have anti-proliferative activity against the cancer cells. PMID:20180533

  14. Peptidyl-prolyl cis-trans isomerase Pin1 in ageing, cancer and Alzheimer disease.

    PubMed

    Lee, Tae Ho; Pastorino, Lucia; Lu, Kun Ping

    2011-06-20

    Phosphorylation of proteins on serine or threonine residues preceding proline is a key signalling mechanism in diverse physiological and pathological processes. Pin1 (peptidyl-prolyl cis-trans isomerase) is the only enzyme known that can isomerise specific Ser/Thr-Pro peptide bonds after phosphorylation and regulate their conformational changes with high efficiency. These Pin1-catalysed conformational changes can have profound effects on phosphorylation signalling by regulating a spectrum of target activities. Interestingly, Pin1 deregulation is implicated in a number of diseases, notably ageing and age-related diseases, including cancer and Alzheimer disease. Pin1 is overexpressed in most human cancers; it activates numerous oncogenes or growth enhancers and also inactivates a large number of tumour suppressors or growth inhibitors. By contrast, ablation of Pin1 prevents cancer, but eventually leads to premature ageing and neurodegeneration. Consistent with its neuroprotective role, Pin1 has been shown to be inactivated in neurons of patients with Alzheimer disease. Therefore, Pin1-mediated phosphorylation-dependent prolyl isomerisation represents a unique signalling mechanism that has a pivotal role in the development of human diseases, and might offer an attractive new diagnostic and therapeutic target.

  15. Mycobacterium tuberculosis Peptidyl-Prolyl Isomerases Are Immunogenic, Alter Cytokine Profile and Aid in Intracellular Survival

    PubMed Central

    Pandey, Saurabh; Tripathi, Deeksha; Khubaib, Mohd; Kumar, Ashutosh; Sheikh, Javaid A.; Sumanlatha, Gaddam; Ehtesham, Nasreen Z.; Hasnain, Seyed E.

    2017-01-01

    Mycobacterium tuberculosis (M. tb) has two peptidyl-prolyl isomerases (Ppiases) PpiA and PpiB, popularly known as cyclophilin A and cyclophilin B. The role of cyclophilins in processes such as signaling, cell surface recognition, chaperoning, and heat shock response has been well-documented. We present evidence that M. tb Ppiases modulate the host immune response. ELISA results revealed significant presence of antibodies to M. tb Ppiases in patient sera as compared to sera from healthy individuals. Treatment of THP-1 cells with increasing concentrations of rPpiA, induced secretion of pro-inflammatory cytokines TNF-α and IL-6. Alternatively, treatment with rPpiB inhibited secretion of TNF-α and induced secretion of IL-10. Furthermore, heterologous expression of M. tb PpiA and PpiB in Mycobacterium smegmatis increased bacterial survival in THP-1 cells as compared to those transformed with the vector control. Our results demonstrate that M. tb Ppiases are immunogenic proteins that can possibly modulate host immune response and enhance persistence of the pathogen within the host by subverting host cell generated stresses. PMID:28261567

  16. Cyclophilin A Peptidyl-Prolyl Isomerase Activity Promotes Zpr1 Nuclear Export

    PubMed Central

    Ansari, Husam; Greco, Giampaolo; Luban, Jeremy

    2002-01-01

    The peptidyl-prolyl isomerase (PPIase) cyclophilin A (Cpr1p) is conserved from eubacteria to mammals, yet its biological function has resisted elucidation. Unable to identify a phenotype that is suggestive of Cpr1p's function in a cpr1Δ Saccharomyces cerevisiae strain, we screened for CPR1-dependent strains. In all cases, dependence was conferred by mutations in ZPR1, a gene encoding an essential zinc finger protein. CPR1 dependence was suppressed by overexpression of EF1α (a translation factor that binds Zpr1p), Cpr6p (another cyclophilin), or Fpr1p (a structurally unrelated PPIase). Suppression by a panel of cyclophilin A mutants correlated with PPIase activity, confirming the relevance of this activity in CPR1-dependent strains. In CPR1+ cells, wild-type Zpr1p was distributed equally between the nucleus and cytoplasm. In contrast, proteins encoded by CPR1-dependent alleles of ZPR1 accumulated in the nucleus, as did wild-type Zpr1p in cpr1Δ cells. Transport kinetic studies indicated that nuclear export of Zpr1p was defective in cpr1Δ cells, and rescue of this defect correlated with PPIase activity. Our results demonstrate a functional interaction between Cpr1p, Zpr1p, and EF1α, a role for Cpr1p in Zpr1p nuclear export, and a biological function for Cpr1p PPIase activity. PMID:12242280

  17. Microbial Peptidyl-Prolyl cis/trans Isomerases (PPIases): Virulence Factors and Potential Alternative Drug Targets

    PubMed Central

    2014-01-01

    SUMMARY Initially discovered in the context of immunomodulation, peptidyl-prolyl cis/trans isomerases (PPIases) were soon identified as enzymes catalyzing the rate-limiting protein folding step at peptidyl bonds preceding proline residues. Intense searches revealed that PPIases are a superfamily of proteins consisting of three structurally distinguishable families with representatives in every described species of prokaryote and eukaryote and, recently, even in some giant viruses. Despite the clear-cut enzymatic activity and ubiquitous distribution of PPIases, reports on solely PPIase-dependent biological roles remain scarce. Nevertheless, they have been found to be involved in a plethora of biological processes, such as gene expression, signal transduction, protein secretion, development, and tissue regeneration, underscoring their general importance. Hence, it is not surprising that PPIases have also been identified as virulence-associated proteins. The extent of contribution to virulence is highly variable and dependent on the pleiotropic roles of a single PPIase in the respective pathogen. The main objective of this review is to discuss this variety in virulence-related bacterial and protozoan PPIases as well as the involvement of host PPIases in infectious processes. Moreover, a special focus is given to Legionella pneumophila macrophage infectivity potentiator (Mip) and Mip-like PPIases of other pathogens, as the best-characterized virulence-related representatives of this family. Finally, the potential of PPIases as alternative drug targets and first tangible results are highlighted. PMID:25184565

  18. Selective inactivation of parvulin-like peptidyl-prolyl cis/trans isomerases by juglone.

    PubMed

    Hennig, L; Christner, C; Kipping, M; Schelbert, B; Rücknagel, K P; Grabley, S; Küllertz, G; Fischer, G

    1998-04-28

    In contrast to FK506 binding proteins and cyclophilins, the parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases; E.C. 5.2.1.8) cannot be inhibited by either FK506 or cyclosporin A. We have found that juglone, 5-hydroxy-1,4-naphthoquinone, irreversibly inhibits the enzymatic activity of several parvulins, like the E. coli parvulin, the yeast Ess1/Ptf1, and human Pin1, in a specific manner, thus allowing selective inactivation of these enzymes in the presence of other PPIases. The mode of action was studied by analyzing the inactivation kinetics and the nature of products of the reaction of E. coli parvulin and its Cys69Ala variant with juglone. For all parvulins investigated, complete inactivation was obtained by a slow process that is characterized by pseudo-first-order rate constants in the range of 5.3 x 10(-)4 to 4. 5 x 10(-)3 s-1. The inactivated parvulin contains two juglone molecules that are covalently bound to the side chains of Cys41 and Cys69 because of a Michael addition of the thiol groups to juglone. Redox reactions did not contribute to the inactivation process. Because thiol group modification was shown to proceed 5-fold faster than the rate of enzyme inactivation, it was considered as a necessary but not sufficient condition for inactivation. When measured by far-UV circular dichroism (CD), the rate of structural alterations following thiol group modification parallels exactly the rate of inactivation. Thus, partial unfolding of the active site of the parvulins was thought to be the cause of the deterioration of PPIase activity.

  19. The Peptidyl-prolyl Isomerase Pin1 Up-regulation and Proapoptotic Function in Dopaminergic Neurons

    PubMed Central

    Ghosh, Anamitra; Saminathan, Hariharan; Kanthasamy, Arthi; Anantharam, Vellareddy; Jin, Huajun; Sondarva, Gautam; Harischandra, Dilshan S.; Qian, Ziqing; Rana, Ajay; Kanthasamy, Anumantha G.

    2013-01-01

    Parkinson disease (PD) is a chronic neurodegenerative disease characterized by a slow and progressive degeneration of dopaminergic neurons in substantia nigra. The pathophysiological mechanisms underlying PD remain unclear. Pin1, a major peptidyl-prolyl isomerase, has recently been associated with certain diseases. Notably, Ryo et al. (Ryo, A., Togo, T., Nakai, T., Hirai, A., Nishi, M., Yamaguchi, A., Suzuki, K., Hirayasu, Y., Kobayashi, H., Perrem, K., Liou, Y. C., and Aoki, I. (2006) J. Biol. Chem. 281, 4117–4125) implicated Pin1 in PD pathology. Therefore, we sought to systematically characterize the role of Pin1 in PD using cell culture and animal models. To our surprise we observed a dramatic up-regulation of Pin1 mRNA and protein levels in dopaminergic MN9D neuronal cells treated with the parkinsonian toxicant 1-methyl-4-phenylpyridinium (MPP+) as well as in the substantia nigra of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. Notably, a marked expression of Pin1 was also observed in the substantia nigra of human PD brains along with a high co-localization of Pin1 within dopaminergic neurons. In functional studies, siRNA-mediated knockdown of Pin1 almost completely prevented MPP+-induced caspase-3 activation and DNA fragmentation, indicating that Pin1 plays a proapoptotic role. Interestingly, multiple pharmacological Pin1 inhibitors, including juglone, attenuated MPP+-induced Pin1 up-regulation, α-synuclein aggregation, caspase-3 activation, and cell death. Furthermore, juglone treatment in the MPTP mouse model of PD suppressed Pin1 levels and improved locomotor deficits, dopamine depletion, and nigral dopaminergic neuronal loss. Collectively, our findings demonstrate for the first time that Pin1 is up-regulated in PD and has a pathophysiological role in the nigrostriatal dopaminergic system and suggest that modulation of Pin1 levels may be a useful translational therapeutic strategy in PD. PMID:23754278

  20. Targeting peptidyl-prolyl isomerase pin1 to inhibit tumor cell aggressiveness.

    PubMed

    Beretta, Giovanni L; De Cesare, Michelandrea; Albano, Luisa; Magnifico, Alessandra; Carenini, Nives; Corna, Elisabetta; Perego, Paola; Gatti, Laura

    2016-01-01

    Because the peptidyl-prolyl isomerase PIN1 interacts with multiple protein kinases and phosphoproteins into a network orchestrating the cellular response to various stimuli, there is an increasing interest in exploiting its potential as therapeutic target. In the present study, the effect of targeting PIN1 was investigated in 2 human cancer cell lines characterized by increased aggressive potential, high expression of erbB receptor family members, and defective p53. PIN1 silencing was carried out in skin squamous cell carcinoma A431 cells displaying elevated EGFR/HER1 levels and in ovarian adenocarcinoma SKOV-3 cells displaying high levels of erbB2 (HER2). Nonoverlapping siRNA duplexes targeting different regions of PIN1 mRNA were transfected in tumor cells, which were analyzed using Western blotting for the expression of selected proteins. In vivo tumorigenicity studies were carried out in athymic nude mice. A431 and SKOV-3 cell systems were found to be a source of cells with increased aggressive potential, i.e., cancer stem cell-like cells, as defined by the capability to grow as spheres. A marked decrease of PIN1 levels and of sphere-forming capability was observed in PIN1-silenced cells. The expression of phospho-p38 decreased following PIN1 silencing in A431 and SKOV-3 cells, as well as phospho-EGFR levels in A431 - silenced cells. PIN1 inhibition prolonged latency and reduced tumor take and growth of SKOV-3 cells in nude mice. Our results support that PIN1 may be a valuable target to hit in cancer cells characterized by increased aggressive potential, overexpression of erbB receptor family members, and defective p53.

  1. Secretion by Trypanosoma cruzi of a peptidyl-prolyl cis-trans isomerase involved in cell infection.

    PubMed Central

    Moro, A; Ruiz-Cabello, F; Fernández-Cano, A; Stock, R P; González, A

    1995-01-01

    Macrophage infectivity potentiators are membrane proteins described as virulence factors in bacterial intracellular parasites, such as Legionella and Chlamydia. These factors share amino acid homology to eukaryotic peptidyl-prolyl cis-trans isomerases that are inhibited by FK506, an inhibitor of signal transduction in mammalian cells with potent immunosuppressor activity. We report here the characterization of a protein released into the culture medium by the infective stage of the protozoan intracellular parasite Trypanosoma cruzi. The protein possesses a peptidyl-prolyl cis-trans isomerase activity that is inhibited by FK506 and its non-immunosuppressing derivative L-685,818. The corresponding gene presents sequence homology with bacterial macrophage infectivity potentiators. The addition of the protein, produced heterologously in Escherichia coli, to cultures of trypomastigotes and simian epithelial or HeLa cells enhances invasion of the mammalian cells by the parasites. Antibodies raised in mice against the T.cruzi isomerase greatly reduce infectivity. A similar reduction of infectivity is obtained by addition to the cultures of FK506 and L-685,818. We concluded that the T.cruzi isomerase is involved in cell invasion. Images PMID:7540135

  2. Synthetic Lethality of Retinoblastoma Mutant Cells in the Drosophila Eye by Mutation of a Novel Peptidyl Prolyl Isomerase Gene

    PubMed Central

    Edgar, Kyle A.; Belvin, Marcia; Parks, Annette L.; Whittaker, Kellie; Mahoney, Matt B.; Nicoll, Monique; Park, Christopher C.; Winter, Christopher G.; Chen, Feng; Lickteig, Kim; Ahmad, Ferhad; Esengil, Hanife; Lorenzi, Matthew V.; Norton, Amanda; Rupnow, Brent A.; Shayesteh, Laleh; Tabios, Mariano; Young, Lynn M.; Carroll, Pamela M.; Kopczynski, Casey; Plowman, Gregory D.; Friedman, Lori S.; Francis-Lang, Helen L.

    2005-01-01

    Mutations that inactivate the retinoblastoma (Rb) pathway are common in human tumors. Such mutations promote tumor growth by deregulating the G1 cell cycle checkpoint. However, uncontrolled cell cycle progression can also produce new liabilities for cell survival. To uncover such liabilities in Rb mutant cells, we performed a clonal screen in the Drosophila eye to identify second-site mutations that eliminate Rbf− cells, but allow Rbf+ cells to survive. Here we report the identification of a mutation in a novel highly conserved peptidyl prolyl isomerase (PPIase) that selectively eliminates Rbf− cells from the Drosophila eye. PMID:15744054

  3. The Wheat Peptidyl Prolyl cis-trans-Isomerase FKBP77 Is Heat Induced and Developmentally Regulated1

    PubMed Central

    Kurek, Isaac; Aviezer, Keren; Erel, Noa; Herman, Eliot; Breiman, Adina

    1999-01-01

    We isolated a cDNA encoding a 568-amino acid, heat-stress-induced peptidyl prolyl isomerase belonging to the FK506-binding-protein (FKBP) family. The open reading frame encodes for a peptidyl prolyl isomerase that possesses three FKBP-12-like domains, a putative tetratricopeptide motif, and a calmodulin-binding domain. Specific antibodies showed that the open reading frame encodes a heat-induced 77-kD protein, the wheat FKBP77 (wFKBP77), which exhibits 84% identity with the wFKBP73 and 42% identity with the human FKBP59. Because of the high similarity in sequence to wFKBP73, wFKBP77 was designated as the heat-induced isoform. The wFKBP77 mRNA steady-state level was 14-fold higher at 37°C than at 25°C. The wFKBP77 transcript abundance was the highest in mature embryos that had imbibed and 2-d-old green shoots exposed to 37°C, and decreased to 6% in 6-d-old green shoots. The transcript level returned to the level detected at 25°C after recovery of the embryos for 90 min at 25°C. We compared wFKBP73 and wFKBP77 with the heat-shock proteins having cognate and heat-stress-induced counterparts. PMID:9952466

  4. Identification and comparative analysis of sixteen fungal peptidyl-prolyl cis/trans isomerase repertoires

    PubMed Central

    Pemberton, Trevor J

    2006-01-01

    Background The peptidyl-prolyl cis/trans isomerase (PPIase) class of proteins is present in all known eukaryotes, prokaryotes, and archaea, and it is comprised of three member families that share the ability to catalyze the cis/trans isomerisation of a prolyl bond. Some fungi have been used as model systems to investigate the role of PPIases within the cell, however how representative these repertoires are of other fungi or humans has not been fully investigated. Results PPIase numbers within these fungal repertoires appears associated with genome size and orthology between repertoires was found to be low. Phylogenetic analysis showed the single-domain FKBPs to evolve prior to the multi-domain FKBPs, whereas the multi-domain cyclophilins appear to evolve throughout cyclophilin evolution. A comparison of their known functions has identified, besides a common role within protein folding, multiple roles for the cyclophilins within pre-mRNA splicing and cellular signalling, and within transcription and cell cycle regulation for the parvulins. However, no such commonality was found with the FKBPs. Twelve of the 17 human cyclophilins and both human parvulins, but only one of the 13 human FKBPs, identified orthologues within these fungi. hPar14 orthologues were restricted to the Pezizomycotina fungi, and R. oryzae is unique in the known fungi in possessing an hCyp33 orthologue and a TPR-containing FKBP. The repertoires of Cryptococcus neoformans, Aspergillus fumigatus, and Aspergillus nidulans were found to exhibit the highest orthology to the human repertoire, and Saccharomyces cerevisiae one of the lowest. Conclusion Given this data, we would hypothesize that: (i) the evolution of the fungal PPIases is driven, at least in part, by the size of the proteome, (ii) evolutionary pressures differ both between the different PPIase families and the different fungi, and (iii) whilst the cyclophilins and parvulins have evolved to perform conserved functions, the FKBPs have

  5. Noncatalytic Role of the FKBP52 Peptidyl-Prolyl Isomerase Domain in the Regulation of Steroid Hormone Signaling▿

    PubMed Central

    Riggs, Daniel L.; Cox, Marc B.; Tardif, Heather L.; Hessling, Martin; Buchner, Johannes; Smith, David F.

    2007-01-01

    Hormone-dependent transactivation by several of the steroid hormone receptors is potentiated by the Hsp90-associated cochaperone FKBP52, although not by the closely related FKBP51. Here we analyze the mechanisms of potentiation and the functional differences between FKBP51 and FKBP52. While both have peptidyl-prolyl isomerase activity, this is not required for potentiation, as mutations abolishing isomerase activity did not affect potentiation. Genetic selection in Saccharomyces cerevisiae for gain of potentiation activity in a library of randomly mutated FKBP51 genes identified a single residue at position 119 in the N-terminal FK1 domain as being a critical difference between these two proteins. In both the yeast model and mammalian cells, the FKBP51 mutation L119P, which is located in a hairpin loop overhanging the catalytic pocket and introduces the proline found in FKBP52, conferred significant potentiation activity, whereas the converse P119L mutation in FKBP52 decreased potentiation. A second residue in this loop, A116, also influences potentiation levels; in fact, the FKBP51-A116V L119P double mutant potentiated hormone signaling as well as wild-type FKBP52 did. These results suggest that the FK1 domain, and in particular the loop overhanging the catalytic pocket, is critically involved in receptor interactions and receptor activity. PMID:17938211

  6. Noncatalytic role of the FKBP52 peptidyl-prolyl isomerase domain in the regulation of steroid hormone signaling.

    PubMed

    Riggs, Daniel L; Cox, Marc B; Tardif, Heather L; Hessling, Martin; Buchner, Johannes; Smith, David F

    2007-12-01

    Hormone-dependent transactivation by several of the steroid hormone receptors is potentiated by the Hsp90-associated cochaperone FKBP52, although not by the closely related FKBP51. Here we analyze the mechanisms of potentiation and the functional differences between FKBP51 and FKBP52. While both have peptidyl-prolyl isomerase activity, this is not required for potentiation, as mutations abolishing isomerase activity did not affect potentiation. Genetic selection in Saccharomyces cerevisiae for gain of potentiation activity in a library of randomly mutated FKBP51 genes identified a single residue at position 119 in the N-terminal FK1 domain as being a critical difference between these two proteins. In both the yeast model and mammalian cells, the FKBP51 mutation L119P, which is located in a hairpin loop overhanging the catalytic pocket and introduces the proline found in FKBP52, conferred significant potentiation activity, whereas the converse P119L mutation in FKBP52 decreased potentiation. A second residue in this loop, A116, also influences potentiation levels; in fact, the FKBP51-A116V L119P double mutant potentiated hormone signaling as well as wild-type FKBP52 did. These results suggest that the FK1 domain, and in particular the loop overhanging the catalytic pocket, is critically involved in receptor interactions and receptor activity.

  7. FKBP65-dependent peptidyl-prolyl isomerase activity potentiates the lysyl hydroxylase 2-driven collagen cross-link switch

    PubMed Central

    Chen, Yulong; Terajima, Masahiko; Banerjee, Priyam; Guo, Houfu; Liu, Xin; Yu, Jiang; Yamauchi, Mitsuo; Kurie, Jonathan M.

    2017-01-01

    Bruck Syndrome is a connective tissue disease associated with inactivating mutations in lysyl hydroxylase 2 (LH2/PLOD2) or FK506 binding protein 65 (FKBP65/FKBP10). However, the functional relationship between LH2 and FKBP65 remains unclear. Here, we postulated that peptidyl prolyl isomerase (PPIase) activity of FKBP65 positively modulates LH2 enzymatic activity and is critical for the formation of hydroxylysine-aldehyde derived intermolecular collagen cross-links (HLCCs). To test this hypothesis, we analyzed collagen cross-links in Fkbp10-null and –wild-type murine embryonic fibroblasts. Although LH2 protein levels did not change, FKBP65 deficiency significantly diminished HLCCs and increased the non-hydroxylated lysine-aldehyde–derived collagen cross-links (LCCs), a pattern consistent with loss of LH2 enzymatic activity. The HLCC-to-LCC ratio was rescued in FKBP65-deficient murine embryonic fibroblasts by reconstitution with wild-type but not mutant FKBP65 that lacks intact PPIase domains. Findings from co-immunoprecipitation, protein-fragment complementation, and co-immunofluorescence assays showed that LH2 and FKBP65 are part of a common protein complex. We conclude that FKBP65 regulates LH2-mediated collagen cross-linking. Because LH2 promotes fibrosis and cancer metastasis, our findings suggest that pharmacologic strategies to target FKBP65 and LH2 may have complementary therapeutic activities. PMID:28378777

  8. The emerging role of peptidyl-prolyl isomerase chaperones in tau oligomerization, amyloid processing and Alzheimer's disease

    PubMed Central

    Blair, Laura J.; Baker, Jeremy D.; Sabbagh, Jonathan J.; Dickey, Chad A.

    2015-01-01

    Peptidyl-prolyl cis/trans isomerases (PPIases), a unique family of molecular chaperones, regulate protein folding at proline residues. These residues are abundant within intrinsically disordered proteins, like the microtubule-associated protein tau. Tau has been shown to become hyperphosphorylated and accumulate as one of the two main pathological hallmarks in Alzheimer's disease (AD), the other being amyloid beta (Aβ). PPIases, including Pin1, FK506-binding protein (FKBP) 52, FKBP51, and FKBP12, have been shown to interact with and regulate tau biology. This interaction is particularly important given the numerous proline-directed phosphorylation sites found on tau and the role phosphorylation has been found to play in pathogenesis. This regulation then affects downstream aggregation and oligomerization of tau. However, many PPIases have yet to be explored for their effects on tau biology, despite the high likelihood of interaction based on proline content. Moreover, Pin1, FKBP12, FKBP52, cyclophilin (Cyp) A, CypB, and CypD have been shown to also regulate Aβ production or the toxicity associated with Aβ pathology. Therefore, PPIases directly and indirectly regulate pathogenic protein multimerization in AD and represent a family rich in targets for modulating the accumulation and toxicity. PMID:25628064

  9. Active site mutants of human cyclophilin A separate peptidyl-prolyl isomerase activity from cyclosporin A binding and calcineurin inhibition.

    PubMed Central

    Zydowsky, L. D.; Etzkorn, F. A.; Chang, H. Y.; Ferguson, S. B.; Stolz, L. A.; Ho, S. I.; Walsh, C. T.

    1992-01-01

    Based on recent X-ray structural information, six site-directed mutants of human cyclophilin A (hCyPA) involving residues in the putative active site--H54, R55, F60, Q111, F113, and H126--have been constructed, overexpressed, and purified from Escherichia coli to homogeneity. The proteins W121A (Liu, J., Chen, C.-M., & Walsh, C.T., 1991a, Biochemistry 30, 2306-2310), H54Q, R55A, F60A, Q111A, F113A, and H126Q were assayed for cis-trans peptidyl-prolyl isomerase (PPIase) activity, their ability to bind the immunosuppressive drug cyclosporin A (CsA), and protein phosphatase 2B (calcineurin) inhibition in the presence of CsA. Results indicate that H54Q, Q111A, F113A, and W121A retain 3-15% of the catalytic efficiency (kcat/Km) of wild-type recombinant hCyPA. The remaining three mutants (R55A, F60A, and H126Q) each retain less than 1% of the wild-type catalytic efficiency, indicating participation by these residues in PPIase catalysis. Each of the mutants bound to a CsA affinity matrix. The mutants R55A, F60A, F113A, and H126Q inhibited calcineurin in the presence of CsA, whereas W121A did not. Although CsA is a competitive inhibitor of PPIase activity, it can complex with enzymatically inactive cyclophilins and inhibit the phosphatase activity of calcineurin. PMID:1338979

  10. Peptidyl-prolyl cis/trans-isomerase A1 (Pin1) is a target for modification by lipid electrophiles.

    PubMed

    Aluise, Christopher D; Rose, Kristie; Boiani, Mariana; Reyzer, Michelle L; Manna, Joseph D; Tallman, Keri; Porter, Ned A; Marnett, Lawrence J

    2013-02-18

    Oxidation of membrane phospholipids is associated with inflammation, neurodegenerative disease, and cancer. Oxyradical damage to phospholipids results in the production of reactive aldehydes that adduct proteins and modulate their function. 4-Hydroxynonenal (HNE), a common product of oxidative damage to lipids, adducts proteins at exposed Cys, His, or Lys residues. Here, we demonstrate that peptidyl-prolyl cis/trans-isomerase A1 (Pin1), an enzyme that catalyzes the conversion of the peptide bond of pSer/pThr-Pro moieties in signaling proteins from cis to trans, is highly susceptible to HNE modification. Incubation of purified Pin1 with HNE followed by MALDI-TOF/TOF mass spectrometry resulted in detection of Michael adducts at the active site residues His-157 and Cys-113. Time and concentration dependencies indicate that Cys-113 is the primary site of HNE modification. Pin1 was adducted in MDA-MB-231 breast cancer cells treated with 8-alkynyl-HNE as judged by click chemistry conjugation with biotin followed by streptavidin-based pulldown and Western blotting with anti-Pin1 antibody. Furthermore, orbitrap MS data support the adduction of Cys-113 in the Pin1 active site upon HNE treatment of MDA-MB-231 cells. siRNA knockdown of Pin1 in MDA-MB-231 cells partially protected the cells from HNE-induced toxicity. Recent studies indicate that Pin1 is an important molecular target for the chemopreventive effects of green tea polyphenols. The present study establishes that it is also a target for electrophilic modification by products of lipid peroxidation.

  11. Ziploc-ing the structure 2.0: Endoplasmic reticulum-resident peptidyl prolyl isomerases show different activities toward hydroxyproline.

    PubMed

    Ishikawa, Yoshihiro; Mizuno, Kazunori; Bächinger, Hans Peter

    2017-06-02

    Extracellular matrix proteins are biosynthesized in the rough endoplasmic reticulum (rER), and the triple-helical protein collagen is the most abundant extracellular matrix component in the human body. Many enzymes, molecular chaperones, and post-translational modifiers facilitate collagen biosynthesis. Collagen contains a large number of proline residues, so the cis/trans isomerization of proline peptide bonds is the rate-limiting step during triple-helix formation. Accordingly, the rER-resident peptidyl prolyl cis/trans isomerases (PPIases) play an important role in the zipper-like triple-helix formation in collagen. We previously described this process as "Ziploc-ing the structure" and now provide additional information on the activity of individual rER PPIases. We investigated the substrate preferences of these PPIases in vitro using type III collagen, the unhydroxylated quarter fragment of type III collagen, and synthetic peptides as substrates. We observed changes in activity of six rER-resident PPIases, cyclophilin B (encoded by the PPIB gene), FKBP13 (FKBP2), FKBP19 (FKBP11), FKBP22 (FKBP14), FKBP23 (FKBP7), and FKBP65 (FKBP10), due to posttranslational modifications of proline residues in the substrate. Cyclophilin B and FKBP13 exhibited much lower activity toward post-translationally modified substrates. In contrast, FKBP19, FKBP22, and FKBP65 showed increased activity toward hydroxyproline-containing peptide substrates. Moreover, FKBP22 showed a hydroxyproline-dependent effect by increasing the amount of refolded type III collagen in vitro and FKBP19 seems to interact with triple helical type I collagen. Therefore, we propose that hydroxyproline modulates the rate of Ziploc-ing of the triple helix of collagen in the rER. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Structure of human peptidyl-prolyl cis–trans isomerase FKBP22 containing two EF-hand motifs

    PubMed Central

    Boudko, Sergei P; Ishikawa, Yoshihiro; Nix, Jay; Chapman, Michael S; Bächinger, Hans Peter

    2014-01-01

    The FK506-binding protein (FKBP) family consists of proteins with a variety of protein–protein interaction domains and versatile cellular functions. It is assumed that all members are peptidyl-prolyl cis–trans isomerases with the enzymatic function attributed to the FKBP domain. Six members of this family localize to the mammalian endoplasmic reticulum (ER). Four of them, FKBP22 (encoded by the FKBP14 gene), FKBP23 (FKBP7), FKBP60 (FKBP9), and FKBP65 (FKBP10), are unique among all FKBPs as they contain the EF-hand motifs. Little is known about the biological roles of these proteins, but emerging genetics studies are attracting great interest to the ER resident FKBPs, as mutations in genes encoding FKBP10 and FKBP14 were shown to cause a variety of matrix disorders. Although the structural organization of the FKBP-type domain as well as of the EF-hand motif has been known for a while, it is difficult to conclude how these structures are combined and how it affects the protein functionality. We have determined a unique 1.9 Å resolution crystal structure for human FKBP22, which can serve as a prototype for other EF hand-containing FKBPs. The EF-hand motifs of two FKBP22 molecules form a dimeric complex with an elongated and predominantly hydrophobic cavity that can potentially be occupied by an aliphatic ligand. The FKBP-type domains are separated by a cleft and their putative active sites can catalyze isomerazation of two bonds within a polypeptide chain in extended conformation. These structural results are of prime interest for understanding biological functions of ER resident FKBPs containing EF-hand motifs. PMID:24272907

  13. A cyclophilin-like peptidyl-prolyl cis/trans isomerase from Legionella pneumophila--characterization, molecular cloning and overexpression.

    PubMed

    Schmidt, B; Tradler, T; Rahfeld, J U; Ludwig, B; Jain, B; Mann, K; Rücknagel, K P; Janowski, B; Schierhorn, A; Küllertz, G; Hacker, J; Fischer, G

    1996-09-01

    Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires' disease). A major virulence factor, the Mip protein (FK506-binding protein, FKBP25mem), belongs to the enzyme family of peptidyl-prolyl cis/trans isomerases (PPlases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPlase at a level of enzyme activity comparable to that of FKBP25mem. The N-terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPlases. The Icy gene (Legionella cyclophilin) was cloned and sequenced. It encodes a putative 164-amino-acid protein with a molecular mass of 17968 Da called L. pneumophila cyclophilin 18 (L.p.Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coli with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19-amino-acid polypeptide extension including a 6-histidine tag and an enterokinase cleavage site exhibits PPlase activity when produced at high levels in E. coli K-12. After removal of the extension by enterokinase, the properties of the recombinant Cyp18 were indistinguishable from those of the authentic enzyme. In order to investigate the influence of Cyp18 on intracellular survival of L. pneumophila an Icy-negative L. pneumophila strain was constructed. Compared with the wild-type strain, the mutant did not exhibit a significant phenotype but was 10-fold less invasive for Acanthamoeba castellanii. Like human cyclophilin, the L. p. Cyp18 exhibits nuclease activity, but this enzymatic activity does not appear to be linked with the native structure of the protein.

  14. Cyclophilin J Is a Novel Peptidyl-Prolyl Isomerase and Target for Repressing the Growth of Hepatocellular Carcinoma

    PubMed Central

    Chen, Jian; Chen, Shuai; Wang, Jiahui; Zhang, Mingjun; Gong, Zhaohua; Wei, Youheng; Li, Li; Zhang, Yuanyuan; Zhao, Xuemei; Jiang, Songmin; Yu, Long

    2015-01-01

    Cyclophilin J (CYPJ) is a new member of the peptidyl-prolyl cis/trans-isomerase (PPIase) identified with upregulated expression in human glioma. However, the biological function of CYPJ remained unclear. We aimed to study the role of CYPJ in hepatocellular carcinoma (HCC) carcinogenesis and its therapeutic potential. We determined the expression of CYPJ in HCC/adjacent normal tissues using Western blot, Northern blot and semi-quantitative RT-PCR, analyzed the biochemical characteristics of CYPJ, and resolved the 3D-structure of CYPJ/Cyclosporin A (CsA) complex. We also studied the roles of CYPJ in cell cycle, cyclin D1 regulation, in vitro and in vivo tumor growth. We found that CYPJ expression was upregulated in over 60% HCC tissues. The PPIase activity of CYPJ could be inhibited by the widely used immunosuppressive drug CsA. CYPJ was found expressed in the whole cell of HCC with preferential location at the cell nucleus. CYPJ promoted the transition of cells from G1 phase to S phase in a PPIase-dependent manner by activating cyclin D1 promoter. CYPJ overexpression accelerated liver cell growth in vitro (cell growth assay, colony formation) and in vivo (xenograft tumor formation). Inhibition of CYPJ by its inhibitor CsA or CYPJ-specific RNAi diminished the growth of liver cancer cells in vitro and in vivo. In conclusion, CYPJ could facilitate HCC growth by promoting cell cycle transition from G1 to S phase through the upregulation of cyclin D1. Suppression of CYPJ could repress the growth of HCC, which makes CYPJ a potential target for the development of new strategies to treat this malignancy. PMID:26020957

  15. Cyclophilin J is a novel peptidyl-prolyl isomerase and target for repressing the growth of hepatocellular carcinoma.

    PubMed

    Chen, Jian; Chen, Shuai; Wang, Jiahui; Zhang, Mingjun; Gong, Zhaohua; Wei, Youheng; Li, Li; Zhang, Yuanyuan; Zhao, Xuemei; Jiang, Songmin; Yu, Long

    2015-01-01

    Cyclophilin J (CYPJ) is a new member of the peptidyl-prolyl cis/trans-isomerase (PPIase) identified with upregulated expression in human glioma. However, the biological function of CYPJ remained unclear. We aimed to study the role of CYPJ in hepatocellular carcinoma (HCC) carcinogenesis and its therapeutic potential. We determined the expression of CYPJ in HCC/adjacent normal tissues using Western blot, Northern blot and semi-quantitative RT-PCR, analyzed the biochemical characteristics of CYPJ, and resolved the 3D-structure of CYPJ/Cyclosporin A (CsA) complex. We also studied the roles of CYPJ in cell cycle, cyclin D1 regulation, in vitro and in vivo tumor growth. We found that CYPJ expression was upregulated in over 60% HCC tissues. The PPIase activity of CYPJ could be inhibited by the widely used immunosuppressive drug CsA. CYPJ was found expressed in the whole cell of HCC with preferential location at the cell nucleus. CYPJ promoted the transition of cells from G1 phase to S phase in a PPIase-dependent manner by activating cyclin D1 promoter. CYPJ overexpression accelerated liver cell growth in vitro (cell growth assay, colony formation) and in vivo (xenograft tumor formation). Inhibition of CYPJ by its inhibitor CsA or CYPJ-specific RNAi diminished the growth of liver cancer cells in vitro and in vivo. In conclusion, CYPJ could facilitate HCC growth by promoting cell cycle transition from G1 to S phase through the upregulation of cyclin D1. Suppression of CYPJ could repress the growth of HCC, which makes CYPJ a potential target for the development of new strategies to treat this malignancy.

  16. Presence of autoantibodies to peptidyl-prolyl cis-trans isomerase (cyclosporin A-binding protein) in systemic lupus erythematosus.

    PubMed

    Harigai, M; Hara, M; Takahashi, N; Kitani, A; Hirose, T; Suzuki, K; Kawakami, M; Hidaka, T; Kawaguchi, Y; Ishizuka, T

    1992-04-01

    Several autoantibodies against cytoplasmic or nuclear components of cells have been reported in autoimmune diseases. We report here a previously unrecognized autoantibody to peptidyl-prolyl cis-trans isomerase (PPIase) in patients with systemic lupus erythematosus (SLE). PPIase, which catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides, has recently been found to be identical to cyclophilin, a specific binding protein of a potent immunosuppressant, cyclosporin A. IgG and IgM anti-PPIase antibodies were detected in 40 and 20% of unselected patients with SLE, respectively, by ELISA. The reactivity of these sera was confirmed by immunoblotting experiments. Sera from rheumatoid arthritis patients showed no reactivity and 1 of 8 sera from systemic sclerosis patients and 1 of 25 sera from normal controls showed only weak reactivity. Unexpectedly, the anti-PPIase antibody was unable to inhibit PPIase activity, indicating that the autoantibody recognizes an epitope of PPIase which is different from the active site of PPIase. The levels of the anti-PPIase antibody in SLE patients correlated with remissions and flares of the disease. The anti-PPIase antibody was higher in patients with active SLE than those with inactive disease. The prevalence of the active stage of the disease was significantly higher in IgG anti-PPIase antibody-positive SLE patients as compared to antibody-negative SLE patients. These data define the presence of a new autoantibody against PPIase and its association with the activity and certain clinical manifestations in SLE.

  17. Structure and Activity of the Peptidyl-Prolyl Isomerase Domain from the Histone Chaperone Fpr4 toward Histone H3 Proline Isomerization*

    PubMed Central

    Monneau, Yoan R.; Soufari, Heddy; Nelson, Christopher J.; Mackereth, Cameron D.

    2013-01-01

    The FK506-binding protein (FKBP) family of peptidyl-prolyl isomerases (PPIases) is characterized by a common catalytic domain that binds to the inhibitors FK506 and rapamycin. As one of four FKBPs within the yeast Saccharomyces cerevisiae, Fpr4 has been described as a histone chaperone, and is in addition implicated in epigenetic function in part due to its mediation of cis-trans conversion of proline residues within histone tails. To better understand the molecular details of this activity, we have determined the solution structure of the Fpr4 C-terminal PPIase domain by using NMR spectroscopy. This canonical FKBP domain actively increases the rate of isomerization of three decapeptides derived from the N terminus of yeast histone H3, whereas maintaining intrinsic cis and trans populations. Observation of the uncatalyzed and Fpr4-catalyzed isomerization rates at equilibrium demonstrate Pro16 and Pro30 of histone H3 as the major proline targets of Fpr4, with little activity shown against Pro38. This alternate ranking of the three target prolines, as compared with affinity determination or the classical chymotrypsin-based fluorescent assay, reveals the mechanistic importance of substrate residues C-terminal to the peptidyl-prolyl bond. PMID:23888048

  18. Structure and activity of the peptidyl-prolyl isomerase domain from the histone chaperone Fpr4 toward histone H3 proline isomerization.

    PubMed

    Monneau, Yoan R; Soufari, Heddy; Nelson, Christopher J; Mackereth, Cameron D

    2013-09-06

    The FK506-binding protein (FKBP) family of peptidyl-prolyl isomerases (PPIases) is characterized by a common catalytic domain that binds to the inhibitors FK506 and rapamycin. As one of four FKBPs within the yeast Saccharomyces cerevisiae, Fpr4 has been described as a histone chaperone, and is in addition implicated in epigenetic function in part due to its mediation of cis-trans conversion of proline residues within histone tails. To better understand the molecular details of this activity, we have determined the solution structure of the Fpr4 C-terminal PPIase domain by using NMR spectroscopy. This canonical FKBP domain actively increases the rate of isomerization of three decapeptides derived from the N terminus of yeast histone H3, whereas maintaining intrinsic cis and trans populations. Observation of the uncatalyzed and Fpr4-catalyzed isomerization rates at equilibrium demonstrate Pro(16) and Pro(30) of histone H3 as the major proline targets of Fpr4, with little activity shown against Pro(38). This alternate ranking of the three target prolines, as compared with affinity determination or the classical chymotrypsin-based fluorescent assay, reveals the mechanistic importance of substrate residues C-terminal to the peptidyl-prolyl bond.

  19. Peptidyl prolyl isomerase Pin1-inhibitory activity of D-glutamic and D-aspartic acid derivatives bearing a cyclic aliphatic amine moiety.

    PubMed

    Nakagawa, Hidehiko; Seike, Suguru; Sugimoto, Masatoshi; Ieda, Naoya; Kawaguchi, Mitsuyasu; Suzuki, Takayoshi; Miyata, Naoki

    2015-12-01

    Pin1 is a peptidyl prolyl isomerase that specifically catalyzes cis-trans isomerization of phosphorylated Thr/Ser-Pro peptide bonds in substrate proteins and peptides. Pin1 is involved in many important cellular processes, including cancer progression, so it is a potential target of cancer therapy. We designed and synthesized a novel series of Pin1 inhibitors based on a glutamic acid or aspartic acid scaffold bearing an aromatic moiety to provide a hydrophobic surface and a cyclic aliphatic amine moiety with affinity for the proline-binding site of Pin1. Glutamic acid derivatives bearing cycloalkylamino and phenylthiazole groups showed potent Pin1-inhibitory activity comparable with that of known inhibitor VER-1. The results indicate that steric interaction of the cyclic alkyl amine moiety with binding site residues plays a key role in enhancing Pin1-inhibitory activity.

  20. Characterization of Peptidyl-Prolyl Cis-Trans Isomerase- and Calmodulin-Binding Activity of a Cytosolic Arabidopsis thaliana Cyclophilin AtCyp19-3

    PubMed Central

    Kaur, Gundeep; Singh, Supreet; Singh, Harpreet; Chawla, Mrinalini; Dutta, Tanima; Kaur, Harsimran; Bender, Kyle; Snedden, W. A.; Kapoor, Sanjay; Pareek, Ashwani; Singh, Prabhjeet

    2015-01-01

    Cyclophilins, which bind to immunosuppressant cyclosporin A (CsA), are ubiquitous proteins and constitute a multigene family in higher organisms. Several members of this family are reported to catalyze cis-trans isomerisation of the peptidyl-prolyl bond, which is a rate limiting step in protein folding. The physiological role of these proteins in plants, with few exceptions, is still a matter of speculation. Although Arabidopsis genome is predicted to contain 35 cyclophilin genes, biochemical characterization, imperative for understanding their cellular function(s), has been carried only for few of the members. The present study reports the biochemical characterization of an Arabidopsis cyclophilin, AtCyp19-3, which demonstrated that this protein is enzymatically active and possesses peptidyl-prolyl cis-trans isomerase (PPIase) activity that is specifically inhibited by CsA with an inhibition constant (Ki) of 18.75 nM. The PPIase activity of AtCyp19-3 was also sensitive to Cu2+, which covalently reacts with the sulfhydryl groups, implying redox regulation. Further, using calmodulin (CaM) gel overlay assays it was demonstrated that in vitro interaction of AtCyp19-3 with CaM is Ca2+-dependent, and CaM-binding domain is localized to 35–70 amino acid residues in the N-terminus. Bimolecular fluorescence complementation assays showed that AtCyp19-3 interacts with CaM in vivo also, thus, validating the in vitro observations. However, the PPIase activity of the Arabidopsis cyclophilin was not affected by CaM. The implications of these findings are discussed in the context of Ca2+ signaling and cyclophilin activity in Arabidopsis. PMID:26317213

  1. Characterization of Peptidyl-Prolyl Cis-Trans Isomerase- and Calmodulin-Binding Activity of a Cytosolic Arabidopsis thaliana Cyclophilin AtCyp19-3.

    PubMed

    Kaur, Gundeep; Singh, Supreet; Singh, Harpreet; Chawla, Mrinalini; Dutta, Tanima; Kaur, Harsimran; Bender, Kyle; Snedden, W A; Kapoor, Sanjay; Pareek, Ashwani; Singh, Prabhjeet

    2015-01-01

    Cyclophilins, which bind to immunosuppressant cyclosporin A (CsA), are ubiquitous proteins and constitute a multigene family in higher organisms. Several members of this family are reported to catalyze cis-trans isomerisation of the peptidyl-prolyl bond, which is a rate limiting step in protein folding. The physiological role of these proteins in plants, with few exceptions, is still a matter of speculation. Although Arabidopsis genome is predicted to contain 35 cyclophilin genes, biochemical characterization, imperative for understanding their cellular function(s), has been carried only for few of the members. The present study reports the biochemical characterization of an Arabidopsis cyclophilin, AtCyp19-3, which demonstrated that this protein is enzymatically active and possesses peptidyl-prolyl cis-trans isomerase (PPIase) activity that is specifically inhibited by CsA with an inhibition constant (Ki) of 18.75 nM. The PPIase activity of AtCyp19-3 was also sensitive to Cu(2+), which covalently reacts with the sulfhydryl groups, implying redox regulation. Further, using calmodulin (CaM) gel overlay assays it was demonstrated that in vitro interaction of AtCyp19-3 with CaM is Ca(2+)-dependent, and CaM-binding domain is localized to 35-70 amino acid residues in the N-terminus. Bimolecular fluorescence complementation assays showed that AtCyp19-3 interacts with CaM in vivo also, thus, validating the in vitro observations. However, the PPIase activity of the Arabidopsis cyclophilin was not affected by CaM. The implications of these findings are discussed in the context of Ca(2+) signaling and cyclophilin activity in Arabidopsis.

  2. Enhancement of antibody fragment secretion into the Escherichia coli periplasm by co-expression with the peptidyl prolyl isomerase, FkpA, in the cytoplasm.

    PubMed

    Levy, Raphael; Ahluwalia, Kiran; Bohmann, David J; Giang, Hoa M; Schwimmer, Lauren J; Issafras, Hassan; Reddy, Nithin B; Chan, Chung; Horwitz, Arnold H; Takeuchi, Toshihiko

    2013-08-30

    Improper protein folding or aggregation can frequently be responsible for low expression and poor functional activity of antibody fragments secreted into the Escherichia coli periplasm. Expression issues also can affect selection of antibody candidates from phage libraries, since antibody fragments displayed on phage also are secreted into the E. coli periplasm. To improve secretion of properly folded antibody fragments into the periplasm, we have developed a novel approach that involves co-expressing the antibody fragments with the peptidyl prolyl cis-trans isomerase, FkpA, lacking its signal sequence (cytFkpA) which consequently is expressed in the E. coli cytosol. Cytoplasmic expression of cytFkpA improved secretion of functional Fab fragments into the periplasm, exceeding even the benefits from co-expressing Fab fragments with native, FkpA localized in the periplasm. In addition, panning and subsequent screening of large Fab and scFv naïve phage libraries in the presence of cytFkpA significantly increased the number of unique clones selected, as well as their functional expression levels and diversity.

  3. Biochemical and Genetic Characterization of an FK506-Sensitive Peptidyl Prolyl cis-trans Isomerase from a Thermophilic Archaeon, Methanococcus thermolithotrophicus

    PubMed Central

    Furutani, Masahiro; Iida, Toshii; Yamano, Shigeyuki; Kamino, Kei; Maruyama, Tadashi

    1998-01-01

    A peptidyl prolyl cis-trans isomerase (PPIase) was purified from a thermophilic methanogen, Methanococcus thermolithotrophicus. The PPIase activity was inhibited by FK506 but not by cyclosporine. The molecular mass of the purified enzyme was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 42 kDa by gel filtration. The enzyme was thermostable, with the half-lives of its activity at 90 and 100°C being 90 and 30 min, respectively. The catalytic efficiencies (kcat/Km) measured at 15°C for the peptidyl substrates, N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, were 0.35 and 0.20 μM−1 s−1, respectively, in chymotrypsin-coupled assays. The purified enzyme was sensitive to FK506 and therefore was called MTFK (M. thermolithotrophicus FK506-binding protein). The MTFK gene (462 bp) was cloned from an M. thermolithotrophicus genomic library. The comparison of the amino acid sequence of MTFK with those of other FK506-binding PPIases revealed that MTFK has a 13-amino-acid insertion in the N-terminal region that is unique to thermophilic archaea. The relationship between the thermostable nature of MTFK and its structure is discussed. PMID:9440528

  4. Fine-tuning the extent and dynamics of binding cleft opening as a potential general regulatory mechanism in parvulin-type peptidyl prolyl isomerases

    NASA Astrophysics Data System (ADS)

    Czajlik, András; Kovács, Bertalan; Permi, Perttu; Gáspári, Zoltán

    2017-03-01

    Parvulins or rotamases form a distinct group within peptidyl prolyl cis-trans isomerases. Their exact mode of action as well as the role of conserved residues in the family are still not unambiguously resolved. Using backbone S2 order parameters and NOEs as restraints, we have generated dynamic structural ensembles of three distinct parvulins, SaPrsA, TbPin1 and CsPinA. The resulting ensembles are in good agreement with the experimental data but reveal important differences between the three enzymes. The largest difference can be attributed to the extent of the opening of the substrate binding cleft, along which motional mode the three molecules occupy distinct regions. Comparison with a wide range of other available parvulin structures highlights structural divergence along the bottom of the binding cleft acting as a hinge during the opening-closing motion. In the prototype WW-domain containing parvulin, Pin1, this region is also important in forming contacts with the WW domain known to modulate enzymatic activity of the catalytic domain. We hypothesize that modulation of the extent and dynamics of the identified ‘breathing motion’ might be one of the factors responsible for functional differences in the distinct parvulin subfamilies.

  5. Fine-tuning the extent and dynamics of binding cleft opening as a potential general regulatory mechanism in parvulin-type peptidyl prolyl isomerases

    PubMed Central

    Czajlik, András; Kovács, Bertalan; Permi, Perttu; Gáspári, Zoltán

    2017-01-01

    Parvulins or rotamases form a distinct group within peptidyl prolyl cis-trans isomerases. Their exact mode of action as well as the role of conserved residues in the family are still not unambiguously resolved. Using backbone S2 order parameters and NOEs as restraints, we have generated dynamic structural ensembles of three distinct parvulins, SaPrsA, TbPin1 and CsPinA. The resulting ensembles are in good agreement with the experimental data but reveal important differences between the three enzymes. The largest difference can be attributed to the extent of the opening of the substrate binding cleft, along which motional mode the three molecules occupy distinct regions. Comparison with a wide range of other available parvulin structures highlights structural divergence along the bottom of the binding cleft acting as a hinge during the opening-closing motion. In the prototype WW-domain containing parvulin, Pin1, this region is also important in forming contacts with the WW domain known to modulate enzymatic activity of the catalytic domain. We hypothesize that modulation of the extent and dynamics of the identified ‘breathing motion’ might be one of the factors responsible for functional differences in the distinct parvulin subfamilies. PMID:28300139

  6. Hepatitis C virus NS5A protein is a substrate for the peptidyl-prolyl cis/trans isomerase activity of cyclophilins A and B.

    PubMed

    Hanoulle, Xavier; Badillo, Aurélie; Wieruszeski, Jean-Michel; Verdegem, Dries; Landrieu, Isabelle; Bartenschlager, Ralf; Penin, François; Lippens, Guy

    2009-05-15

    We report here a biochemical and structural characterization of domain 2 of the nonstructural 5A protein (NS5A) from the JFH1 Hepatitis C virus strain and its interactions with cyclophilins A and B (CypA and CypB). Gel filtration chromatography, circular dichroism spectroscopy, and finally NMR spectroscopy all indicate the natively unfolded nature of this NS5A-D2 domain. Because mutations in this domain have been linked to cyclosporin A resistance, we used NMR spectroscopy to investigate potential interactions between NS5A-D2 and cellular CypA and CypB. We observed a direct molecular interaction between NS5A-D2 and both cyclophilins. The interaction surface on the cyclophilins corresponds to their active site, whereas on NS5A-D2, it proved to be distributed over the many proline residues of the domain. NMR heteronuclear exchange spectroscopy yielded direct evidence that many proline residues in NS5A-D2 form a valid substrate for the enzymatic peptidyl-prolyl cis/trans isomerase (PPIase) activity of CypA and CypB.

  7. Hepatitis C Virus NS5A Protein Is a Substrate for the Peptidyl-prolyl cis/trans Isomerase Activity of Cyclophilins A and B*S⃞

    PubMed Central

    Hanoulle, Xavier; Badillo, Aurélie; Wieruszeski, Jean-Michel; Verdegem, Dries; Landrieu, Isabelle; Bartenschlager, Ralf; Penin, François; Lippens, Guy

    2009-01-01

    We report here a biochemical and structural characterization of domain 2 of the nonstructural 5A protein (NS5A) from the JFH1 Hepatitis C virus strain and its interactions with cyclophilins A and B (CypA and CypB). Gel filtration chromatography, circular dichroism spectroscopy, and finally NMR spectroscopy all indicate the natively unfolded nature of this NS5A-D2 domain. Because mutations in this domain have been linked to cyclosporin A resistance, we used NMR spectroscopy to investigate potential interactions between NS5A-D2 and cellular CypA and CypB. We observed a direct molecular interaction between NS5A-D2 and both cyclophilins. The interaction surface on the cyclophilins corresponds to their active site, whereas on NS5A-D2, it proved to be distributed over the many proline residues of the domain. NMR heteronuclear exchange spectroscopy yielded direct evidence that many proline residues in NS5A-D2 form a valid substrate for the enzymatic peptidyl-prolyl cis/trans isomerase (PPIase) activity of CypA and CypB. PMID:19297321

  8. 1.88 A crystal structure of the C domain of hCyP33: A novel domain of peptidyl-prolyl cis-trans isomerase

    SciTech Connect

    Wang Tao; Yun Caihong; Gu Shenyan; Chang Wenrui; Liang Dongcai . E-mail: dcliang@sun5.ibp.ac.cn

    2005-08-05

    Cyclophilins (CyPs) are a widespreading protein family in living organisms and possess the activity of peptidyl-prolyl cis-trans isomerase (PPIase), which is inhibited by cyclosporin A (CsA). The human nuclear cyclophilin (hCyP33) is the first protein which was found to contain two RNA binding domains at the amino-terminus and a PPIase domain at the carboxyl-terminus. We isolated the hCyP33 gene from the human hematopoietic stem/progenitor cells and expressed it in Escherichia coli, and determined the crystal structure of the C domain of hCyP33 at 1.88 A resolution. The core structure is a {beta}-barrel covered by two {alpha}-helices. Superposition of the structure of the C domain of hCyP33 with the structure of CypA suggests that the C domain contains PPIase active site which binds to CsA. Furthermore, C domain seems to be able to bind with the Gag-encoded capsid (CA) of HIV-1 and may affect the viral replication of HIV-1. A key residue of the active site is changed from Ala-103-CypA to Ser-239-hCyP33, which may affect the PPIase domain/substrates interactions.

  9. Inhibition of the peptidyl-prolyl-isomerase Pin1 enhances the responses of acute myeloid leukemia cells to retinoic acid via stabilization of RARalpha and PML-RARalpha.

    PubMed

    Gianni', Maurizio; Boldetti, Andrea; Guarnaccia, Valeria; Rambaldi, Alessandro; Parrella, Edoardo; Raska, Ivan; Rochette-Egly, Cecile; Del Sal, Giannino; Rustighi, Alessandra; Terao, Mineko; Garattini, Enrico

    2009-02-01

    The peptidyl-prolyl-isomerase Pin1 interacts with phosphorylated proteins, altering their conformation. The retinoic acid receptor RARalpha and the acute-promyelocytic-leukemia-specific counterpart PML-RARalpha directly interact with Pin1. Overexpression of Pin1 inhibits ligand-dependent activation of RARalpha and PML-RARalpha. Inhibition is relieved by Pin1-targeted short interfering RNAs and by pharmacologic inhibition of the catalytic activity of the protein. Mutants of Pin1 catalytically inactive or defective for client-protein-binding activity are incapable of inhibiting ligand-dependent RARalpha transcriptional activity. Functional inhibition of RARalpha and PML-RARalpha by Pin1 correlates with degradation of the nuclear receptors via the proteasome-dependent pathway. In the acute myelogenous leukemia cell lines HL-60 and NB4, Pin1 interacts with RARalpha in a constitutive fashion. Suppression of Pin1 by a specific short hairpin RNA in HL-60 or NB4 cells stabilizes RARalpha and PML-RARalpha, resulting in increased sensitivity to the cytodifferentiating and antiproliferative activities of all-trans retinoic acid. Treatment of the two cell lines and freshly isolated acute myelogenous leukemia blasts (M1 to M4) with ATRA and a pharmacologic inhibitor of Pin1 causes similar effects. Our results add a further layer of complexity to the regulation of nuclear retinoic acid receptors and suggest that Pin1 represents an important target for strategies aimed at increasing the therapeutic index of retinoids.

  10. Subtractive hybridization analysis of gastric diseases-associated Helicobacter pylori identifies peptidyl-prolyl isomerase as a potential marker for gastric cancer.

    PubMed

    Gong, Yue-Hua; Chen, Moye; Xu, Ying; Dong, Nannan; Sang, Zhikun; Liu, Jun; Yuan, Yuan

    2011-07-01

    Helicobacter pylori, a microaerophilic Gram-negative bacterium, is known to cause chronic gastritis, peptic ulcer and gastric cancer. Genes that are present in certain isolates may determine strain-specific traits such as disease association and drug resistance. In order to understand the pathogenic mechanisms of gastric diseases, identify molecular markers of the diseases associated with H. pylori strains and provide clues for target treatment of H. pylori-related diseases, a subtracted DNA library was constructed from a gastric cancer-associated H. pylori strain and a superficial gastritis-associated H. pylori strain by suppression subtractive hybridization. The presence of gastric cancer-specific genes was identified by dot blot hybridization, DNA sequencing and PCR-based screening. Twelve gastric cancer-specific high-copy genes and nine low-copy genes were found in gastric cancer compared with the superficial gastritis strain. These genes were confirmed by PCR analysis of H. pylori isolates. Notably, peptidyl-prolyl cis-trans isomerase (PPIase) was detected positively in 11 out of 22 (50%) gastric cancer-associated H. pylori strains. In contrast, <24% of the H. pylori strains from superficial gastritis showed positive results. Given the potential role of PPIases in cell growth, apoptosis and oncogenic transformation, our results suggest that PPIase may represent a novel marker and potential therapeutic target for gastric cancer. © 2011 The First Affiliated Hospital, China Medical University. FEMS Microbiology Letters © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.

  11. The peptidyl-prolyl isomerase Pin1 up-regulation and proapoptotic function in dopaminergic neurons: relevance to the pathogenesis of Parkinson disease.

    PubMed

    Ghosh, Anamitra; Saminathan, Hariharan; Kanthasamy, Arthi; Anantharam, Vellareddy; Jin, Huajun; Sondarva, Gautam; Harischandra, Dilshan S; Qian, Ziqing; Rana, Ajay; Kanthasamy, Anumantha G

    2013-07-26

    Parkinson disease (PD) is a chronic neurodegenerative disease characterized by a slow and progressive degeneration of dopaminergic neurons in substantia nigra. The pathophysiological mechanisms underlying PD remain unclear. Pin1, a major peptidyl-prolyl isomerase, has recently been associated with certain diseases. Notably, Ryo et al. (Ryo, A., Togo, T., Nakai, T., Hirai, A., Nishi, M., Yamaguchi, A., Suzuki, K., Hirayasu, Y., Kobayashi, H., Perrem, K., Liou, Y. C., and Aoki, I. (2006) J. Biol. Chem. 281, 4117-4125) implicated Pin1 in PD pathology. Therefore, we sought to systematically characterize the role of Pin1 in PD using cell culture and animal models. To our surprise we observed a dramatic up-regulation of Pin1 mRNA and protein levels in dopaminergic MN9D neuronal cells treated with the parkinsonian toxicant 1-methyl-4-phenylpyridinium (MPP(+)) as well as in the substantia nigra of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. Notably, a marked expression of Pin1 was also observed in the substantia nigra of human PD brains along with a high co-localization of Pin1 within dopaminergic neurons. In functional studies, siRNA-mediated knockdown of Pin1 almost completely prevented MPP(+)-induced caspase-3 activation and DNA fragmentation, indicating that Pin1 plays a proapoptotic role. Interestingly, multiple pharmacological Pin1 inhibitors, including juglone, attenuated MPP(+)-induced Pin1 up-regulation, α-synuclein aggregation, caspase-3 activation, and cell death. Furthermore, juglone treatment in the MPTP mouse model of PD suppressed Pin1 levels and improved locomotor deficits, dopamine depletion, and nigral dopaminergic neuronal loss. Collectively, our findings demonstrate for the first time that Pin1 is up-regulated in PD and has a pathophysiological role in the nigrostriatal dopaminergic system and suggest that modulation of Pin1 levels may be a useful translational therapeutic strategy in PD.

  12. Identification and Comparative Analysis of the Peptidyl-Prolyl cis/trans Isomerase Repertoires of H. sapiens, D. melanogaster, C. elegans, S. cerevisiae and Sz. pombe

    PubMed Central

    Kay, John E.

    2005-01-01

    The peptidyl-prolyl cis/trans isomerase (PPIase) class of proteins comprises three member families that are found throughout nature and are present in all the major compartments of the cell. Their numbers appear to be linked to the number of genes in their respective genomes, although we have found the human repertoire to be smaller than expected due to a reduced cyclophilin repertoire. We show here that whilst the members of the cyclophilin family (which are predominantly found in the nucleus and cytoplasm) and the parvulin family (which are predominantly nuclear) are largely conserved between different repertoires, the FKBPs (which are predominantly found in the cytoplasm and endoplasmic reticulum) are not. It therefore appears that the cyclophilins and parvulins have evolved to perform conserved functions, while the FKBPs have evolved to fill ever-changing niches within the constantly evolving organisms. Many orthologous subgroups within the different PPIase families appear to have evolved from a distinct common ancestor, whereas others, such as the mitochondrial cyclophilins, appear to have evolved independently of one another. We have also identified a novel parvulin within Drosophila melanogaster that is unique to the fruit fly, indicating a recent evolutionary emergence. Interestingly, the fission yeast repertoire, which contains no unique cyclophilins and parvulins, shares no PPIases solely with the budding yeast but it does share a majority with the higher eukaryotes in this study, unlike the budding yeast. It therefore appears that, in comparison with Schizosaccharomyces pombe, Saccharomyces cerevisiae is a poor representation of the higher eukaryotes for the study of PPIases. PMID:18629211

  13. Single-Domain Peptidyl-Prolyl cis/trans Isomerase FkpA from Corynebacterium glutamicum Improves the Biomass Yield at Increased Growth Temperatures.

    PubMed

    Kallscheuer, Nicolai; Bott, Michael; van Ooyen, Jan; Polen, Tino

    2015-11-01

    Peptidyl-prolyl cis/trans isomerases (PPIases) catalyze the rate-limiting protein folding step at peptidyl bonds preceding proline residues and were found to be involved in several biological processes, including gene expression, signal transduction, and protein secretion. Representative enzymes were found in almost all sequenced genomes, including Corynebacterium glutamicum, a facultative anaerobic Gram-positive and industrial workhorse for the production of amino acids. In C. glutamicum, a predicted single-domain FK-506 (tacrolimus) binding protein (FKBP)-type PPIase (FkpA) is encoded directly downstream of gltA, which encodes citrate synthase (CS). This gene cluster is also present in other Actinobacteria. Here we carried out in vitro and in vivo experiments to study the function and influence of predicted FkpA in C. glutamicum. In vitro, FkpA indeed shows typical PPIase activity with artificial substrates and is inhibited by FK-506. Furthermore, FkpA delays the aggregation of CS, which is also inhibited by FK-506. Surprisingly, FkpA has a positive effect on the activity and temperature range of CS in vitro. Deletion of fkpA causes a 50% reduced biomass yield compared to that of the wild type when grown at 37°C, whereas there is only a 10% reduced biomass yield at the optimal growth temperature of 30°C accompanied by accumulation of 7 mM l-glutamate and 22 mM 2-oxoglutarate. Thus, FkpA may be exploited for improved product formation in biotechnical processes. Comparative transcriptome analysis revealed 69 genes which exhibit ≥2-fold mRNA level changes in C. glutamicum ΔfkpA, giving insight into the transcriptional response upon mild heat stress when FkpA is absent. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  14. Single-Domain Peptidyl-Prolyl cis/trans Isomerase FkpA from Corynebacterium glutamicum Improves the Biomass Yield at Increased Growth Temperatures

    PubMed Central

    van Ooyen, Jan

    2015-01-01

    Peptidyl-prolyl cis/trans isomerases (PPIases) catalyze the rate-limiting protein folding step at peptidyl bonds preceding proline residues and were found to be involved in several biological processes, including gene expression, signal transduction, and protein secretion. Representative enzymes were found in almost all sequenced genomes, including Corynebacterium glutamicum, a facultative anaerobic Gram-positive and industrial workhorse for the production of amino acids. In C. glutamicum, a predicted single-domain FK-506 (tacrolimus) binding protein (FKBP)-type PPIase (FkpA) is encoded directly downstream of gltA, which encodes citrate synthase (CS). This gene cluster is also present in other Actinobacteria. Here we carried out in vitro and in vivo experiments to study the function and influence of predicted FkpA in C. glutamicum. In vitro, FkpA indeed shows typical PPIase activity with artificial substrates and is inhibited by FK-506. Furthermore, FkpA delays the aggregation of CS, which is also inhibited by FK-506. Surprisingly, FkpA has a positive effect on the activity and temperature range of CS in vitro. Deletion of fkpA causes a 50% reduced biomass yield compared to that of the wild type when grown at 37°C, whereas there is only a 10% reduced biomass yield at the optimal growth temperature of 30°C accompanied by accumulation of 7 mM l-glutamate and 22 mM 2-oxoglutarate. Thus, FkpA may be exploited for improved product formation in biotechnical processes. Comparative transcriptome analysis revealed 69 genes which exhibit ≥2-fold mRNA level changes in C. glutamicum ΔfkpA, giving insight into the transcriptional response upon mild heat stress when FkpA is absent. PMID:26341203

  15. Parvulin (Par14), a peptidyl-prolyl cis-trans isomerase, is a novel rRNA processing factor that evolved in the metazoan lineage.

    PubMed

    Fujiyama-Nakamura, Sally; Yoshikawa, Harunori; Homma, Keiichi; Hayano, Toshiya; Tsujimura-Takahashi, Teruko; Izumikawa, Keiichi; Ishikawa, Hideaki; Miyazawa, Naoki; Yanagida, Mitsuaki; Miura, Yutaka; Shinkawa, Takashi; Yamauchi, Yoshio; Isobe, Toshiaki; Takahashi, Nobuhiro

    2009-07-01

    Although parvulin (Par14/eukaryotic parvulin homolog), a peptidyl-prolyl cis-trans isomerase, is found associated with the preribosomal ribonucleoprotein (pre-rRNP) complexes, its roles in ribosome biogenesis remain undetermined. In this study, we describe a comprehensive proteomics analysis of the Par14-associated pre-rRNP complexes using LC-MS/MS and a knockdown analysis of Par14. Together with our previous results, we finally identified 115 protein components of the complexes, including 39 ribosomal proteins and 54 potential trans-acting factors whose yeast homologs are found in the pre-rRNP complexes formed at various stages of ribosome biogenesis. We give evidence that, although Par14 exists in both the phosphorylated and unphosphorylated forms in the cell, only the latter form is associated with the pre-40 S and pre-60 S ribosomal complexes. We also show that Par14 co-localizes with the nucleolar protein B23 during the interphase and in the spindle apparatus during mitosis and that actinomycin D treatment results in the exclusion of Par14 from the nucleolus. Finally we demonstrate that knockdown of Par14 mRNA decelerates the processing of pre-rRNA to 18 and 28 S rRNAs. We propose that Par14 is a component of the pre-rRNA complexes and functions as an rRNA processing factor in ribosome biogenesis. As the amino acid sequence of Par14 including that in the amino-terminal pre-rRNP binding region is conserved only in metazoan homologs, we suggest that its roles in ribosome biogenesis have evolved in the metazoan lineage.

  16. Peptidyl prolyl cis/trans-isomerases: comparative reactivities of cyclophilins, FK506-binding proteins, and parvulins with fluorinated oligopeptide and protein substrates.

    PubMed

    Golbik, Ralph; Yu, Chao; Weyher-Stingl, Elisabeth; Huber, Robert; Moroder, Luis; Budisa, Nediljko; Schiene-Fischer, Cordelia

    2005-12-13

    Peptidyl prolyl cis/trans-isomerases catalyze the cis-trans isomerization of prolyl bonds in oligopeptides and various folding states of proteins. The proline residue in PPIase substrates at the P1' subsite, which follows the isomerizing peptide bond, appears to be the common recognition element for all subfamilies of this enzyme class. The molecular principles that govern substrate specificity at the P1' subsite were analyzed using 4-fluoroproline-containing tetrapeptide 4-nitroanilides and barstar Cys40Ala/Cys82Ala/Pro27Ala/Pro48-->4-fluoroproline quadruple variants. Generally, PPIase catalysis demonstrated stereospecificity for monofluoro substitutions at the 4-position of the pyrrolidine ring. However, the replacement of hydrogens with fluoro atoms did not impair productive interactions for the majority of PPIase-substrate complexes. Comparison of specificity constants for oligopeptide and protein substrates revealed striking differences in the 4-fluoroproline substituent effects between members of the PPIase families. Introduction of 4(R)-fluoroproline resulted in an oligopeptide substrate completely resistant to catalytic effects of FKBP-like PPIases. By contrast, the 4(R)-fluoroproline barstar variant demonstrated only slightly reduced or even better catalytic susceptibility when compared to the parent barstar Cys40Ala/Cys82Ala/Pro27Ala/Pro48 substrate. On the other hand, Suc-Ala-Ser-4(S)-FPro-Phe-pNA exhibits a discriminating specificity toward the prototypic parvulin, the Escherichia coli Par10. The E. coli trigger factor, in the extreme, catalyzes Cys40Ala/Cys82Ala/Pro27Ala/4-F(2)Pro48 with a more than 20-fold higher efficiency when compared to the proline-containing congener. These findings support the combined subsite concept for PPIase catalysis in which the positioning of a substrate in the active cleft must activate a still unknown number of remote subsites in the transition state of the reaction. The number of critical subsites was shown to vary

  17. The peptidyl-prolyl isomerase motif is lacking in PmpA, the PrsA-like protein involved in the secretion machinery of Lactococcus lactis.

    PubMed

    Drouault, Sophie; Anba, Jamila; Bonneau, Sophie; Bolotin, Alexander; Ehrlich, S Dusko; Renault, Pierre

    2002-08-01

    The prsA-like gene from Lactococcus lactis encoding its single homologue to PrsA, an essential protein triggering the folding of secreted proteins in Bacillus subtilis, was characterized. This gene, annotated pmpA, encodes a lipoprotein of 309 residues whose expression is increased 7- to 10-fold when the source of nitrogen is limited. A slight increase in the expression of the PrsA-like protein (PLP) in L. lactis removed the degradation products previously observed with the Staphylococcus hyicus lipase used as a model secreted protein. This shows that PmpA either triggers the folding of the secreted lipase or activates its degradation by the cell surface protease HtrA. Unlike the case for B. subtilis, the inactivation of the gene encoding PmpA reduced only slightly the growth rate of L. lactis in standard conditions. However, it almost stopped its growth when the lipase was overexpressed in the presence of salt in the medium. Like PrsA of B. subtilis and PrtM of L. lactis, the L. lactis PmpA protein could thus have a foldase activity that facilitates protein secretion. These proteins belong to the third family of peptidyl-prolyl cis/trans-isomerases (PPIases) for which parvulin is the prototype. Almost all PLP from gram-positive bacteria contain a domain with the PPIase signature. An exception to this situation was found only in Streptococcaceae, the family to which L. lactis belongs. PLP from Streptococcus pneumoniae and Enterococcus faecalis possess this signature, but those of L. lactis, Streptococcus pyogenes, and Streptococcus mutans do not. However, secondary structure predictions suggest that the folding of PLP is conserved over the entire length of the proteins, including the unconserved signature region. The activity associated with the expression of PmpA in L. lactis and these genomic data show that either the PPIase motif is not necessary for PPIase activity or, more likely, PmpA foldase activity does not necessarily require PPIase activity.

  18. Identification of homologs for thioredoxin, peptidyl prolyl cis-trans isomerase, and glycerophosphodiester phosphodiesterase in outer membrane fractions from Treponema pallidum, the syphilis spirochete.

    PubMed Central

    Shevchenko, D V; Akins, D R; Robinson, E J; Li, M; Shevchenko, O V; Radolf, J D

    1997-01-01

    In this study, we characterized candidate rare outer membrane (OM) proteins with apparent molecular masses of 19, 27, 38, and 38.5 kDa, which had been identified previously in OM fractions from Treponema pallidum (J. D. Radolf et al., Infect. Immun. 63:4244-4252, 1995). Using N-terminal and internal amino acid sequences, a probe for the 19-kDa candidate was PCR amplified and used to screen a T. pallidum genomic library in Lambda Zap II. The corresponding gene (tlp) encoded a homolog for periplasmic thioredoxin-like proteins (Tlp), which reduce c-type cytochromes. A degenerate oligonucleotide derived from the N terminus of the 27-kDa protein was used to PCR amplify a duplex probe from a T. pallidum genomic library in pBluescript II SK+. With this probe, the corresponding gene (ppiB) was identified and found to code for a presumptive periplasmic cyclophilin B-type peptidyl prolyl cis-trans isomerase (PpiB). We postulate that PpiB assists the folding of proteins within the T. pallidum periplasmic space. The N terminus of the 38-kDa candidate was blocked to Edman degradation. However, internal sequence data revealed that it was basic membrane protein (Bmp), a previously characterized, signal peptidase I-processed protein. Triton X-114 phase partitioning revealed that despite its name, Bmp is hydrophilic and therefore likely to be periplasmic. The final candidate was also blocked to Edman degradation; as before, a duplex probe was PCR amplified with degenerate primers derived from internal sequences. The corresponding gene (glpQ) coded for a presumptively lipid-modified homolog of glycerophosphodiester phosphodiesterase (GlpQ). Based upon findings with other treponemal lipoproteins, the hydrophilic GlpQ polypeptide is thought to be anchored by N-terminal lipids to the periplasmic leaflet(s) of the cytoplasmic membrane and/or OM. The discovery of T. pallidum periplasmic proteins with potentially defined functions provides fresh insights into a poorly understood aspect of

  19. An Intracellular Peptidyl-Prolyl cis/trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease.

    PubMed

    Wiemels, Richard E; Cech, Stephanie M; Meyer, Nikki M; Burke, Caleb A; Weiss, Andy; Parks, Anastacia R; Shaw, Lindsey N; Carroll, Ronan K

    2017-01-01

    Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus IMPORTANCE: Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently

  20. Peptidyl-prolyl cis-trans isomerase of Bacillus subtilis: identification of residues involved in cyclosporin A affinity and catalytic efficiency.

    PubMed

    Göthel, S F; Herrler, M; Marahiel, M A

    1996-03-19

    The 17-kDa peptidyl-prolyl cis-trans-isomerase from Bacillus subtilis (PPiB) is a member of the cyclophilin family and shows strong homology to PPIases of eukaryotic origin (40%) and less identify to PPIase sequences of Gram-negative bacteria (27-32%). Although the majority of residues that form the PPIase active site are highly conserved, three residues, V52, H90, and H109 in the sequence of the B.subtilis PPIase, were found to differ from the sequences found in human (hCyP) and Escherichia coli (eCyP). Also, the binding affinity of cyclosporin A (CsA) to the different PPIases varies in IC(50) values from 6 nM for human PPIase hCyPA and 84 nM for the human hCyPB to over 120 nM for B. subtilis and 3000 nM for E. coli. In addition, a variety of k(cat)/K(m) values, ranging from 1.1 mM(-1) s(-1) for the B. subtilis PPIase to over 10 mM(-1) s(-1) for human and 13 mM(-1) s(-1) for E. coli, were detected using the common substrate suc-Ala-Ala-Pro-Phe-pNA. Through site-specific mutagenesis we demonstrate that the differences in the three mentioned residues are mainly responsible for the variations in IC(50) and k(cat)/K(m) values. Replacement of H90 to N90, or H109 to W109, resembling the amino acid sequence of human hCyPA, resulted in more efficient CsA binding (IC(50) value for H90N, 60 nM, and for H109W, 95 nm), whereas replacement of H90 to R90, or H109 to F109, resembling the amino acid sequence of E. coli eCyP, resulted in less efficient CsA binding (IC(50) value for H90R, 2000nM, and for H109F, 5000 nM). In addition to lower CsA affinity, mutant protein H109F shows a k(cat)/K(m) value of 10.5 mM(-1) s(-1), comparably high to that of the wild-type E. coli protein. In contrast, other mutants like C57F, H90N, H90R, and H109W do not differ significantly in k(cat)/K(m) values from wild-type PPiB. Replacement of V52 to M52, which is conserved in E. coli and all known eukaryotic PPIases, does not show any effect in CsA binding affinity (IC(50) value for V52M, 120 nM), but

  1. Interaction of the transmembrane domain of lysis protein E from bacteriophage phiX174 with bacterial translocase MraY and peptidyl-prolyl isomerase SlyD.

    PubMed

    Mendel, Sharon; Holbourn, Joanne M; Schouten, James A; Bugg, Timothy D H

    2006-10-01

    The molecular target for the bacteriolytic E protein from bacteriophage X174, responsible for host cell lysis, is known to be the enzyme phospho-MurNAc-pentapeptide translocase (MraY), an integral membrane protein involved in bacterial cell wall peptidoglycan biosynthesis, with an essential role being played by peptidyl-prolyl isomerase SlyD. A synthetic 37 aa peptide E(pep), containing the N-terminal transmembrane alpha-helix of E, was found to be bacteriolytic against Bacillus licheniformis, and inhibited membrane-bound MraY. The solution conformation of E(pep) was found by circular dichroism (CD) spectroscopy to be 100 % alpha-helical. No change in the CD spectrum was observed upon addition of purified Escherichia coli SlyD, implying that SlyD does not catalyse prolyl isomerization upon E. However, E(pep) was found to be a potent inhibitor of SlyD-catalysed peptidyl-prolyl isomerization (IC(50) 0.15 microM), implying a strong interaction between E and SlyD. E(pep) was found to inhibit E. coli MraY activity when assayed in membranes (IC(50) 0.8 microM); however, no inhibition of solubilized MraY was observed, unlike nucleoside natural product inhibitor tunicamycin. These results imply that the interaction of E with MraY is not at the MraY active site, and suggest that a protein-protein interaction is formed between E and MraY at a site within the transmembrane region.

  2. Posttranslocation chaperone PrsA2 regulates the maturation and secretion of Listeria monocytogenes proprotein virulence factors.

    PubMed

    Forster, Brian M; Zemansky, Jason; Portnoy, Daniel A; Marquis, Hélène

    2011-11-01

    PrsA2 is a conserved posttranslocation chaperone and a peptidyl prolyl cis-trans isomerase (PPIase) that contributes to the virulence of the Gram-positive intracellular pathogen Listeria monocytogenes. One of the phenotypes associated with a prsA2 mutant is decreased activity of the broad-range phospholipase C (PC-PLC). PC-PLC is made as a proenzyme whose maturation is mediated by a metalloprotease (Mpl). The proforms of PC-PLC and Mpl accumulate at the membrane-cell wall interface until a decrease in pH triggers their maturation and rapid secretion into the host cell. In this study, we examined the mechanism by which PrsA2 regulates the activity of PC-PLC. We observed that in the absence of PrsA2, the proenzymes are secreted at physiological pH and do not mature upon a decrease in pH. The sensitivity of the prsA2 mutant to cell wall hydrolases was modified. However, no apparent changes in cell wall porosity were detected. Interestingly, synthesis of PC-PLC in the absence of its propeptide lead to the secretion of a fully active enzyme in the cytosol of host cells independent of PrsA2, indicating that neither the propeptide of PC-PLC nor PrsA2 is required for native folding of the catalytic domain, although both influence secretion of the enzyme. Taken together, these results suggest that PrsA2 regulates compartmentalization of Mpl and PC-PLC, possibly by influencing cell wall properties and interacting with the PC-PLC propeptide. Moreover, the ability of these proproteins to respond to a decrease in pH during intracellular growth depends on their localization at the membrane-cell wall interface.

  3. Posttranslocation Chaperone PrsA2 Regulates the Maturation and Secretion of Listeria monocytogenes Proprotein Virulence Factors ▿

    PubMed Central

    Forster, Brian M.; Zemansky, Jason; Portnoy, Daniel A.; Marquis, Hélène

    2011-01-01

    PrsA2 is a conserved posttranslocation chaperone and a peptidyl prolyl cis-trans isomerase (PPIase) that contributes to the virulence of the Gram-positive intracellular pathogen Listeria monocytogenes. One of the phenotypes associated with a prsA2 mutant is decreased activity of the broad-range phospholipase C (PC-PLC). PC-PLC is made as a proenzyme whose maturation is mediated by a metalloprotease (Mpl). The proforms of PC-PLC and Mpl accumulate at the membrane-cell wall interface until a decrease in pH triggers their maturation and rapid secretion into the host cell. In this study, we examined the mechanism by which PrsA2 regulates the activity of PC-PLC. We observed that in the absence of PrsA2, the proenzymes are secreted at physiological pH and do not mature upon a decrease in pH. The sensitivity of the prsA2 mutant to cell wall hydrolases was modified. However, no apparent changes in cell wall porosity were detected. Interestingly, synthesis of PC-PLC in the absence of its propeptide lead to the secretion of a fully active enzyme in the cytosol of host cells independent of PrsA2, indicating that neither the propeptide of PC-PLC nor PrsA2 is required for native folding of the catalytic domain, although both influence secretion of the enzyme. Taken together, these results suggest that PrsA2 regulates compartmentalization of Mpl and PC-PLC, possibly by influencing cell wall properties and interacting with the PC-PLC propeptide. Moreover, the ability of these proproteins to respond to a decrease in pH during intracellular growth depends on their localization at the membrane-cell wall interface. PMID:21908675

  4. Identification of Conserved and Species-Specific Functions of the Listeria monocytogenes PrsA2 Secretion Chaperone

    PubMed Central

    Cahoon, Laty A.

    2015-01-01

    The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen that relies on the regulated secretion and activity of a variety of proteins that sustain life within diverse environments. PrsA2 has recently been identified as a secreted peptidyl-prolyl cis/trans isomerase and chaperone that is dispensable for bacterial growth in broth culture but essential for L. monocytogenes virulence. Following host infection, PrsA2 contributes to the proper folding and activity of secreted proteins that are required for bacterial replication within the host cytosol and for bacterial spread to adjacent cells. PrsA2 is one member of a family of Gram-positive secretion chaperones that appear to play important roles in bacterial physiology; however, it is not known how these proteins recognize their substrate proteins or the degree to which their function is conserved across diverse Gram-positive species. We therefore examined PrsA proteins encoded by a variety of Gram-positive bacteria for functional complementation of L. monocytogenes mutants lacking prsA2. PrsA homologues encoded by Bacillus subtilis, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus mutans, Staphylococcus aureus, and Lactococcus lactis were examined for functional complementation of a variety of L. monocytogenes PrsA2-associated phenotypes central to L. monocytogenes pathogenesis and bacterial cell physiology. Our results indicate that while selected aspects of PrsA2 function are broadly conserved among diverse Gram-positive bacteria, PrsA2 exhibits unique specificity for L. monocytogenes target proteins required for pathogenesis. The L. monocytogenes PrsA2 chaperone thus appears evolutionarily optimized for virulence factor secretion within the host cell cytosol while still maintaining aspects of activity relevant to more general features of Gram-positive protein translocation. PMID:26216425

  5. The Posttranslocation Chaperone PrsA2 Contributes to Multiple Facets of Listeria monocytogenes Pathogenesis▿ †

    PubMed Central

    Alonzo, Francis; Port, Gary C.; Cao, Min; Freitag, Nancy E.

    2009-01-01

    Listeria monocytogenes is an intracellular bacterial pathogen whose virulence depends on the regulated expression of numerous secreted bacterial factors. As for other gram-positive bacteria, many proteins secreted by L. monocytogenes are translocated across the bacterial membrane in an unfolded state to the compartment existing between the membrane and the cell wall. This compartment presents a challenging environment for protein folding due to its high density of negative charge, high concentrations of cations, and low pH. We recently identified PrsA2 as a gene product required for L. monocytogenes virulence. PrsA2 was identified based on its increased secretion by strains containing a mutationally activated form of prfA, the key regulator of L. monocytogenes virulence gene expression. The prsA2 gene product is one of at least two predicted peptidyl-prolyl cis/trans-isomerases encoded by L. monocytogenes; these proteins function as posttranslocation protein chaperones and/or foldases. In this study, we demonstrate that PrsA2 plays a unique and important role in L. monocytogenes pathogenesis by promoting the activity and stability of at least two critical secreted virulence factors: listeriolysin O (LLO) and a broad-specificity phospholipase. Loss of PrsA2 activity severely attenuated virulence in mice and impaired bacterial cell-to-cell spread in host cells. In contrast, mutants lacking prsA1 resembled wild-type bacteria with respect to intracellular growth and cell-to-cell spread as well as virulence in mice. PrsA2 is thus distinct from PrsA1 in its unique requirement for the stability and full activity of L. monocytogenes-secreted factors that contribute to host infection. PMID:19451247

  6. Aluminum(III) interferes with the structure and the activity of the peptidyl-prolyl cis-trans isomerase (Pin1): a new mechanism contributing to the pathogenesis of Alzheimer's disease and cancers?

    PubMed

    Wang, Jing-Zhang; Liu, Ji; Lin, Tao; Han, Yong-Guang; Luo, Yue; Xi, Lei; Du, Lin-Fang

    2013-09-01

    The enzyme peptidyl-prolyl cis-trans isomerase (Pin1) may play an important role in preventing the development of Alzheimer's disease (AD). The structural and functional stability of Pin1 is extremely important. Previously, we have determined the stability of Pin1 under stressed conditions, such as thermal treatment and acidic-pH. Considering that aluminum (Al(III)) is well known for its potential neurotoxicity in the pathogenesis of AD, we examined whether Al(III) affects the structure and function of Pin1, by means of a PPIase activity assay, intrinsic fluorescence, circular dichroism (CD) spectroscopy, FTIR, and differential scanning calorimetry (DSC). The intrinsic tryptophan fluorescence measurements mainly show that Al(III) may bind to the clusters nearby W11 and W34 in the WW domain of Pin1, quenching the intrinsic fluorescence of the two tryptophan residues, which possibly results in the decreased binding affinity of Pin1 to substrates. The secondary structural analysis as revealed by FTIR and CD measurements indicate that Al(III) induces the increase in β-sheet and the decrease in α-helix in Pin1. Furthermore, the changes of the thermodynamic parameters for Pin1 as monitored by DSC confirm that the thermal stability of Pin1 significantly increases in the presence of Al(III). The Al(III)-induced structural changes of Pin1 result in a sharp decrease of the PPIase activity of Pin1. To some extent, our research is suggestive that Al(III) may inhibit the isomerization activity of Pin1 in vivo, which may contribute to the pathogenesis of AD. Copyright © 2013. Published by Elsevier Inc.

  7. NF-κB transcriptional activity is modulated by FK506-binding proteins FKBP51 and FKBP52: a role for peptidyl-prolyl isomerase activity.

    PubMed

    Erlejman, Alejandra G; De Leo, Sonia A; Mazaira, Gisela I; Molinari, Alejandro M; Camisay, María Fernanda; Fontana, Vanina; Cox, Marc B; Piwien-Pilipuk, Graciela; Galigniana, Mario D

    2014-09-19

    Hsp90 binding immunophilins FKBP51 and FKBP52 modulate steroid receptor trafficking and hormone-dependent biological responses. With the purpose to expand this model to other nuclear factors that are also subject to nuclear-cytoplasmic shuttling, we analyzed whether these immunophilins modulate NF-κB signaling. It is demonstrated that FKBP51 impairs both the nuclear translocation rate of NF-κB and its transcriptional activity. The inhibitory action of FKBP51 requires neither the peptidylprolyl-isomerase activity of the immunophilin nor its association with Hsp90. The TPR domain of FKBP51 is essential. On the other hand, FKBP52 favors the nuclear retention time of RelA, its association to a DNA consensus binding sequence, and NF-κB transcriptional activity, the latter effect being strongly dependent on the peptidylprolyl-isomerase activity and also on the TPR domain of FKBP52, but its interaction with Hsp90 is not required. In unstimulated cells, FKBP51 forms endogenous complexes with cytoplasmic RelA. Upon cell stimulation with phorbol ester, the NF-κB soluble complex exchanges FKBP51 for FKBP52, and the NF-κB biological effect is triggered. Importantly, FKBP52 is functionally recruited to the promoter region of NF-κB target genes, whereas FKBP51 is released. Competition assays demonstrated that both immunophilins antagonize one another, and binding assays with purified proteins suggest that the association of RelA and immunophilins could be direct. These observations suggest that the biological action of NF-κB in different cell types could be positively regulated by a high FKBP52/FKBP51 expression ratio by favoring NF-κB nuclear retention, recruitment to the promoter regions of target genes, and transcriptional activity. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Inhibition of Ca2(+)-induced large-amplitude swelling of liver and heart mitochondria by cyclosporin is probably caused by the inhibitor binding to mitochondrial-matrix peptidyl-prolyl cis-trans isomerase and preventing it interacting with the adenine nucleotide translocase.

    PubMed Central

    Halestrap, A P; Davidson, A M

    1990-01-01

    1. Isolated rat liver and heart mitochondria incubated in 150 mM-KSCN or sucrose medium in the presence of respiratory-chain inhibitors showed a large increase in swelling when exposed to 250 microM-Ca2+. Swelling was inhibited by bongkrekic acid and cyclosporin A in both media and by ADP in KSCN medium; the effect of ADP was reversed by carboxyatractyloside. These results demonstrate that this is a suitable technique with which to study the opening of the Ca2(+)-induced non-specific pore of the mitochondrial inner membrane and implicate the adenine nucleotide carrier in this process. 2. Titration of the rate of swelling with increasing concentrations of cyclosporin showed the number of cyclosporin-binding sites (+/- S.E.M.) in liver and heart mitochondria to be respectively 113.7 +/- 5.0 (n = 9) and 124.3 +/- 11.2 (n = 10) pmol/mg of protein, with a Ki of about 5 nM. 3. Liver and heart mitochondrial-matrix fractions were prepared free of membrane and cytosolic contamination and shown to contain cyclosporin-sensitive peptidyl-prolyl cis-trans isomerase (cyclophilin) activity. Titration of isomerase activity with cyclosporin gave values (+/- S.E.M.) of 110.6 +/- 10.1 (n = 5) and 165.4 +/- 15.0 (n = 3) pmol of enzyme/mg of liver and heart mitochondrial protein respectively, with a Ki of 2.5 nM. The similarity of these results to those from the swelling experiments suggest that the isomerase may be involved in the Ca2(+)-induced swelling. 4. The rapid light-scattering change induced in energized heart mitochondria exposed to submicromolar Ca2+ [Halestrap (1987) Biochem. J. 244, 159-164] was inhibited by ADP and bongkrekic acid, the former effect being reversed by carboxyatractyloside. These results suggest an interaction of Ca2+ with the adenine nucleotide carrier when the 'c' conformation. 5. A model is proposed in which mitochondrial peptidyl-prolyl cis-trans isomerase interacts with the adenine nucleotide carrier in the presence of Ca2+ to cause non-specific pore

  9. Folding of barstar C40A/C82A/P27A and catalysis of the peptidyl-prolyl cis/trans isomerization by human cytosolic cyclophilin (Cyp18).

    PubMed Central

    Golbik, R.; Fischer, G.; Fersht, A. R.

    1999-01-01

    Refolding of b*C40A/C82A/P27A is comprised of several kinetically detectable folding phases. The slowest phase in refolding originates from trans-->cis isomerization of the Tyr47-Pro48 peptide bond being in cis conformation in the native state. This refolding phase can be accelerated by the peptidyl-prolyl cis/trans isomerase human cytosolic cyclophilin (Cyp18) with a kcat/K(M) of 254,000 M(-1) s(-1). The fast refolding phase is not influenced by the enzyme. PMID:10422840

  10. Functional characterisation of parvulin-type peptidyl prolyl cis-trans isomerase, PinA in Dictyostelium discoideum.

    PubMed

    Haokip, Nemneineng; Naorem, Aruna

    2017-01-08

    Pin1-type parvulins are unique among PPIases that can catalyse an otherwise slow cis-trans isomerisation of phosphorylated peptide bond preceding proline in target proteins. This prolyl isomerisation process can regulate activity, stability and localisation of target proteins and thus control cellular processes like eukaryotic cell proliferation, cell cycle progression and gene regulation. Towards understanding the function of Pin1-type prolyl isomerisation in Dictyostelium discoideum, a slime mould with distinct growth and developmental phases, we identified PinA as a novel Pin1-type parvulin by its ability to complement the temperature sensitivity phenotype associated with a mutation in ESS1 in S. cerevisiae. In D. discoideum, pinA is temporally and spatially regulated during growth and development. PinA is both nuclear as well as cytoplasmic in the growing cells. We further show that loss of pinA (pinA(-)) leads to decreased growth rate, reduced spore formation and abnormal prespore-prestalk patterning. We conclude that PinA is required for normal growth as well as development in D. discoideum.

  11. Peptidyl-prolyl isomerase 1 (Pin1) preserves the phosphorylation state of tissue factor and prolongs its release within microvesicles.

    PubMed

    Ettelaie, Camille; Collier, Mary E W; Featherby, Sophie; Greenman, John; Maraveyas, Anthony

    2017-09-26

    The exposure and release of TF is regulated by post-translational modifications of its cytoplasmic domain. Here, the potential of Pin1 to interact with the cytoplasmic domain of TF, and the outcome on TF function was examined. MDA-MB-231 and transfected-primary endothelial cells were incubated with either Pin1 deactivator Juglone, or its control Plumbagin, as well as transfected with Pin1-specific or control siRNA. TF release into microvesicles following activation, and also phosphorylation and ubiquitination states of cellular-TF were then assessed. Furthermore, the ability of Pin1 to bind wild-type and mutant forms of overexpressed TF-tGFP was investigated by co-immunoprecipitation. Additionally, the ability of recombinant or cellular Pin1 to bind to peptides of the C-terminus of TF, synthesised in different phosphorylation states was examined by binding assays and spectroscopically. Finally, the influence of recombinant Pin1 on the ubiquitination and dephosphorylation of the TF-peptides was examined. Pre-incubation of Pin1 with Juglone but not Plumbagin, reduced TF release as microvesicles and was also achievable following transfection with Pin1-siRNA. This was concurrent with early ubiquitination and dephosphorylation of cellular TF at Ser253. Pin1 co-immunoprecipitated with overexpressed wild-type TF-tGFP but not Ser258→Ala or Pro259→Ala substituted mutants. Pin1 did interact with Ser258-phosphorylated and double-phosphorylated TF-peptides, with the former having higher affinity. Finally, recombinant Pin1 was capable of interfering with the ubiquitination and dephosphorylation of TF-derived peptides. In conclusion, Pin1 is a fast-acting enzyme which may be utilised by cells to protect the phosphorylation state of TF in activated cells prolonging TF activity and release, and therefore ensuring adequate haemostasis. Copyright © 2017. Published by Elsevier B.V.

  12. Peptidyl-prolyl isomerase Pin1-mediated abrogation of APC-β-catenin interaction in squamous cell carcinoma of cervix.

    PubMed

    Jawanjal, Poonam; Salhan, Sudha; Dhawan, Indrani; Tripathi, Richa; Rath, Gayatri

    2014-01-01

    The present study was aimed to investigate the importance of Pin1 expression in Squamous Cell Carcinoma (SCC) of cervix and to assess its level with β-catenin and APC to understand the possible involvement of Pin1 in the regulation of these proteins and subsequent activation of Wnt/β-catenin signaling. Expression of Pin1, β-catenin and APC was examined in 153 SCC patients by immunohistochemistry and revalidated by western blotting. Of the 153 SCC analyzed, Pin1 was overexpressed in 73 (47.71%) cases. Loss of membranous β-catenin was noticed in 117 (76.47%) SCCs, whereas 66/153 (43.13%) and 93/153 (60.78%) cases showed its distinct cytoplasmic as well as nuclear accumulation respectively. Down regulation/loss of APC was observed in 69 (45.09%) cases, suggesting the activation of Wnt/β-catenin pathway in SCCs. Pin1 showed the significant association with nuclear β-catenin (r=.349, p<0.0001) and cytoplasmic loss of APC (r=-.287, p<0.0001). Both Pin1 as well as nuclear β-catenin were found to be associated with tumor stage (p=0.004, p=0.031) and tumor size (p=0.022, p=0.003). The Pin1 overexpression showed the significant association with disease free survival (p=0.002) but not with overall survival (p=0.421) of SCC patients. Current results explore the expressional relationship between Pin1, β-catenin and APC suggesting that Pin1 regulates the activation of Wnt/β-catenin pathway in SCCs via modulating the interaction between β-catenin and APC. Furthermore, the significant association of Pin1 and β-catenin with tumor variables underscores the clinical utility of these proteins in cervical cancer.

  13. Structural plasticity of peptidyl-prolyl isomerase sFkpA is a key to its chaperone function as revealed by solution NMR.

    PubMed

    Hu, Kaifeng; Galius, Veniamin; Pervushin, Konstantin

    2006-10-03

    Intramolecular dynamics of periplasmic chaperone FkpA-deltaCT (sFkpA) and its complexes with partially structured substrates are studied by NMR in solution. The backbone amide 15N relaxation of sFkpA reveals flexibility in the relative orientation between the dimerization domain and two juxtaposed catalytic domains identified in the X-ray structure of sFkpA. This flexibility is attributed to the structural plasticity within the long alpha-helical arm (helix III) consisting of residues 84 and 91. Residual dipolar couplings (RDCs) indicate an absence of fixed orientation between the sFkpA domains. The substrate binding surface of sFkpA is defined on the X-ray structure by mapping of chemical shift perturbations introduced by complexation of sFkpA with its corresponding protein substrates: partially folded RNase A S-protein and reduced carboxymethylated bovine alpha-lactalbumin (RCM-la). A comparison of 15N relaxation of apo-sFkpA and its complex with RNase A S-protein indicates an increased rigidity within the long alpha-helix III and decreased interdomain mobility of the complex. We speculate that these dynamic properties may play a key role in the chaperone activity of sFkpA, since ability to bind different substrates potentially requires structural adaptations of the chaperone protein. We show that binding of sFkpA to RNase A S-protein greatly reduces the population of aggregated oligomeric species of RNase A S-protein. Finally, a molecular model, the so-called "mother's arms" model, is proposed to illustrate the mechanism of chaperone activity by FkpA.

  14. Prolyl isomerases in gene transcription

    PubMed Central

    Hanes, Steven D.

    2014-01-01

    Background Peptidyl-prolyl isomerases (PPIases) are enzymes that assist in the folding of newly-synthesized proteins and regulate the stability, localization, and activity of mature proteins. They do so by catalyzing reversible (cis-trans) rotation about the peptide bond that precedes proline, inducing conformational changes in target proteins. Scope of Review This review will discuss how PPIases regulate gene transcription by controlling the activity of (1) DNA-binding transcription regulatory proteins, (2) RNA polymerase II, and (3) chromatin and histone modifying enzymes. Major Conclusions Members of each family of PPIase (cyclophilins, FKBPs, and parvulins) regulate gene transcription at multiple levels. In all but a few cases, the exact mechanisms remain elusive. Structure studies, development of specific inhibitors, and new methodologies for studying cis/trans isomerization in vivo represent some of the challenges in this new frontier that merges two important fields. General Significance Prolyl isomerases have been found to play key regulatory roles in all phases of the transcription process. Moreover, PPIases control upstream signaling pathways that regulate gene-specific transcription during development, hormone response and environmental stress. More broadly, although transcription is often rate-limiting in the production of enzymes and structural proteins, post-transcriptional modifications are also critical, and PPIases play key roles here as well (see other reviews in this issue). PMID:25450176

  15. Listeria monocytogenes PrsA2 Is Required for Virulence Factor Secretion and Bacterial Viability within the Host Cell Cytosol▿

    PubMed Central

    Alonzo, Francis; Freitag, Nancy E.

    2010-01-01

    In the course of establishing its replication niche within the cytosol of infected host cells, the facultative intracellular bacterial pathogen Listeria monocytogenes must efficiently regulate the secretion and activity of multiple virulence factors. L. monocytogenes encodes two predicted posttranslocation secretion chaperones, PrsA1 and PrsA2, and evidence suggests that PrsA2 has been specifically adapted for bacterial pathogenesis. PrsA-like chaperones have been identified in a number of Gram-positive bacteria, where they are reported to function at the bacterial membrane-cell wall interface to assist in the folding of proteins translocated across the membrane; in some cases, these proteins have been found to be essential for bacterial viability. In this study, the contributions of PrsA2 and PrsA1 to L. monocytogenes growth and protein secretion were investigated in vitro and in vivo. Neither PrsA2 nor PrsA1 was found to be essential for L. monocytogenes growth in broth culture; however, optimal bacterial viability was found to be dependent upon PrsA2 for L. monocytogenes located within the cytosol of host cells. Proteomic analyses of prsA2 mutant strains in the presence of a mutationally activated allele of the virulence regulator PrfA revealed a critical requirement for PrsA2 activity under conditions of PrfA activation, an event which normally takes place within the host cell cytosol. Despite a high degree of amino acid similarity, no detectable degree of functional overlap was observed between PrsA2 and PrsA1. Our results indicate a critical requirement for PrsA2 under conditions relevant to host cell infection. PMID:20823208

  16. Prolyl isomerase Pin1 regulates the osteogenic activity of Osterix.

    PubMed

    Lee, Sung Ho; Jeong, Hyung Min; Han, Younho; Cheong, Heesun; Kang, Bok Yun; Lee, Kwang Youl

    2015-01-15

    Osterix is an essential transcription factor for osteoblast differentiation and bone formation. The mechanism of regulation of Osterix by post-translational modification remains unknown. Peptidyl-prolyl isomerase 1 (Pin1) catalyzes the isomerization of pSer/Thr-Pro bonds and induces a conformational change in its substrates, subsequently regulating diverse cellular processes. In this study, we demonstrated that Pin1 interacts with Osterix and influences its protein stability and transcriptional activity. This regulation is likely due to the suppression of poly-ubiquitination-mediated proteasomal degradation of Osterix. Collectively, our data demonstrate that Pin1 is a novel regulator of Osterix and may play an essential role in the regulation of osteogenic differentiation.

  17. Prolyl isomerase Pin1 promotes proplatelet formation of megakaryocytes via tau.

    PubMed

    Shimizu, Taiki; Uchida, Chiyoko; Shimizu, Ritsuko; Motohashi, Hozumi; Uchida, Takafumi

    2017-11-18

    Here we show that Pin1, a peptidyl-prolyl cis/trans isomerase which catalyzes the isomerization of phosphorylated Ser/Thr-Pro, is a regulatory molecule of thrombopoiesis. We found that mice lacking the Pin1 gene (Pin1(-⁄-) mice) formed more megakaryocytes (MKs) than wild type mice (WT mice), and that the proplatelet formation of MKs was poorer in Pin1(-⁄-) mice than WT mice. Treatment of Meg-01 cells, a megakaryoblastic floating cell line, with shRNA against Pin1 suppressed the proplatelet formation. Expression of tau, a microtubule associated protein was induced in MKs during proplatelet formation. Pin1 bound tau and promoted microtubule polymerization. Our results show that Pin1 serves as a positive regulatory molecule of proplatelet formation of MKs by enhancing the function of phosphorylated tau. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. A high-throughput screen for inhibitors of the prolyl isomerase, Pin1, identifies a seaweed polyphenol that reduces adipose cell differentiation.

    PubMed

    Mori, Tadashi; Hidaka, Masafumi; Ikuji, Hiroko; Yoshizawa, Ibuki; Toyohara, Haruhiko; Okuda, Toru; Uchida, Chiyoko; Asano, Tomoichiro; Yotsu-Yamashita, Mari; Uchida, Takafumi

    2014-01-01

    The peptidyl prolyl cis/trans isomerase Pin1 enhances the uptake of triglycerides and the differentiation of fibroblasts into adipose cells in response to insulin stimulation. Pin1 downregulation could be a potential approach to prevent and treat obesity-related disorders. In order to identify an inhibitor of Pin1 that exhibited minimal cytotoxicity, we established a high-throughput screen for Pin1 inhibitors and used this method to identify an inhibitor from 1,056 crude fractions of two natural product libraries. The candidate, a phlorotannin called 974-B, was isolated from the seaweed, Ecklonia kurome. 974-B inhibited the differentiation of mouse embryonic fibroblasts and 3T3-L1 cells into adipose cells without inducing cytotoxicity. We discovered the Pin1 inhibitor, 974-B, from the seaweed, E. kurome, and showed that it blocks the differentiation of fibroblasts into adipose cells, suggesting that 974-B could be a lead drug candidate for obesity-related disorders.

  19. Roles of Prolyl Isomerases in RNA-Mediated Gene Expression

    PubMed Central

    Thapar, Roopa

    2015-01-01

    The peptidyl-prolyl cis-trans isomerases (PPIases) that include immunophilins (cyclophilins and FKBPs) and parvulins (Pin1, Par14, Par17) participate in cell signaling, transcription, pre-mRNA processing and mRNA decay. The human genome encodes 19 cyclophilins, 18 FKBPs and three parvulins. Immunophilins are receptors for the immunosuppressive drugs cyclosporin A, FK506, and rapamycin that are used in organ transplantation. Pin1 has also been targeted in the treatment of Alzheimer’s disease, asthma, and a number of cancers. While these PPIases are characterized as molecular chaperones, they also act in a nonchaperone manner to promote protein-protein interactions using surfaces outside their active sites. The immunosuppressive drugs act by a gain-of-function mechanism by promoting protein-protein interactions in vivo. Several immunophilins have been identified as components of the spliceosome and are essential for alternative splicing. Pin1 plays roles in transcription and RNA processing by catalyzing conformational changes in the RNA Pol II C-terminal domain. Pin1 also binds several RNA binding proteins such as AUF1, KSRP, HuR, and SLBP that regulate mRNA decay by remodeling mRNP complexes. The functions of ribonucleoprotein associated PPIases are largely unknown. This review highlights PPIases that play roles in RNA-mediated gene expression, providing insight into their structures, functions and mechanisms of action in mRNP remodeling in vivo. PMID:25992900

  20. Theileria parasites secrete a prolyl isomerase to maintain host leukocyte transformation.

    PubMed

    Marsolier, J; Perichon, M; DeBarry, J D; Villoutreix, B O; Chluba, J; Lopez, T; Garrido, C; Zhou, X Z; Lu, K P; Fritsch, L; Ait-Si-Ali, S; Mhadhbi, M; Medjkane, S; Weitzman, J B

    2015-04-16

    Infectious agents develop intricate mechanisms to interact with host cell pathways and hijack their genetic and epigenetic machinery to change host cell phenotypic states. Among the Apicomplexa phylum of obligate intracellular parasites, which cause veterinary and human diseases, Theileria is the only genus that transforms its mammalian host cells. Theileria infection of bovine leukocytes induces proliferative and invasive phenotypes associated with activated signalling pathways, notably JNK and AP-1 (ref. 2). The transformed phenotypes are reversed by treatment with the theilericidal drug buparvaquone. We used comparative genomics to identify a homologue of the peptidyl-prolyl isomerase PIN1 in T. annulata (TaPIN1) that is secreted into the host cell and modulates oncogenic signalling pathways. Here we show that TaPIN1 is a bona fide prolyl isomerase and that it interacts with the host ubiquitin ligase FBW7, leading to its degradation and subsequent stabilization of c-JUN, which promotes transformation. We performed in vitro and in silico analysis and in vivo zebrafish xenograft experiments to demonstrate that TaPIN1 is directly inhibited by the anti-parasite drug buparvaquone (and other known PIN1 inhibitors) and is mutated in a drug-resistant strain. Prolyl isomerization is thus a conserved mechanism that is important in cancer and is used by Theileria parasites to manipulate host oncogenic signalling.

  1. Theileria parasites secrete a prolyl isomerase to maintain host leukocyte transformation

    PubMed Central

    Marsolier, J.; Perichon, M.; DeBarry, JD.; Villoutreix, BO.; Chluba, J.; Lopez, T.; Garrido, C.; Zhou, XZ.; Lu, KP.; Fritsch, L.; Ait-Si-Ali, S.; Mhadhbi, M; Medjkane, S.; Weitzman, JB.

    2014-01-01

    Infectious agents develop intricate mechanisms to interact with host cell pathways and hijack the genetic and epigenetic machinery to change phenotypic states. Amongst the Apicomplexa phylum of obligate intracellular parasites which cause veterinary and human diseases, Theileria is the only genus which transforms its mammalian host cells1. Theileria infection of bovine leukocytes induces proliferative and invasive phenotypes associated with activated signalling pathways, notably JNK and AP-12. The transformed phenotypes are reversed by treatment with the theilericidal drug Buparvaquone3. We used comparative genomics to identify a homologue of the Peptidyl Prolyl Isomerase Pin1 (designated TaPin1) in T. annulata which is secreted into the host cell and modulates oncogenic signalling pathways. Here we show that TaPin1 is a bona fide prolyl isomerase and that it interacts with the host ubiquitin ligase FBW7 leading to its degradation and subsequent stabilization of c-Jun which promotes transformation. We performed in vitro analysis and in vivo zebrafish xenograft experiments to demonstrate that TaPin1 is directly inhibited by the anti-parasite drug Buparvaquone (and other known Pin1 inhibitors) and is mutated in a drug-resistant strain. Prolyl isomerisation is thus a conserved mechanism which is important in cancer and is used by Theileria parasites to manipulate host oncogenic signaling. PMID:25624101

  2. Activation of Colicin M by the FkpA Prolyl Cis-Trans Isomerase/Chaperone*

    PubMed Central

    Helbig, Stephanie; Patzer, Silke I.; Schiene-Fischer, Cordelia; Zeth, Kornelius; Braun, Volkmar

    2011-01-01

    Colicin M (Cma) is specifically imported into the periplasm of Escherichia coli and kills the cells. Killing depends on the periplasmic peptidyl prolyl cis-trans isomerase/chaperone FkpA. To identify the Cma prolyl bonds targeted by FkpA, we replaced the 15 proline residues individually with alanine. Seven mutant proteins were fully active; Cma(P129A), Cma(P176A), and Cma(P260A) displayed 1%, and Cma(P107A) displayed 10% of the wild-type activity. Cma(P107A), Cma(P129A), and Cma(P260A), but not Cma(P176A), killed cells after entering the periplasm via osmotic shock, indicating that the former mutants were translocation-deficient; Cma(P129A) did not bind to the FhuA outer membrane receptor. The crystal structures of Cma and Cma(P176A) were identical, excluding inactivation of the activity domain located far from Pro-176. In a new peptidyl prolyl cis-trans isomerase assay, FkpA isomerized the Cma prolyl bond in peptide Phe-Pro-176 at a high rate, but Lys-Pro-107 and Leu-Pro-260 isomerized at only <10% of that rate. The four mutant proteins secreted into the periplasm via a fused signal sequence were toxic but much less than wild-type Cma. Wild-type and mutant Cma proteins secreted or translocated across the outer membrane by energy-coupled import or unspecific osmotic shock were only active in the presence of FkpA. We propose that Cma unfolds during transfer across the outer or cytoplasmic membrane and refolds to the active form in the periplasm assisted by FkpA. Weak refolding of Cma(P176A) would explain its low activity in all assays. Of the four proline residues identified as being important for Cma activity, Phe-Pro-176 is most likely targeted by FkpA. PMID:21149455

  3. Secretion Chaperones PrsA2 and HtrA Are Required for Listeria monocytogenes Replication following Intracellular Induction of Virulence Factor Secretion

    PubMed Central

    Ahmed, Jana K.

    2016-01-01

    The Gram-positive bacterium Listeria monocytogenes transitions from an environmental organism to an intracellular pathogen following its ingestion by susceptible mammalian hosts. Bacterial replication within the cytosol of infected cells requires activation of the central virulence regulator PrfA followed by a PrfA-dependent induction of secreted virulence factors. The PrfA-induced secreted chaperone PrsA2 and the chaperone/protease HtrA contribute to the folding and stability of select proteins translocated across the bacterial membrane. L. monocytogenes strains that lack both prsA2 and htrA exhibit near-normal patterns of growth in broth culture but are severely attenuated in vivo. We hypothesized that, in the absence of PrsA2 and HtrA, the increase in PrfA-dependent protein secretion that occurs following bacterial entry into the cytosol results in misfolded proteins accumulating at the bacterial membrane with a subsequent reduction in intracellular bacterial viability. Consistent with this hypothesis, the introduction of a constitutively activated allele of prfA (prfA*) into ΔprsA2 ΔhtrA strains was found to essentially inhibit bacterial growth at 37°C in broth culture. ΔprsA2 ΔhtrA strains were additionally found to be defective for cell invasion and vacuole escape in selected cell types, steps that precede full PrfA activation. These data establish the essential requirement for PrsA2 and HtrA in maintaining bacterial growth under conditions of PrfA activation. In addition, chaperone function is required for efficient bacterial invasion and rapid vacuole lysis within select host cell types, indicating roles for PrsA2/HtrA prior to cytosolic PrfA activation and the subsequent induction of virulence factor secretion. PMID:27481256

  4. Phosphate–Induced Renal Fibrosis Requires the Prolyl Isomerase Pin1

    PubMed Central

    Shiizaki, Kazuhiro; Kuro-o, Makoto; Malter, James S.

    2016-01-01

    Tubulo-interstitial fibrosis is a common, destructive endpoint for a variety of kidney diseases. Fibrosis is well correlated with the loss of kidney function in both humans and rodents. The identification of modulators of fibrosis could provide novel therapeutic approaches to reducing disease progression or severity. Here, we show that the peptidyl-prolyl isomerase Pin1 is an important molecular contributor that facilitates renal fibrosis in a well-characterized animal model. While wild-type mice fed a high phosphate diet (HPD) for 8–12 weeks developed calcium deposition, macrophage infiltration and extracellular matrix (ECM) accumulation in the kidney interstitium, Pin1 null mice showed significantly less pathology. The serum Pi in both WT and KO mice were significantly increased by the HPD, but the serum Ca was slightly decreased in KO compared to WT. In addition, both WT and KO HPD mice had less weight gain but exhibited normal organ mass (kidney, lung, spleen, liver and heart). Unexpectedly, renal function was not initially impaired in either genotype irrespective of the HPD. Our results suggest that diet containing high Pi induces rapid renal fibrosis before a significant impact on renal function and that Pin1 plays an important role in the fibrotic process. PMID:26914452

  5. Brown Algae Polyphenol, a Prolyl Isomerase Pin1 Inhibitor, Prevents Obesity by Inhibiting the Differentiation of Stem Cells into Adipocytes.

    PubMed

    Suzuki, Atsuko; Saeki, Toshiyuki; Ikuji, Hiroko; Uchida, Chiyoko; Uchida, Takafumi

    2016-01-01

    While screening for an inhibitor of the peptidyl prolyl cis/trans isomerase, Pin1, we came across a brown algae polyphenol that blocks the differentiation of fibroblasts into adipocytes. However, its effectiveness on the accumulation of fat in the body has never been studied. Oral administration of brown algae polyphenol to mice fed with a high fat diet, suppressed the increase in fat volume to a level observed in mice fed with a normal diet. We speculate that Pin1 might be required for the differentiation of stem cell to adipocytes. We established wild type (WT) and Pin1-/- (Pin1-KO) adipose-derived mesenchymal stem cell (ASC) lines and found that WT ASCs differentiate to adipocytes but Pin1-KO ASCs do not. Oral administration of brown algae polyphenol, a Pin1 inhibitor, reduced fat buildup in mice. We showed that Pin1 is required for the differentiation of stem cells into adipocytes. We propose that oral intake of brown algae polyphenol is useful for the treatment of obesity.

  6. Brown Algae Polyphenol, a Prolyl Isomerase Pin1 Inhibitor, Prevents Obesity by Inhibiting the Differentiation of Stem Cells into Adipocytes

    PubMed Central

    Suzuki, Atsuko; Saeki, Toshiyuki; Ikuji, Hiroko; Uchida, Chiyoko; Uchida, Takafumi

    2016-01-01

    Background While screening for an inhibitor of the peptidyl prolyl cis/trans isomerase, Pin1, we came across a brown algae polyphenol that blocks the differentiation of fibroblasts into adipocytes. However, its effectiveness on the accumulation of fat in the body has never been studied. Methodology/Principal Findings Oral administration of brown algae polyphenol to mice fed with a high fat diet, suppressed the increase in fat volume to a level observed in mice fed with a normal diet. We speculate that Pin1 might be required for the differentiation of stem cell to adipocytes. We established wild type (WT) and Pin1-/- (Pin1-KO) adipose-derived mesenchymal stem cell (ASC) lines and found that WT ASCs differentiate to adipocytes but Pin1-KO ASCs do not. Conclusion and Significance Oral administration of brown algae polyphenol, a Pin1 inhibitor, reduced fat buildup in mice. We showed that Pin1 is required for the differentiation of stem cells into adipocytes. We propose that oral intake of brown algae polyphenol is useful for the treatment of obesity. PMID:28036348

  7. Cyclophilin40 isomerase activity is regulated by a temperature-dependent allosteric interaction with Hsp90.

    PubMed

    Blackburn, Elizabeth A; Wear, Martin A; Landré, Vivian; Narayan, Vikram; Ning, Jia; Erman, Burak; Ball, Kathryn L; Walkinshaw, Malcolm D

    2015-09-01

    Cyclophilin 40 (Cyp40) comprises an N-terminal cyclophilin domain with peptidyl-prolyl isomerase (PPIase) activity and a C-terminal tetratricopeptide repeat (TPR) domain that binds to the C-terminal-EEVD sequence common to both heat shock protein 70 (Hsp70) and Hsp90. We show in the present study that binding of peptides containing the MEEVD motif reduces the PPIase activity by ∼30%. CD and fluorescence assays show that the TPR domain is less stable than the cyclophilin domain and is stabilized by peptide binding. Isothermal titration calorimetry (ITC) shows that the affinity for the-MEEVD peptide is temperature sensitive in the physiological temperature range. Results from these biophysical studies fit with the MD simulations of the apo and holo (peptide-bound) structures which show a significant reduction in root mean square (RMS) fluctuation in both TPR and cyclophilin domains when-MEEVD is bound. The MD simulations of the apo-protein also highlight strong anti-correlated motions between residues around the PPIase-active site and a band of residues running across four of the seven helices in the TPR domain. Peptide binding leads to a distortion in the shape of the active site and a significant reduction in these strongly anti-correlated motions, providing an explanation for the allosteric effect of ligand binding and loss of PPIase activity. Together the experimental and MD results suggest that on heat shock, dissociation of Cyp40 from complexes mediated by the TPR domain leads to an increased pool of free Cyp40 capable of acting as an isomerase/chaperone in conditions of cellular stress.

  8. A dual inhibitor against prolyl isomerase Pin1 and cyclophilin discovered by a novel real-time fluorescence detection method

    SciTech Connect

    Mori, Tadashi; Hidaka, Masafumi; Lin, Yi-Chin; Yoshizawa, Ibuki; Okabe, Takayoshi; Egashira, Shinichiro; Kojima, Hirotatsu; Nagano, Tetsuo; Koketsu, Mamoru; Takamiya, Mari; Uchida, Takafumi

    2011-03-18

    Research highlights: {yields} A Pin1 (prolyl isomerase) inhibitor, TME-001, has been discovered by using a new established high-throughput screening method. {yields} The TME-001 showed a cell-active inhibition with lower cytotoxic effect than known Pin1 inhibitors. {yields} Kinetic analyses revealed that the TME-001 is the first compound that exhibits dual inhibition of Pin1 and another type of prolyl isomerase, cyclophilin. {yields} Thus, similarities of structure and reaction mechanism between Pin1 and cyclophilin are proposed. -- Abstract: Pin1, a peptidyl prolyl cis/trans isomerase (PPIase), is a potential target molecule for cancer, infectious disease, and Alzheimer's disease. We established a high-throughput screening method for Pin1 inhibitors, which employs a real-time fluorescence detector. This screening method identified 66 compounds that inhibit Pin1 out of 9756 compounds from structurally diverse chemical libraries. Further evaluations of surface plasmon resonance methods and a cell proliferation assay were performed. We discovered a cell-active inhibitor, TME-001 (2-(3-chloro-4-fluoro-phenyl)-isothiazol-3-one). Surprisingly, kinetic analyses revealed that TME-001 is the first compound that exhibits dual inhibition of Pin1 (IC{sub 50} = 6.1 {mu}M) and cyclophilin, another type of PPIase, (IC{sub 50} = 13.7 {mu}M). This compound does not inhibit FKBP. This finding suggests the existence of similarities of structure and reaction mechanism between Pin1 and cyclophilin, and may lead to a more complete understanding of the active sites of PPIases.

  9. Specific cross-linking of the proline isomerase cyclophilin to a non-proline-containing peptide.

    PubMed Central

    McNew, J A; Sykes, K; Goodman, J M

    1993-01-01

    A peptide corresponding to an efficient peroxisomal targeting sequence, the carboxy terminal 12 amino acids of PMP20 from Candida boidinii, was employed as an affinity ligand to search for a peroxisomal targeting receptor. Two proteins from yeast extracts with apparent molecular masses of 20 and 80 kDa were detected by chemical cross-linking to radioiodinated peptide. Both proteins were present in cytosolic supernatants. The 20-kDa species did not cross-link to a control peptide with reversed sequence, whereas the 80-kDa protein cross-linked to both peptides. The cross-linking assay was used to purify the 20-kDa protein from Saccharomyces cerevisiae. Partial protein sequencing identified this protein as cyclophilin, the product of the CYP1 gene. This protein, a peptidyl-prolyl cis-trans isomerase, is the yeast homologue of the protein that mediates the immunosuppressant effects of the drug cyclosporin A (CsA). Cross-linking of peptide to cyclophilin was inhibited by CsA. The cross-linking of cyclophilin to the PMP20-derived peptide was unanticipated because the peptide contains no prolines. The CYP1-encoded protein was not required to target proteins to peroxisomes because this organelle appeared to be assembled normally in a CYP1-disrupted strain. Furthermore, the final three amino acids of the peptide, which are critical for peroxisomal sorting, were not required for cross-linking to cyclophilin. We conclude that either cyclophilin is playing a nonessential facilitating role in peroxisomal targeting or that the interaction of the targeting peptide to cyclophilin is mimicking an interaction with an unidentified substrate or effector of cyclophilin. Images PMID:8443418

  10. Specific cross-linking of the proline isomerase cyclophilin to a non-proline-containing peptide.

    PubMed

    McNew, J A; Sykes, K; Goodman, J M

    1993-02-01

    A peptide corresponding to an efficient peroxisomal targeting sequence, the carboxy terminal 12 amino acids of PMP20 from Candida boidinii, was employed as an affinity ligand to search for a peroxisomal targeting receptor. Two proteins from yeast extracts with apparent molecular masses of 20 and 80 kDa were detected by chemical cross-linking to radioiodinated peptide. Both proteins were present in cytosolic supernatants. The 20-kDa species did not cross-link to a control peptide with reversed sequence, whereas the 80-kDa protein cross-linked to both peptides. The cross-linking assay was used to purify the 20-kDa protein from Saccharomyces cerevisiae. Partial protein sequencing identified this protein as cyclophilin, the product of the CYP1 gene. This protein, a peptidyl-prolyl cis-trans isomerase, is the yeast homologue of the protein that mediates the immunosuppressant effects of the drug cyclosporin A (CsA). Cross-linking of peptide to cyclophilin was inhibited by CsA. The cross-linking of cyclophilin to the PMP20-derived peptide was unanticipated because the peptide contains no prolines. The CYP1-encoded protein was not required to target proteins to peroxisomes because this organelle appeared to be assembled normally in a CYP1-disrupted strain. Furthermore, the final three amino acids of the peptide, which are critical for peroxisomal sorting, were not required for cross-linking to cyclophilin. We conclude that either cyclophilin is playing a nonessential facilitating role in peroxisomal targeting or that the interaction of the targeting peptide to cyclophilin is mimicking an interaction with an unidentified substrate or effector of cyclophilin.

  11. The chaperonin cycle cannot substitute for prolyl isomerase activity, but GroEL alone promotes productive folding of a cyclophilin-sensitive substrate to a cyclophilin-resistant form.

    PubMed

    von Ahsen, O; Tropschug, M; Pfanner, N; Rassow, J

    1997-08-01

    The chaperonin GroEL and the peptidyl-prolyl cis-trans isomerase cyclophilin are major representatives of two distinct cellular systems that help proteins to adopt their native three-dimensional structure: molecular chaperones and folding catalysts. Little is known about whether and how these proteins cooperate in protein folding. In this study, we have examined the action of GroEL and cyclophilin on a substrate protein in two distinct prolyl isomerization states. Our results indicate that: (i) GroEL binds the same substrate in different prolyl isomerization states. (ii) GroEL-ES does not promote prolyl isomerizations, but even retards isomerizations. (iii) Cyclophilin cannot promote the correct isomerization of prolyl bonds of a GroEL-bound substrate, but acts sequentially after release of the substrate from GroEL. (iv) A denatured substrate with all-native prolyl bonds is delayed in folding by cyclophilin due to isomerization to non-native prolyl bonds; a substrate that has proceeded in folding beyond a stage where it can be bound by GroEL is still sensitive to cyclophilin. (v) If a denatured cyclophilin-sensitive substrate is first bound to GroEL, however, productive folding to a cyclophilin-resistant form can be promoted, even without GroES. We conclude that GroEL and cyclophilin act sequentially and exert complementary functions in protein folding.

  12. Involvement of prolyl isomerase PIN1 in the cell cycle progression and proliferation of hepatic oval cells.

    PubMed

    Risal, Prabodh; Shrestha, Nirajan; Chand, Lokendra; Sylvester, Karl G; Jeong, Yeon Jun

    2017-04-01

    Liver regenerates remarkably after toxic injury or surgical resection. In the case of failure of resident hepatocytes to restore loss, repopulation is carried out by induction, proliferation, and differentiation of the progenitor cell. Although, some signaling pathways have been verified to contribute oval cell-mediated liver regeneration, role of Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1(Pin1) in the oval cells proliferation is unknown. In the present study, we evaluate the role of Pin1 in oval cells proliferation. In our study, the expression of Pin1 in the mice liver increased after three weeks feeding of 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) diet along with the proliferation of oval cells. The expression of Pin1 was higher in oval cells compared to the hepatocytes.Pin1 inhibition by Juglone reduced oval cell proliferation, which was restored to normal when oval cells were treated with IGF-1. Consistent with increased cell growth, expression of Pin1, β-catenin and PCNA were increased in IGF-1 treated cells in a time dependent manner. In FACS analysis, siRNA-mediated knockdown of the Pin1 protein in the oval cells significantly increased the numbers of cells in G0/G1 phase. Furthermore, hepatocyte when treated with TGF-β showed marked reduction in cell proliferation and expression of Pin1 whereas this effect was not seen in the oval cells treated with TGF-β. In conclusion, Pin1 plays important role in the cell cycle progression and increase oval cells proliferation which may be crucial in chronic liver injury. Copyright © 2017 Elsevier GmbH. All rights reserved.

  13. An additional function of the rough endoplasmic reticulum protein complex prolyl 3-hydroxylase 1·cartilage-associated protein·cyclophilin B: the CXXXC motif reveals disulfide isomerase activity in vitro.

    PubMed

    Ishikawa, Yoshihiro; Bächinger, Hans Peter

    2013-11-01

    Collagen biosynthesis occurs in the rough endoplasmic reticulum, and many molecular chaperones and folding enzymes are involved in this process. The folding mechanism of type I procollagen has been well characterized, and protein disulfide isomerase (PDI) has been suggested as a key player in the formation of the correct disulfide bonds in the noncollagenous carboxyl-terminal and amino-terminal propeptides. Prolyl 3-hydroxylase 1 (P3H1) forms a hetero-trimeric complex with cartilage-associated protein and cyclophilin B (CypB). This complex is a multifunctional complex acting as a prolyl 3-hydroxylase, a peptidyl prolyl cis-trans isomerase, and a molecular chaperone. Two major domains are predicted from the primary sequence of P3H1: an amino-terminal domain and a carboxyl-terminal domain corresponding to the 2-oxoglutarate- and iron-dependent dioxygenase domains similar to the α-subunit of prolyl 4-hydroxylase and lysyl hydroxylases. The amino-terminal domain contains four CXXXC sequence repeats. The primary sequence of cartilage-associated protein is homologous to the amino-terminal domain of P3H1 and also contains four CXXXC sequence repeats. However, the function of the CXXXC sequence repeats is not known. Several publications have reported that short peptides containing a CXC or a CXXC sequence show oxido-reductase activity similar to PDI in vitro. We hypothesize that CXXXC motifs have oxido-reductase activity similar to the CXXC motif in PDI. We have tested the enzyme activities on model substrates in vitro using a GCRALCG peptide and the P3H1 complex. Our results suggest that this complex could function as a disulfide isomerase in the rough endoplasmic reticulum.

  14. TAL Effectors Target the C-Terminal Domain of RNA Polymerase II (CTD) by Inhibiting the Prolyl-Isomerase Activity of a CTD-Associated Cyclophilin

    PubMed Central

    de Oliveira, Maria Luiza Peixoto; de Mello, Uli Quirino; Benedetti, Celso Eduardo

    2012-01-01

    Transcriptional activator-like (TAL) effectors of plant pathogenic bacteria function as transcription factors in plant cells. However, how TAL effectors control transcription in the host is presently unknown. Previously, we showed that TAL effectors of the citrus canker pathogen Xanthomonas citri, named PthAs, targeted the citrus protein complex comprising the thioredoxin CsTdx, ubiquitin-conjugating enzymes CsUev/Ubc13 and cyclophilin CsCyp. Here we show that CsCyp complements the function of Cpr1 and Ess1, two yeast cyclophilins that regulate transcription by the isomerization of proline residues of the regulatory C-terminal domain (CTD) of RNA polymerase II. We also demonstrate that CsCyp, CsTdx, CsUev and four PthA variants interact with the citrus CTD and that CsCyp co-immunoprecipitate with the CTD in citrus cell extracts and with PthA2 transiently expressed in sweet orange epicotyls. The interactions of CsCyp with the CTD and PthA2 were inhibited by cyclosporin A (CsA), a cyclophilin inhibitor. Moreover, we present evidence that PthA2 inhibits the peptidyl-prolyl cis-trans isomerase (PPIase) activity of CsCyp in a similar fashion as CsA, and that silencing of CsCyp, as well as treatments with CsA, enhance canker lesions in X. citri-infected leaves. Given that CsCyp appears to function as a negative regulator of cell growth and that Ess1 negatively regulates transcription elongation in yeast, we propose that PthAs activate host transcription by inhibiting the PPIase activity of CsCyp on the CTD. PMID:22911812

  15. Rhein exhibits antitumorigenic effects by interfering with the interaction between prolyl isomerase Pin1 and c-Jun.

    PubMed

    Cho, Jin Hyoung; Chae, Jung-Il; Shim, Jung-Hyun

    2017-03-01

    The Pin1 protein (or peptidyl-prolyl cis/trans isomerase) specifically catalyzes the cis/trans isomerization of phosphorylated serine/threonine-proline (Ser/Thr-Pro) bonds and plays an important role in many cellular events through the effects of conformational change in the function of c-Jun, its biological substrate. Pin1 expression is involved in essential cellular pathways that mediate cell proliferation, cell cycle progression, tumorigenesis and apoptosis by altering their stability and function, and it is overexpressed in various types of tumors. Pin1 phosphorylation has been regarded as a marker of Pin1 isomerase activity, and the phosphorylation of Ser/Thr-Pro on protein substrates is prerequisite for its binding activity with Pin1 and subsequent isomerization. Since phosphorylation of proteins on Ser/Thr-Pro is a key regulatory mechanism in the control of cell proliferation and transformation, Pin1 has become an attractive molecule in cancer research. Many inhibitors of Pin1 have been discovered, including several classes of both designed inhibitors and natural products. Anthraquinone compounds possess antitumor properties and have therefore been applied in human and veterinary therapeutics as active substances in medicinal products. Among the anthraquinones, rhein (4,5-dihydroxy-9,10-dioxoanthracene-2-carboxylic acid) is a monomeric anthraquinone derivative found mainly in plants in the Polygonaceae family, such as rhubarb and Polygonum cuspidatum. Recent studies have shown that rhein has numerous pharmacological activities, including antitumor effects. Here, we demonstrated the antitumorigenic effect of rhein using cell proliferation assay, anchorage-independent cell transformation, pull-down assay, luciferase promoter activity, fluorescence-activated cell sorting and western blot analysis. The rhein/Pin1 association was found to play a regulatory role in cell proliferation and neoplastic cell transformation and the binding of phosphorylated c-Jun (Ser

  16. FK506-binding protein mutational analysis: defining the active-site residue contributions to catalysis and the stability of ligand complexes.

    PubMed

    DeCenzo, M T; Park, S T; Jarrett, B P; Aldape, R A; Futer, O; Murcko, M A; Livingston, D J

    1996-02-01

    The 12 kDa FK506-binding protein FKBP12 is a cis-trans peptidyl-prolyl isomerase that binds the macrolides FK506 and rapamycin. We have examined the role of the binding pocket residues of FKBP12 in protein-ligand interactions by making conservative substitutions of 12 of these residues by site-directed mutagenesis. For each mutant FKBP12, we measured the affinity for FK506 and rapamycin and the catalytic efficiency in the cis-frans peptidyl-prolyl isomerase reaction. The mutation of Trp59 or Phe99 generates an FKBP12 with a significantly lower affinity for FK506 than wild-type protein. Tyr26 and Tyr82 mutants are enzymatically active, demonstrating that hydrogen bonding by these residues is not required for catalysis of the cis-trans peptidyl-prolyl isomerase reaction, although these mutations alter the substrate specificity of the enzyme. We conclude that hydrophobic interactions in the active site dominate in the stabilization of FKBP12 binding to macrolide ligands and to the twisted-amide peptidyl-prolyl substrate intermediate.

  17. Genetics Home Reference: glucose phosphate isomerase deficiency

    MedlinePlus

    ... Me Understand Genetics Home Health Conditions GPI deficiency glucose phosphate isomerase deficiency Enable Javascript to view the ... boxes. Download PDF Open All Close All Description Glucose phosphate isomerase (GPI) deficiency is an inherited disorder ...

  18. Development of Novel Sugar Isomerases by Optimization of Active Sites in Phosphosugar Isomerases for Monosaccharides

    PubMed Central

    Yeom, Soo-Jin; Kim, Yeong-Su

    2013-01-01

    Phosphosugar isomerases can catalyze the isomerization of not only phosphosugar but also of monosaccharides, suggesting that the phosphosugar isomerases can be used as sugar isomerases that do not exist in nature. Determination of active-site residues of phosphosugar isomerases, including ribose-5-phosphate isomerase from Clostridium difficile (CDRPI), mannose-6-phosphate isomerase from Bacillus subtilis (BSMPI), and glucose-6-phosphate isomerase from Pyrococcus furiosus (PFGPI), was accomplished by docking of monosaccharides onto the structure models of the isomerases. The determinant residues, including Arg133 of CDRPI, Arg192 of BSMPI, and Thr85 of PFGPI, were subjected to alanine substitutions and found to act as phosphate-binding sites. R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI displayed the highest catalytic efficiencies for monosaccharides at each position. These residues exhibited 1.8-, 3.5-, and 4.9-fold higher catalytic efficiencies, respectively, for the monosaccharides than the wild-type enzyme. However, the activities of these 3 variant enzymes for phosphosugars as the original substrates disappeared. Thus, R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI are no longer phosphosugar isomerases; instead, they are changed to a d-ribose isomerase, an l-ribose isomerase, and an l-talose isomerase, respectively. In this study, we used substrate-tailored optimization to develop novel sugar isomerases which are not found in nature based on phosphosugar isomerases. PMID:23204422

  19. Elucidating cdc25’s Oncogenic Mechanism in Breast Cancer Using Pin1, a Negative Mitotic Regulator

    DTIC Science & Technology

    2001-07-01

    Davies, P., and Lu, K. P. (1999a). The prolyl isomerase Pinl restores the function of Alzheimer-associated phosphorylated tau protein. Nature 399, 784...characterization of a 14 kDa human protein as a novel parvulin-like peptidyl prolyl cis/trans isomerase. Febs Letters 446, 278- 82. Verdecia, M. A., Bowman...isomerization regulates dephosphorylation of Cdc25C and tau proteins. Mol Cell 6, 873-83. 18 Contains unpublished and proprietary information. - Not

  20. A family of cyclophilin-like molecular chaperones in Plasmodium falciparum.

    PubMed

    Marín-Menéndez, Alejandro; Monaghan, Paul; Bell, Angus

    2012-07-01

    The cyclophilins are a large family of proteins implicated in folding, transport and regulation of other proteins and are potential drug targets in cancer and in some viral and parasitic infections. The functionality of cyclophilins appears to depend on peptidyl-prolyl cis-trans isomerase (foldase) and/or molecular chaperone activities. In this study we assessed the peptidyl-prolyl isomerase and chaperone activities of 8 members of the Plasmodium falciparum cyclophilin family, all produced recombinantly using a common host/vector system. While only two of these proteins had isomerase activity, all of them displayed chaperone function as judged by the ability to prevent the thermal aggregation of model substrates. We suggest that the cyclophilins constitute a family of molecular chaperones in malarial parasites that complement the functions of other chaperones such as the heat-shock proteins. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. 21 CFR 862.1720 - Triose phosphate isomerase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... this device are used in the diagnosis and treatment of congenital triose phosphate isomerase enzyme...

  2. 21 CFR 862.1720 - Triose phosphate isomerase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... this device are used in the diagnosis and treatment of congenital triose phosphate isomerase enzyme...

  3. 21 CFR 862.1720 - Triose phosphate isomerase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... this device are used in the diagnosis and treatment of congenital triose phosphate isomerase enzyme...

  4. 21 CFR 862.1720 - Triose phosphate isomerase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... this device are used in the diagnosis and treatment of congenital triose phosphate isomerase enzyme...

  5. 21 CFR 862.1720 - Triose phosphate isomerase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... this device are used in the diagnosis and treatment of congenital triose phosphate isomerase enzyme...

  6. An endoplasmic reticulum-specific cyclophilin.

    PubMed Central

    Hasel, K W; Glass, J R; Godbout, M; Sutcliffe, J G

    1991-01-01

    Cyclophilin is a ubiquitously expressed cytosolic peptidyl-prolyl cis-trans isomerase that is inhibited by the immunosuppressive drug cyclosporin A. A degenerate oligonucleotide based on a conserved cyclophilin sequence was used to isolate cDNA clones representing a ubiquitously expressed mRNA from mice and humans. This mRNA encodes a novel 20-kDa protein, CPH2, that shares 64% sequence identity with cyclophilin. Bacterially expressed CPH2 binds cyclosporin A and is a cyclosporin A-inhibitable peptidyl-prolyl cis-trans isomerase. Cell fractionation of rat liver followed by Western blot (immunoblot) analysis indicated that CPH2 is not cytosolic but rather is located exclusively in the endoplasmic reticulum. These results suggest that cyclosporin A mediates its effect on cells through more than one cyclophilin and that cyclosporin A-induced misfolding of T-cell membrane proteins normally mediated by CPH2 plays a role in immunosuppression. Images PMID:1710767

  7. Thermoinactivation Mechanism of Glucose Isomerase

    NASA Astrophysics Data System (ADS)

    Lim, Leng Hong; Saville, Bradley A.

    In this article, the mechanisms of thermoinactivation of glucose isomerase (GI) from Streptomyces rubiginosus (in soluble and immobilized forms) were investigated, particularly the contributions of thiol oxidation of the enzyme's cysteine residue and a "Maillard-like" reaction between the enzyme and sugars in high fructose corn syrup (HFCS). Soluble GI (SGI) was successfully immobilized on silica gel (13.5 μm particle size), with an activity yield between 20 and 40%. The immobilized GI (IGI) has high enzyme retention on the support during the glucose isomerization process. In batch reactors, SGI (half-life =145 h) was more stable than IGI (half-life=27 h) at 60°C in HFCS, whereas at 80°C, IGI (half-life=12 h) was more stable than SGI (half-life=5.2 h). IGI was subject to thiol oxidation at 60°C, which contributed to the enzyme's deactivation. IGI was subject to thiol oxidation at 80°C, but this did not contribute to the deactivation of the enzyme. SGI did not undergo thiol oxidation at 60°C, but at 80°C SGI underwent severe precipitation and thiol oxidation, which caused the enzyme to deactivate. Experimental results show that immobilization suppresses the destablizing effect of thiol oxidation on GI. A "Maillard-like" reaction between SGI and the sugars also caused SGI thermoinactivation at 60, 70, and 80°C, but had minimal effect on IGI. At 60 and 80°C, IGI had higher thermostability in continuous reactors than in batch reactors, possibily because of reduced contact with deleterious compounds in HFCS.

  8. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Insoluble glucose isomerase enzyme preparations... RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1372 Insoluble glucose isomerase enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the production...

  9. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Insoluble glucose isomerase enzyme preparations... RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1372 Insoluble glucose isomerase enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the production of...

  10. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Insoluble glucose isomerase enzyme preparations... RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1372 Insoluble glucose isomerase enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the production of...

  11. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Insoluble glucose isomerase enzyme preparations... RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1372 Insoluble glucose isomerase enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the production of...

  12. A case study of proline isomerization in cell signaling.

    PubMed

    Min, Lie; Fulton, D Bruce; Andreotti, Amy H

    2005-01-01

    Protein-mediated interactions and enzymatic function provide the foundation upon which cellular signaling cascades control all of the activities of a cell. Post-translational modifications such as phosphorylation or ubiquitiation are well known means for modulating protein activity within the cell. These chemical modifications create new recognition motifs on proteins or shift conformational preferences such that protein catalytic and binding functions are altered in response to external stimuli. Moreover, detection of such modifications is often straightforward by conventional biochemical methods leading investigators toward mechanistic models of cell signaling involving post-translational modifications such as phosphorylation/dephosphorylation. While there is little doubt that such modifications play a significant role in transmission of information throughout the cell, there are certainly other mechanisms at work that are not as well understood at this time. Of particular interest in the context of this review is the intrinsic conformational switch afforded to a polypeptide by peptidyl prolyl cis/trans isomerization. Proline isomerization is emerging as a critical component of certain cell signaling cascades. In addition to serving as a conformational switch that enables a protein to adopt functionally distinct states, proline isomerization may serve as a recognition element for the ubiquitous peptidyl prolyl isomerases. This overview takes a close look at one particular signaling protein, the T cell specific tyrosine kinase Itk, and examines the role of proline isomerization and the peptidyl prolyl isomerase cyclophilin A in mediating Itk function following T cell receptor engagement.

  13. 21 CFR 862.1570 - Phosphohexose isomerase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    .... Measurements of phosphohexose isomerase are used in the diagnosis and treatment of muscle diseases such as muscular dystrophy, liver diseases such as hepatitis or cirrhosis, and metastatic carcinoma....

  14. 21 CFR 862.1570 - Phosphohexose isomerase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    .... Measurements of phosphohexose isomerase are used in the diagnosis and treatment of muscle diseases such as muscular dystrophy, liver diseases such as hepatitis or cirrhosis, and metastatic carcinoma....

  15. 21 CFR 862.1570 - Phosphohexose isomerase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    .... Measurements of phosphohexose isomerase are used in the diagnosis and treatment of muscle diseases such as muscular dystrophy, liver diseases such as hepatitis or cirrhosis, and metastatic carcinoma....

  16. 21 CFR 862.1570 - Phosphohexose isomerase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    .... Measurements of phosphohexose isomerase are used in the diagnosis and treatment of muscle diseases such as muscular dystrophy, liver diseases such as hepatitis or cirrhosis, and metastatic carcinoma....

  17. Dynamical role of phosphorylation on serine/threonine-proline Pin1 substrates from constant force molecular dynamics simulations.

    PubMed

    Velazquez, Hector A; Hamelberg, Donald

    2015-02-21

    Cis-trans isomerization of peptidyl-prolyl bonds of the protein backbone plays an important role in numerous biological processes. Cis-trans isomerization can be the rate-limiting step due its extremely slow dynamics, compared to the millisecond time scale of many processes, and is catalyzed by a widely studied family of peptidyl-prolyl cis-trans isomerase enzymes. Also, mechanical forces along the peptide chain can speed up the rate of isomerization, resulting in "mechanical catalysis," and have been used to study peptidyl-prolyl cis-trans isomerization and other mechanical properties of proteins. Here, we use constant force molecular dynamics simulations to study the dynamical effects of phosphorylation on serine/threonine-proline protein motifs that are involved in the function of many proteins and have been implicated in many aberrant biological processes. We show that the rate of cis-trans isomerization is slowed down by phosphorylation, in excellent agreement with experiments. We use a well-grounded theory to describe the force dependent rate of isomerization. The calculated rates at zero force are also in excellent agreement with experimentally measured rates, providing additional validation of the models and force field parameters. Our results suggest that the slowdown in the rate upon phosphorylation is mainly due to an increase in the friction along the peptidyl-prolyl bond angle during isomerization. Our results provide a microscopic description of the dynamical effects of post-translational phosphorylation on cis-trans isomerization and insights into the properties of proteins under tension.

  18. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Insoluble glucose isomerase enzyme preparations... Substances Affirmed as GRAS § 184.1372 Insoluble glucose isomerase enzyme preparations. (a) Insoluble glucose... defined in § 170.3(o)(9) of this chapter, to convert glucose to fructose. (2) The ingredient is used in...

  19. Current studies on sucrose isomerase and biological isomaltulose production using sucrose isomerase.

    PubMed

    Mu, Wanmeng; Li, Wenjing; Wang, Xiao; Zhang, Tao; Jiang, Bo

    2014-08-01

    Isomaltulose is a natural isomer of sucrose. It is widely used as a functional sweetener with promising properties, including slower digestion, lower glycemic index, prolonged energy release, lower insulin reaction, and less cariogenicity. It has been approved as a safe sucrose substitute by the Food and Drug Administration of the US; Ministry of Health, Labor and Welfare of Japan; and the Commission of the European Communities. This article presents a review of recent studies on the properties, physiological effects, and food application of isomaltulose. In addition, the biochemical properties of sucrose isomerases producing isomaltulose are compared; the heterologous expression, fermentation optimization, structural determination, and catalysis mechanism of sucrose isomerase are reviewed; and the biotechnological production of isomaltulose from sucrose is summarized.

  20. Molecular and industrial aspects of glucose isomerase.

    PubMed Central

    Bhosale, S H; Rao, M B; Deshpande, V V

    1996-01-01

    Glucose isomerase (GI) (D-xylose ketol-isomerase; EC. 5.3.1.5) catalyzes the reversible isomerization of D-glucose and D-xylose to D-fructose and D-xylulose, respectively. The enzyme has the largest market in the food industry because of its application in the production of high-fructose corn syrup (HFCS). HFCS, an equilibrium mixture of glucose and fructose, is 1.3 times sweeter than sucrose and serves as a sweetener for use by diabetics. Interconversion of xylose to xylulose by GI serves a nutritional requirement in saprophytic bacteria and has a potential application in the bioconversion of hemicellulose to ethanol. The enzyme is widely distributed in prokaryotes. Intensive research efforts are directed toward improving its suitability for industrial application. Development of microbial strains capable of utilizing xylan-containing raw materials for growth or screening for constitutive mutants of GI is expected to lead to discontinuation of the use of xylose as an inducer for the production of the enzyme. Elimination of Co2+ from the fermentation medium is desirable for avoiding health problems arising from human consumption of HFCS. Immobilization of GI provides an efficient means for its easy recovery and reuse and lowers the cost of its use. X-ray crystallographic and genetic engineering studies support a hydride shift mechanism for the action of GI. Cloning of GI in homologous as well as heterologous hosts has been carried out, with the prime aim of overproducing the enzyme and deciphering the genetic organization of individual genes (xylA, xylB, and xylR) in the xyl operon of different microorganisms. The organization of xylA and xylB seems to be highly conserved in all bacteria. The two genes are transcribed from the same strand in Escherichia coli and Bacillus and Lactobacillus species, whereas they are transcribed divergently on different strands in Streptomyces species. A comparison of the xylA sequences from several bacterial sources revealed the

  1. Kemp Eliminase Activity of Ketosteroid Isomerase.

    PubMed

    Lamba, Vandana; Sanchez, Enis; Fanning, Lauren Rose; Howe, Kathryn; Alvarez, Maria Alejandra; Herschlag, Daniel; Forconi, Marcello

    2017-01-31

    Kemp eliminases represent the most successful class of computationally designed enzymes, with rate accelerations of up to 10(9)-fold relative to the rate of the same reaction in aqueous solution. Nevertheless, several other systems such as micelles, catalytic antibodies, and cavitands are known to accelerate the Kemp elimination by several orders of magnitude. We found that the naturally occurring enzyme ketosteroid isomerase (KSI) also catalyzes the Kemp elimination. Surprisingly, mutations of D38, the residue that acts as a general base for its natural substrate, produced variants that catalyze the Kemp elimination up to 7000-fold better than wild-type KSI does, and some of these variants accelerate the Kemp elimination more than the computationally designed Kemp eliminases. Analysis of the D38N general base KSI variant suggests that a different active site carboxylate residue, D99, performs the proton abstraction. Docking simulations and analysis of inhibition by active site binders suggest that the Kemp elimination takes place in the active site of KSI and that KSI uses the same catalytic strategies of the computationally designed enzymes. In agreement with prior observations, our results strengthen the conclusion that significant rate accelerations of the Kemp elimination can be achieved with very few, nonspecific interactions with the substrate if a suitable catalytic base is present in a hydrophobic environment. Computational design can fulfill these requirements, and the design of more complex and precise environments represents the next level of challenges for protein design.

  2. Plant Triose Phosphate Isomerase Isozymes 1

    PubMed Central

    Pichersky, Eran; Gottlieb, Leslie D.

    1984-01-01

    We report the first complete purifications of the cytosolic and plastid isozymes of triose phosphate isomerase (TPI; EC 5.3.1.1) from higher plants including spinach (Spinacia oleracea), lettuce (Lactuca sativa), and celery (Apium graveolens). Both isozymes are composed of two isosubunits with approximate molecular weight of 27,000; in spinach and lettuce the plastid isozyme is 200 to 400 larger than the cytosolic isozyme. The two isozymes, purified from lettuce, had closely similar amino acid compositions with the exception of methionine which was four times more prevalent in the cytosolic isozyme. Partial amino acid sequences from the N-terminus were also obtained for both lettuce TPIs. Nine of the 13 positions sequenced in the two proteins had identical amino acid residues. The partial sequences of the plant proteins showed high similarity to previously sequenced animal TPIs. Immunological studies, using antisera prepared independently against the purified plastid and cytosolic isozymes from spinach, revealed that the cytosolic isozymes from a variety of species formed an immunologically distinct group as did the plastid isozymes. However, both plastid and cytosolic TPIs shared some antigenic determinants. The overall similarity of the two isozymes and the high similarity of their partial amino acid sequences to those of several animals indicate that TPI is a very highly conserved protein. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:16663420

  3. Mining the Arabidopsis and rice genomes for cyclophilin protein families.

    PubMed

    Opiyo, S O; Moriyama, E N

    2009-01-01

    Cyclophilins, which possess peptidyl-prolyl isomerase activity, are cellular targets of immunosuppressant drugs and involved in a wide variety of functions. While the Arabidopsis thaliana genome contains the largest number of cyclophilins, the number of plant cyclophilins available in databases is small compared to that of other organisms. It implies that many cyclophilins are yet to be identified in plants. In order to identify cyclophilin candidates from available plant sequence data, we examined alignment-free methods based on Partial Least Squares (PLS). PLS classifier performed better than profile hidden Markov models and PSI-BLAST in identifying cyclophilins from the Arabidopsis and rice genomes.

  4. Yellow lupine cyclophilin interacts with nucleic acids.

    PubMed

    Nuc, Katarzyna; Leśniewicz, Krzysztof; Nuc, Przemysław; Słomski, Ryszard

    2008-01-01

    To investigate properties of yellow lupine cytosolic cyclophilin, an expression vector pET15CYP was constructed. The CyP cDNA (GenBank accession no.Y16088) reveals an open reading frame of 172 amino acids with the conserved tryptophan residue at position 128 and an insertion of seven amino acids spanning positions 48-54. Yellow lupine cyclophilin, purified after expression in E. coli cells, exhibits peptidyl-prolyl cis/trans isomerase activity when assayed with a synthetic oligopeptide. We have demonstrated that the recombinant cyclophilin is able to interact with nucleic acids, both single and double stranded DNA fragments as well as RNA.

  5. Computational insights into the suicide inhibition of Plasmodium falciparum Fk506-binding protein 35.

    PubMed

    MacDonald, Corey A; Boyd, Russell J

    2015-08-15

    Malaria is a parasite affecting millions of people worldwide. With the risk of malarial resistance reaching catastrophic levels, novel methods into the inhibition of this disease need to be prioritized. The exploitation of active site differences between parasitic and human peptidyl-prolyl cis/trans isomerases can be used for suicide inhibition, effectively poisoning the parasite without affecting the patient. This method of inhibition was explored using Plasmodium falciparum and Homo sapiens Fk506-binding proteins as templates for quantum mechanics/molecular mechanics calculations. Modification of the natural substrate has shown suicide inhibition is a valid approach for novel anti-malarials with little risk for parasitic resistance.

  6. Thermolabile triose phosphate isomerase in a psychrophilic Clostridium.

    NASA Technical Reports Server (NTRS)

    Shing, Y. W.; Akagi, J. M.; Himes, R. H.

    1972-01-01

    It was found that a psychrophilic Clostridium contains a triose phosphate isomerase which is very labile at moderate temperatures. An investigation showed that the optimal growth temperature of the psychrophile was between 15 and 20 deg C. No growth occurred at 25 deg C. The thermostability of the glycolytic enzymes in the cell-free extracts of Clostridium sp. strain 69 was studied. The data obtained show that the triose phosphate isomerase is quite labile at moderate temperatures. The instability of the enzyme is sufficient to explain the low maximum growth temperature of the psychrophile.

  7. Thermolabile triose phosphate isomerase in a psychrophilic Clostridium.

    NASA Technical Reports Server (NTRS)

    Shing, Y. W.; Akagi, J. M.; Himes, R. H.

    1972-01-01

    It was found that a psychrophilic Clostridium contains a triose phosphate isomerase which is very labile at moderate temperatures. An investigation showed that the optimal growth temperature of the psychrophile was between 15 and 20 deg C. No growth occurred at 25 deg C. The thermostability of the glycolytic enzymes in the cell-free extracts of Clostridium sp. strain 69 was studied. The data obtained show that the triose phosphate isomerase is quite labile at moderate temperatures. The instability of the enzyme is sufficient to explain the low maximum growth temperature of the psychrophile.

  8. Protein disulfide isomerase a multifunctional protein with multiple physiological roles

    NASA Astrophysics Data System (ADS)

    Ali Khan, Hyder; Mutus, Bulent

    2014-08-01

    Protein disulfide isomerase (PDI), is a member of the thioredoxin superfamily of redox proteins. PDI has three catalytic activities including, thiol-disulfide oxireductase, disulfide isomerase and redox-dependent chaperone. Originally, PDI was identified in the lumen of the endoplasmic reticulum and subsequently detected at additional locations, such as cell surfaces and the cytosol. This review will provide an overview of the recent advances in relating the structural features of PDI to its multiple catalytic roles as well as its physiological and pathophysiological functions related to redox regulation and protein folding.

  9. Structural determinants of product specificity of sucrose isomerases.

    PubMed

    Ravaud, Stéphanie; Robert, Xavier; Watzlawick, Hildegard; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

    2009-06-18

    The healthy sweetener isomaltulose is industrially produced from the conversion of sucrose by the sucrose isomerase SmuA from Protaminobacter rubrum. Crystal structures of SmuA in native and deoxynojirimycin complexed forms completed with modeling studies unravel the characteristics of the isomaltulose synthases catalytic pocket and their substrate binding mode. Comparison with the trehalulose synthase MutB highlights the role of Arg(298) and Arg(306) active site residues and surface charges in controlling product specificity of sucrose isomerases (isomaltulose versus trehalulose). The results provide a rationale for the specific design of optimized enzymes.

  10. Ribose 5-Phosphate Isomerase Investigations for the Undergraduate Biochemistry Laboratory

    ERIC Educational Resources Information Center

    Jewett, Kathy; Sandwick, Roger K.

    2011-01-01

    The enzyme ribose 5-phosphate isomerase (RpiA) has many features that make it attractive as a focal point of a semester-long, advanced biochemistry laboratory for undergraduate students. The protein can easily and inexpensively be isolated from spinach using traditional purification techniques. Characterization of RpiA enzyme activity can be…

  11. 21 CFR 862.1570 - Phosphohexose isomerase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Phosphohexose isomerase test system. 862.1570 Section 862.1570 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... muscular dystrophy, liver diseases such as hepatitis or cirrhosis, and metastatic carcinoma. (b...

  12. Ribose 5-Phosphate Isomerase Investigations for the Undergraduate Biochemistry Laboratory

    ERIC Educational Resources Information Center

    Jewett, Kathy; Sandwick, Roger K.

    2011-01-01

    The enzyme ribose 5-phosphate isomerase (RpiA) has many features that make it attractive as a focal point of a semester-long, advanced biochemistry laboratory for undergraduate students. The protein can easily and inexpensively be isolated from spinach using traditional purification techniques. Characterization of RpiA enzyme activity can be…

  13. L-Ribose isomerase and mannose-6-phosphate isomerase: properties and applications for L-ribose production.

    PubMed

    Xu, Zheng; Sha, Yuanyuan; Liu, Chao; Li, Sha; Liang, Jinfeng; Zhou, Jiahai; Xu, Hong

    2016-11-01

    L-Ribose is a synthetic L-form monosaccharide. It is a building block of many novel nucleotide analog anti-viral drugs. Bio-production of L-ribose relies on a two-step reaction: (i) conversion of L-arabinose to L-ribulose by the catalytic action of L-arabinose isomerase (L-AI) and (ii) conversion of L-ribulose to L-ribose by the catalytic action of L-ribose isomerase (L-RI, EC 5.3.1.B3) or mannose-6-phosphate isomerase (MPI, EC 5.3.1.8, alternately named as phosphomannose isomerase). Between the two enzymes, L-RI is a rare enzyme that was discovered in 1996 by Professor Izumori's group, whereas MPI is an essential enzyme in metabolic pathways in humans and microorganisms. Recent studies have focused on their potentials for industrial production of L-ribose. This review summarizes the applications of L-RI and MPI for L-ribose production.

  14. Evidence for distinct dehydrogenase and isomerase sites within a single 3. beta. -hydroxysteroid dehydrogenase/5-ene-4-ene isomerase protein

    SciTech Connect

    Luu-The, V.; Takahashi, Masakazu; de Launoit, Y.; Dumont, M.; Lachance, Y.; Labrie, F. )

    1991-09-10

    Complementary DNA encoding human 3{beta}-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3-{beta}-HSD) has been expressed in transfected GH{sub 4}C{sub 1} with use of the cytomegalovirus promoter. The activity of the expressed protein clearly shows that both dehydrogenase and isomerase enzymatic activities are present within a single protein. However, such findings do not indicate whether the two activities reside within one or two closely related catalytic sites. With use of ({sup 3}H)-5-androstenedione, the intermediate compound in dehydroepiandrosterone (DHEA) transformation into 4-androstenedione by 3{beta}-HSD, the present study shows that 4MA (N,N-diethyl-4-methyl-3-oxo-4-aza-5{alpha}-androstane-17{beta}-carboxamide) and its analogues of 5-androstenedione to 4-androstenedione with an approximately 1,000-fold higher K{sub i} value. The present results thus strongly suggest that dehydrogenase and isomerase activities are present at separate sites on the 3-{beta}-HSD protein. Such data suggest that the irreversible step in the transformation of DHEA to 4-androstenedione is due to a separate site possessing isomerase activity that converts the 5-ene-3-keto to a much more stable 4-ene-3-keto configuration.

  15. A role for trigger factor and an rgg-like regulator in the transcription, secretion and processing of the cysteine proteinase of Streptococcus pyogenes.

    PubMed Central

    Lyon, W R; Gibson, C M; Caparon, M G

    1998-01-01

    The ability of numerous microorganisms to cause disease relies upon the highly regulated expression of secreted proteinases. In this study, mutagenesis with a novel derivative of Tn4001 was used to identify genes required for the expression of the secreted cysteine proteinase (SCP) of the pathogenic Gram-positive bacterium Streptococcus pyogenes. Designated as Rop loci (regulation of proteinase), ropB is a rgg-like transcriptional activator required for transcription of the gene which encodes the proteinase. In contrast, ropA contributes post-transcriptionally to the secretion and processing of SCP and encodes a homologue of Trigger Factor, a peptidyl-prolyl isomerase and putative chaparone which is highly conserved in most bacterial species, but of unknown function. Analysis of additional ropA mutants demonstrated that RopA acts both to assist in targeting SCP to the secretory pathway and to promote the ability of the proprotein to establish an active conformation upon secretion. This latter function was dependent upon the peptidyl-prolyl isomerase domain of RopA and mutants that lacked this domain exhibited a bipartite deficiency manifested as a kinetic defect in autologous processing of the proprotein to the mature proteinase, and as a catalytic defect in the mature proteinase. These results provide insight into the function of Trigger Factor, the regulation of proteinase activity and the mechanism of secretion in Gram-positive bacteria. PMID:9799235

  16. FKBP12 physically and functionally interacts with aspartokinase in Saccharomyces cerevisiae.

    PubMed Central

    Alarcón, C M; Heitman, J

    1997-01-01

    The peptidyl-prolyl isomerase FKBP12 was originally identified as the intracellular receptor for the immunosuppressive drugs FK506 (tacrolimus) and rapamycin (sirolimus). Although peptidyl-prolyl isomerases have been implicated in catalyzing protein folding, the cellular functions of FKBP12 in Saccharomyces cerevisiae and other organisms are largely unknown. Using the yeast two-hybrid system, we identified aspartokinase, an enzyme that catalyzes an intermediate step in threonine and methionine biosynthesis, as an in vivo binding target of FKBP12. Aspartokinase also binds FKBP12 in vitro, and drugs that bind the FKBP12 active site, or mutations in FKBP12 surface and active site residues, disrupt the FKBP12-aspartokinase complex in vivo and in vitro.fpr1 mutants lacking FKBP12 are viable, are not threonine or methionine auxotrophs, and express wild-type levels of aspartokinase protein and activity; thus, FKBP12 is not essential for aspartokinase activity. The activity of aspartokinase is regulated by feedback inhibition by product, and genetic analyses reveal that FKBP12 is important for this feedback inhibition, possibly by catalyzing aspartokinase conformational changes in response to product binding. PMID:9315655

  17. Immunophilins interact with calcineurin in the absence of exogenous immunosuppressive ligands.

    PubMed Central

    Cardenas, M E; Hemenway, C; Muir, R S; Ye, R; Fiorentino, D; Heitman, J

    1994-01-01

    The peptidyl-prolyl isomerases FKBP12 and cyclophilin A (immunophilins) form complexes with the immunosuppressants FK506 and cyclosporin A that inhibit the phosphatase calcineurin. With the yeast two hybrid system, we detect complexes between FKBP12 and the calcineurin A catalytic subunit in both the presence and absence of FK506. Mutations in FKBP12 surface residues or the absence of the calcineurin B regulatory subunit perturb the FK506-dependent, but not the ligand-independent, FKBP12-calcineurin complex. By affinity chromatography, both FKBP12 and cyclophilin A bind calcineurin A in the absence of ligand, and FK506 and cyclosporin A respectively potentiate these interactions. Both in vivo and in vitro, the peptidyl-prolyl isomerase active sites are dispensable for ligand-independent immunophilin-calcineurin complexes. Lastly, by genetic analyses we demonstrate that FKBP12 modulates calcineurin functions in vivo. These findings reveal that immunophilins interact with calcineurin in the absence of exogenous ligands and suggest that immunosuppressants may take advantage of the inherent ability of immunophilins to interact with calcineurin. Images PMID:7529175

  18. Structural Analysis of Protein Folding by the Long-Chain Archaeal Chaperone FKBP26

    SciTech Connect

    E Martinez-Hackert; W Hendrickson

    2011-12-31

    In the cell, protein folding is mediated by folding catalysts and chaperones. The two functions are often linked, especially when the catalytic module forms part of a multidomain protein, as in Methanococcus jannaschii peptidyl-prolyl cis/trans isomerase FKBP26. Here, we show that FKBP26 chaperone activity requires both a 50-residue insertion in the catalytic FKBP domain, also called 'Insert-in-Flap' or IF domain, and an 80-residue C-terminal domain. We determined FKBP26 structures from four crystal forms and analyzed chaperone domains in light of their ability to mediate protein-protein interactions. FKBP26 is a crescent-shaped homodimer. We reason that folding proteins are bound inside the large crescent cleft, thus enabling their access to inward-facing peptidyl-prolyl cis/trans isomerase catalytic sites and ipsilateral chaperone domain surfaces. As these chaperone surfaces participate extensively in crystal lattice contacts, we speculate that the observed lattice contacts reflect a proclivity for protein associations and represent substrate interactions by FKBP26 chaperone domains. Finally, we find that FKBP26 is an exceptionally flexible molecule, suggesting a mechanism for nonspecific substrate recognition.

  19. Grb7 Protein Stability Modulated by Pin1 in Association with Cell Cycle Progression

    PubMed Central

    Tai, Yu-Ling; Tung, Li-Hsuan; Lin, Yu-Chi; Lu, Pei-Jung; Chu, Pei-Yu; Wang, Ming-Yang; Huang, Wei-Pang; Chen, Ko-Chien; Lee, Hsinyu; Shen, Tang-Long

    2016-01-01

    Growth factor receptor bound protein-7 (Grb7) is a multi-domain adaptor protein that is co-opted by numerous tyrosine kinases involved in various cellular signaling and functions. The molecular mechanisms underlying the regulation of Grb7 remain unclear. Here, we revealed a novel negative post-translational regulation of Grb7 by the peptidyl-prolyl cis/trans isomerase, Pin1. Our data show that phosphorylation of Grb7 protein on the Ser194-Pro motif by c-Jun N-terminal kinase facilitates its binding with the WW domain of Pin1. Subsequently, Grb7 is degraded by the ubiquitin- and proteasome-dependent proteolytic pathway. Indeed, we found that Pin1 exerts its peptidyl-prolyl cis/trans isomerase activity in the modulation of Grb7 protein stability in regulation of cell cycle progression at the G2-M phase. This study illustrates a novel regulatory mechanism in modulating Grb7-mediated signaling, which may take part in pathophysiological consequences. PMID:27658202

  20. Cyclophilin 20 is involved in mitochondrial protein folding in cooperation with molecular chaperones Hsp70 and Hsp60.

    PubMed

    Rassow, J; Mohrs, K; Koidl, S; Barthelmess, I B; Pfanner, N; Tropschug, M

    1995-05-01

    We studied the role of mitochondrial cyclophilin 20 (CyP20), a peptidyl-prolyl cis-trans isomerase, in preprotein translocation across the mitochondrial membranes and protein folding inside the organelle. The inhibitory drug cyclosporin A did not impair membrane translocation of preproteins, but it delayed the folding of an imported protein in wild-type mitochondria. Similarly, Neurospora crassa mitochondria lacking CyP20 efficiently imported preproteins into the matrix, but folding of an imported protein was significantly delayed, indicating that CyP20 is involved in protein folding in the matrix. The slow folding in the mutant mitochondria was not inhibited by cyclosporin A. Folding intermediates of precursor molecules reversibly accumulated at the molecular chaperones Hsp70 and Hsp60 in the matrix. We conclude that CyP20 is a component of the mitochondrial protein folding machinery and that it cooperates with Hsp70 and Hsp60. It is speculated that peptidyl-prolyl cis-trans isomerases in other cellular compartments may similarly promote protein folding in cooperation with chaperone proteins.

  1. Molecular characterization of a cyclosporin A-insensitive cyclophilin from the parasitic nematode Brugia malayi.

    PubMed

    Page, A P; Landry, D; Wilson, G G; Carlow, C K

    1995-09-12

    The cyclophilins are a family of proteins that exhibit peptidyl-prolyl cis-trans isomerase (PPIase, EC 5.2.1.8) activity and bind the immunosuppressive agent cyclosporin A (CsA) to varying degrees. We have isolated a cDNA clone encoding a novel cyclophilin from the human filarial parasite Brugia malayi. This gene possesses an N-terminal domain homologous to cyclophilins from diverse phyla (49-60% amino acid sequence identity) and a hydrophilic C-terminal domain. The cyclophilin domain was overexpressed in Escherichia coli and found to possess peptidyl-prolyl cis-trans isomerase (PPIase) activity, with a kcat/Km value of 7.9 x 10(6) M-1 s-1. A histidine residue in lieu of tryptophan in the highly conserved CsA-binding site suggests that B. malayi cyclophilin is more closely related to the cyclophilin-like proteins described recently from natural killer (NK) cells, plants, and the 40 kDa cyclophilins from mammals. In accordance with the histidine-containing CsA-binding domain, the B. malayi enzyme was relatively insensitive to inhibition by CsA, since an IC50 value of 860 nM (compared to 19 nM for human cyclophilin A) was determined.

  2. A membrane-anchored Theileria parva cyclophilin with a non-cleaved amino-terminal signal peptide for entry into the endoplasmic reticulum.

    PubMed

    Ebel, Thomas; Pellé, Roger; Janoo, Rozmin; Lipp, Joachim; Bishop, Richard

    2004-05-07

    Recent studies suggest that peptidyl-prolyl isomerases of the cyclophilin family, that access the secretory pathway, can be involved in the interaction of parasitic protozoa with mammalian host cells. The amino acid sequence of a cDNA encoding a cyclophilin family member of the intracellular protozoan parasite of cattle Theileria parva contains a conserved C-terminal domain that exhibits 70% amino acid identity to cyclophilin proteins from other organisms, and a unique 60 amino acid novel N-terminal extension. Cell-free expression of the cDNA revealed a 26kDa amino translation product, indicating expression of the N-terminal domain. The protein-coding region contains three short introns, less than 100 base pairs in length and Northern blot analysis demonstrates expression of a single 0.9 kb transcript in the piroplasm and schizont stages. The transcript is present in high abundance in the intra-lymphocytic schizont stage. The recombinant protein binds to immobilized cyclosporin A, a finding consistent with peptidyl-prolyl cis-trans isomerase function in vivo. A predicted N-terminal signal peptide was functional for entry into the eukaryotic secretory transport pathway in a cell-free in vitro transcription/translation system. The C-terminal cyclophilin domain was translocated across the membrane of the endoplasmic reticulum and the uncleaved signal peptide functioned as a membrane anchor. Copyright 2004 Elsevier B.V.

  3. FKBP51, a novel T-cell-specific immunophilin capable of calcineurin inhibition.

    PubMed Central

    Baughman, G; Wiederrecht, G J; Campbell, N F; Martin, M M; Bourgeois, S

    1995-01-01

    The immunosuppressive drugs FK506 and cyclosporin A block T-lymphocyte proliferation by inhibiting calcineurin, a critical signaling molecule for activation. Multiple intracellular receptors (immunophilins) for these drugs that specifically bind either FK506 and rapamycin (FK506-binding proteins [FKBPs]) or cyclosporin A (cyclophilins) have been identified. We report the cloning and characterization of a new 51-kDa member of the FKBP family from murine T cells. The novel immunophilin, FKBP51, is distinct from the previously isolated and sequenced 52-kDa murine FKBP, demonstrating 53% identity overall. Importantly, Western blot (immunoblot) analysis showed that unlike all other FKBPs characterized to date, FKBP51 expression was largely restricted to T cells. Drug binding to recombinant FKBP51 was demonstrated by inhibition of peptidyl prolyl isomerase activity. As judged from peptidyl prolyl isomerase activity, FKBP51 had a slightly higher affinity for rapamycin than for FK520, an FK506 analog. FKBP51, when complexed with FK520, was capable of inhibiting calcineurin phosphatase activity in an in vitro assay system. Inhibition of calcineurin phosphatase activity has been implicated both in the mechanism of immunosuppression and in the observed toxic side effects of FK506 in nonlymphoid cells. Identification of a new FKBP that can mediate calcineurin inhibition and is restricted in its expression to T cells suggests that new immunosuppressive drugs may be identified that, by virtue of their specific interaction with FKBP51, would be targeted in their site of action. PMID:7542743

  4. Exploring the chemistry and evolution of the isomerases

    PubMed Central

    2016-01-01

    Isomerization reactions are fundamental in biology, and isomers usually differ in their biological role and pharmacological effects. In this study, we have cataloged the isomerization reactions known to occur in biology using a combination of manual and computational approaches. This method provides a robust basis for comparison and clustering of the reactions into classes. Comparing our results with the Enzyme Commission (EC) classification, the standard approach to represent enzyme function on the basis of the overall chemistry of the catalyzed reaction, expands our understanding of the biochemistry of isomerization. The grouping of reactions involving stereoisomerism is straightforward with two distinct types (racemases/epimerases and cis-trans isomerases), but reactions entailing structural isomerism are diverse and challenging to classify using a hierarchical approach. This study provides an overview of which isomerases occur in nature, how we should describe and classify them, and their diversity. PMID:26842835

  5. Isopentenyl diphosphate isomerase: A checkpoint to isoprenoid biosynthesis.

    PubMed

    Berthelot, Karine; Estevez, Yannick; Deffieux, Alain; Peruch, Frédéric

    2012-08-01

    Even if the isopentenyl diphosphate (IPP) isomerases have been discovered in the 50s, it is only in the last decade that the genetical, enzymatical, structural richness and cellular importance of this large family of crucial enzymes has been uncovered. Present in all living kingdoms, they can be classified in two subfamilies: type 1 and type 2 IPP isomerases, which show clearly distinct characteristics. They all perform the regulatory isomerization of isopentenyl diphosphate into dimethylallyl diphosphate, a key rate-limiting step of the terpenoid biosynthesis, via a protonation/deprotonation mechanism. Due to their importance in the isoprenoid metabolism and the increasing interest of industry devoted to terpenoid production, it is foreseen that the biotechnological development of such enzymes should be under intense scrutiny in the near future.

  6. Solubility of glucose isomerase in ammonium sulphate solutions

    NASA Astrophysics Data System (ADS)

    Chayen, N.; Akins, J.; Campbell-Smith, S.; Blow, D. M.

    1988-07-01

    In order to quantify protein crystallization techniques, a method for measuring protein solubility in high salt concentration has been developed. It is based on a sensitive protein concentration assay, using binding to Coomassie blue dye. The protein concentration in a supernatant from which glucose isomerase is crystallising has been studied as a function of time. Equilibrium is established in 3-5 weeks, and the protein concentration remaining in solution is defined as the solubility of the protein. The solubility of glucose isomerase has been determined as a function of ammonium sulphate concentration; its variation with pH in 1.50M ammonium sulphate has also been studied. A remarkable dependence on pH over the range of 5.5 to 6.5 has been observed.

  7. Methods of measuring Protein Disulfide Isomerase activity: a critical overview

    NASA Astrophysics Data System (ADS)

    Watanabe, Monica; Laurindo, Francisco; Fernandes, Denise

    2014-09-01

    Protein disulfide isomerase is an essential redox chaperone from the endoplasmic reticulum (ER) and is responsible for correct disulfide bond formation in nascent proteins. PDI is also found in other cellular locations in the cell, particularly the cell surface. Overall, PDI contributes to ER and global cell redox homeostasis and signaling. The knowledge about PDI structure and function progressed substantially based on in vitro studies using recombinant PDI and chimeric proteins. In these experimental scenarios, PDI reductase and chaperone activities are readily approachable. In contrast, assays to measure PDI isomerase activity, the hallmark of PDI family, are more complex. Assessment of PDI roles in cells and tissues mainly relies on gain- or loss-of-function studies. However, there is limited information regarding correlation of experimental readouts with the distinct types of PDI activities. In this mini-review, we evaluate the main methods described for measuring the different kinds of PDI activity: thiol reductase, thiol oxidase, thiol isomerase and chaperone. We emphasize the need to use appropriate controls and the role of critical interferents (e.g., detergent, presence of reducing agents). We also discuss the translation of results from in vitro studies with purified recombinant PDI to cellular and tissue samples, with critical comments on the interpretation of results.

  8. Escherichia coli arabinose isomerase and Staphylococcus aureus tagatose-6-phosphate isomerase: which is a better template for directed evolution of non-natural substrate isomerization?

    PubMed

    Kim, Hye Jung; Uhm, Tae Guk; Kim, Seong Bo; Kim, Pil

    2010-06-01

    Metallic and non-metallic isomerases can be used to produce commercially important monosaccharides. To determine which category of isomerase is more suitable as a template for directed evolution to improve enzymes for galactose isomerization, L-arabinose isomerase from Escherichia coli (ECAI; E.C. 5.3.1.4) and tagatose-6-phosphate isomerase from Staphylococcus aureus (SATI; E.C. 5.3.1.26) were chosen as models of a metallic and non-metallic isomerase, respectively. Random mutations were introduced into the genes encoding ECAI and SATI at the same rate, resulting in the generation of 515 mutants of each isomerase. The isomerization activity of each of the mutants toward a non-natural substrate (galactose) was then measured. With an average mutation rate of 0.2 mutations/kb, 47.5% of the mutated ECAIs showed an increase in activity compared with wild-type ECAI, and the remaining 52.5% showed a decrease in activity. Among the mutated SATIs, 58.6% showed an increase in activity, whereas 41.4% showed a decrease in activity. Mutant clones showing a significant change in relative activity were sequenced and specific increases in activity were measured. The maximum increase in activity achieved by mutation of ECAI was 130%, and that for SATI was 190%. Based on these results, the characteristics of the different isomerases are discussed in terms of their usefulness for directed evolution of non-natural substrate isomerization.

  9. Hmo1p, a high mobility group 1/2 homolog, genetically and physically interacts with the yeast FKBP12 prolyl isomerase.

    PubMed Central

    Dolinski, K J; Heitman, J

    1999-01-01

    The immunosuppressive drugs FK506 and rapamycin bind to the cellular protein FKBP12, and the resulting FKBP12-drug complexes inhibit signal transduction. FKBP12 is a ubiquitous, highly conserved, abundant enzyme that catalyzes a rate-limiting step in protein folding: peptidyl-prolyl cis-trans isomerization. However, FKBP12 is dispensible for viability in both yeast and mice, and therefore does not play an essential role in protein folding. The functions of FKBP12 may involve interactions with a number of partner proteins, and a few proteins that interact with FKBP12 in the absence of FK506 or rapamycin have been identified, including the ryanodine receptor, aspartokinase, and the type II TGF-beta receptor; however, none of these are conserved from yeast to humans. To identify other targets and functions of FKBP12, we have screened for mutations that are synthetically lethal with an FKBP12 mutation in yeast. We find that mutations in HMO1, which encodes a high mobility group 1/2 homolog, are synthetically lethal with mutations in the yeast FPR1 gene encoding FKBP12. Deltahmo1 and Deltafpr1 mutants share two phenotypes: an increased rate of plasmid loss and slow growth. In addition, Hmo1p and FKBP12 physically interact in FKBP12 affinity chromatography experiments, and two-hybrid experiments suggest that FKBP12 regulates Hmo1p-Hmo1p or Hmo1p-DNA interactions. Because HMG1/2 proteins are conserved from yeast to humans, our findings suggest that FKBP12-HMG1/2 interactions could represent the first conserved function of FKBP12 other than mediating FK506 and rapamycin actions. PMID:10049913

  10. xylA cloning and sequencing and biochemical characterization of xylose isomerase from Thermotoga neapolitana.

    PubMed Central

    Vieille, C; Hess, J M; Kelly, R M; Zeikus, J G

    1995-01-01

    The xylA gene coding for xylose isomerase from the hyperthermophile Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 444 residues with a calculated molecular weight of 50,892. The native enzyme was a homotetramer with a molecular weight of 200,000. This xylose isomerase was a member of the family II enzymes (these differ from family I isomerases by the presence of approximately 50 additional residues at the amino terminus). The enzyme was extremely thermostable, with optimal activity above 95 degrees C. The xylose isomerase showed maximum activity at pH 7.1, but it had high relative activity over a broad pH range. The catalytic efficiency (kcat/Km) of the enzyme was essentially constant between 60 and 90 degrees C, and the catalytic efficiency decreased between 90 and 98 degrees C primarily because of a large increase in Km. The T. neapolitana xylose isomerase had a higher turnover number and a lower Km for glucose than other family II xylose isomerases. Comparisons with other xylose isomerases showed that the catalytic and cation binding regions were well conserved. Comparison of different xylose isomerase sequences showed that numbers of asparagine and glutamine residues decreased with increasing enzyme thermostability, presumably as a thermophilic strategy for diminishing the potential for chemical denaturation through deamidation at elevated temperatures. PMID:7646024

  11. Thermal-unfolding reaction of triosephosphate isomerase from Trypanosoma cruzi.

    PubMed

    Mixcoha-Hernández, Edgar; Moreno-Vargas, Liliana M; Rojo-Domínguez, Arturo; Benítez-Cardoza, Claudia G

    2007-10-01

    Thermal denaturation of triosephosphate isomerase from Trypanosoma cruzi was studied by circular dicrhoism and fluorescence spectroscopies. The unfolding transition was found to be highly irreversible even at the very early stages of the reaction. Kinetic studies, allowed us to identify consecutive reactions. Firstly, only the tryptophan environment is altered. Next, changes on the secondary structure and hydrophobic surface exposure measured by 1-anilino-8-naphthalenesulfonate (ANS) binding were observed. Further conformational changes imply additional modifications on the secondary and tertiary structures and release of the hydrophobic dye leading to the formation of the unfolded state that is prone to aggregate.

  12. GPI Mount Scopus--a variant of glucosephosphate isomerase deficiency.

    PubMed

    Shalev, O; Shalev, R S; Forman, L; Beutler, E

    1993-10-01

    Glucosephosphate isomerase (GPI) deficiency is an unusual cause of hereditary nonspherocytic hemolytic anemia. The disease, inherited as an autosomal recessive disorder, is most often manifested by symptoms and signs of chronic hemolysis, ameliorated by splenectomy. We recently diagnosed GPI deficiency in a 23-year-old Ashkenazi Jewish man who displayed the typical clinical course of this disorder. The biophysical characteristics of the GPI variant are slow electrophoretic mobility, presence of only one of the two bands normally present, and extreme thermolability. To the best of our knowledge, this is the first report of GPI deficiency in a patient of Jewish descent, and we propose to designate this enzyme variant "GPI Mount Scopus".

  13. The immunosuppressant SR 31747 blocks cell proliferation by inhibiting a steroid isomerase in Saccharomyces cerevisiae.

    PubMed Central

    Silve, S; Leplatois, P; Josse, A; Dupuy, P H; Lanau, C; Kaghad, M; Dhers, C; Picard, C; Rahier, A; Taton, M; Le Fur, G; Caput, D; Ferrara, P; Loison, G

    1996-01-01

    SR 31747 is a novel immunosuppressant agent that arrests cell proliferation in the yeast Saccharomyces cerevisiae, SR 31747-treated cells accumulate the same aberrant sterols as those found in a mutant impaired in delta 8- delta 7-sterol isomerase. Sterol isomerase activity is also inhibited by SR 31747 in in vitro assays. Overexpression of the sterol isomerase-encoding gene, ERG2, confers enhanced SR resistance. Cells growing anaerobically on ergosterol-containing medium are not sensitive to SR. Disruption of the sterol isomerase-encoding gene is lethal in cells growing in the absence of exogenous ergosterol, except in SR-resistant mutants lacking either the SUR4 or the FEN1 gene product. The results suggest that sterol isomerase is the target of SR 31747 and that both the SUR4 and FEN1 gene products are required to mediate the proliferation arrest induced by ergosterol depletion. PMID:8649379

  14. Characterization of a mutant glucose isomerase from Thermoanaerobacterium saccharolyticum.

    PubMed

    Xu, Heng; Shen, Dong; Wu, Xue-Qiang; Liu, Zhi-Wei; Yang, Qi-He

    2014-10-01

    A series of site-directed mutant glucose isomerase at tryptophan 139 from Thermoanaerobacterium saccharolyticum strain B6A were purified to gel electrophoretic homogeneity, and the biochemical properties were determined. W139F mutation is the most efficient mutant derivative with a tenfold increase in its catalytic efficiency toward glucose compared with the native GI. With a maximal activity at 80 °C of 59.58 U/mg on glucose, this mutant derivative is the most active type ever reported. The enzyme activity was maximal at 90 °C and like other glucose isomerase, this mutant enzyme required Co(2+) or Mg(2+) for enzyme activity and thermal stability (stable for 20 h at 80 °C in the absence of substrate). Its optimum pH was around 7.0, and it had 86 % of its maximum activity at pH 6.0 incubated for 12 h at 60 °C. This enzyme was determined as thermostable and weak-acid stable. These findings indicated that the mutant GI W139F from T. saccharolyticum strain B6A is appropriate for use as a potential candidate for high-fructose corn syrup producing enzyme.

  15. Converting a Sulfenic Acid Reductase into a Disulfide Bond Isomerase

    PubMed Central

    Chatelle, Claire; Kraemer, Stéphanie; Ren, Guoping; Chmura, Hannah; Marechal, Nils; Boyd, Dana; Roggemans, Caroline; Ke, Na; Riggs, Paul; Bardwell, James

    2015-01-01

    Abstract Aims: Posttranslational formation of disulfide bonds is essential for the folding of many secreted proteins. Formation of disulfide bonds in a protein with more than two cysteines is inherently fraught with error and can result in incorrect disulfide bond pairing and, consequently, misfolded protein. Protein disulfide bond isomerases, such as DsbC of Escherichia coli, can recognize mis-oxidized proteins and shuffle the disulfide bonds of the substrate protein into their native folded state. Results: We have developed a simple blue/white screen that can detect disulfide bond isomerization in vivo, using a mutant alkaline phosphatase (PhoA*) in E. coli. We utilized this screen to isolate mutants of the sulfenic acid reductase (DsbG) that allowed this protein to act as a disulfide bond isomerase. Characterization of the isolated mutants in vivo and in vitro allowed us to identify key amino acid residues responsible for oxidoreductase properties of thioredoxin-like proteins such as DsbC or DsbG. Innovation and Conclusions: Using these key residues, we also identified and characterized interesting environmental homologs of DsbG with novel properties, thus demonstrating the capacity of this screen to discover and elucidate mechanistic details of in vivo disulfide bond isomerization. Antioxid. Redox Signal. 23, 945–957. PMID:26191605

  16. Loss of secretory pathway FK506-binding proteins results in cold-sensitive lethality and associate extracellular matrix defects in the nematode Caenorhabditis elegans.

    PubMed

    Winter, Alan D; Eschenlauer, Sylvain C P; McCormack, Gillian; Page, Antony P

    2007-04-27

    The FK506-binding proteins (FKBs) represent ubiquitous enzymes that catalyze the rate-limiting peptidyl prolyl cis-trans isomerization step in protein folding. The nematode Caenorhabditis elegans has eight FKBs, three of which (FKB-3, -4, and -5) have dual peptidyl prolyl cis-trans isomerase (PPIase) domains, signal peptides and ER retention signals. PPIase activity has been detected for recombinant FKB-3. Both FKB-3 and -5 are expressed in the exoskeleton-synthesizing hypodermis with transcript peaks that correspond to the molting and collagen synthesis cycles. FKB-4 is expressed at a low level throughout development. No phenotypes were observed in deletion mutants in each of the secretory pathway FKBs. Combined triple and fkb-4, -5 double deletion mutants were however found to arrest at 12 degrees C, but developed normally at 15-25 degrees C. This cold-sensitive larval lethal effect was not maternally derived, occurred during embryogenesis, and could be rescued following the transgenic introduction of a wild type copy of either fkb-4 or fkb-5. The temperature-sensitive defects also affected molting, cuticle collagen expression, hypodermal seam cell morphology, and the structural integrity of the cuticular extracellular matrix. This study establishes that the secretory pathway FK506-binding PPIase enzymes are essential for normal nematode development, collagen biogenesis, and the formation of an intact exoskeleton under adverse physiological conditions.

  17. Microbial cyclophilins: specialized functions in virulence and beyond.

    PubMed

    Dimou, Maria; Venieraki, Anastasia; Katinakis, Panagiotis

    2017-08-08

    Cyclophilins belong to the superfamily of peptidyl-prolyl cis/trans isomerases (PPIases, EC: 5.2.1.8), the enzymes that catalyze the cis/trans isomerization of peptidyl-prolyl peptide bonds in unfolded and partially folded polypeptide chains and native state proteins. Cyclophilins have been extensively studied, since they are involved in multiple cellular processes related to human pathologies, such as neurodegenerative disorders, infectious diseases, and cancer. However, the presence of cyclophilins in all domains of life indicates a broader biological importance. In this mini-review, we summarize current advances in the study of microbial cyclophilins. Apart from their anticipated role in protein folding and chaperoning, cyclophilins are involved in several other biological processes, such as cellular signal transduction, adaptation to stress, control of pathogens virulence, and modulation of host immune response. Since many existing family members do not have well-defined functions and novel ones are being characterized, the requirement for further studies on their biological role and molecular mechanism of action is apparent.

  18. Soybean cyclophilin GmCYP1 interacts with an isoflavonoid regulator GmMYB176

    PubMed Central

    Mainali, Hemanta Raj; Vadivel, Arun Kumaran Anguraj; Li, Xuyan; Gijzen, Mark; Dhaubhadel, Sangeeta

    2017-01-01

    Cyclophilins (CYPs) belong to the immunophilin superfamily with peptidyl-prolyl cis-trans isomerase (PPIase) activity. They catalyze the interconversion of the cis- and trans-rotamers of the peptidyl-prolyl amide bond of peptides. A yeast-two-hybrid screening using the isoflavonoid regulator GmMYB176 as bait identified GmCYP1 as one of the interacting proteins in soybean embryos. GmCYP1 localizes both in the nucleus and cytoplasm, and interacts in planta with GmMYB176, in the nucleus, and with SGF14l (a soybean 14-3-3 protein) in the nucleus and the cytoplasm. GmCYP1 contains a single cyclophilin-like domain and displays a high sequence identity with other plant CYPs that are known to have stress-specific function. Tissue-specific expression of GmCYP1 revealed higher expression in developing seeds compared to other vegetative tissues, suggesting their seed-specific role. Furthermore, GmCYP1 transcript level was reduced in response to stress. Since isoflavonoids are involved in plant stress resistance against biotic and abiotic factors, the interaction of GmCYP1 with the isoflavonoid regulators GmMYB176 and 14-3-3 protein suggests its role in defense in soybean. PMID:28074922

  19. [Cloning of Escherichia coli K12 xylose isomerase (glucose isomerase) and studying the enzymatic properties of its expression product].

    PubMed

    Rozanov, A S; Zagrebel'nyĭ, S N; Beklemishchev, A B

    2009-01-01

    The coding region of Escherichia coli K12 xylose (glucose) isomerase gene was inserted into the pRAC expression vector and cloned in E. coli BL21 (DE3) cells. After induction of expression of the cloned gene, the proportion of recombinant xylose isomerase accounted for 40% of the total protein content. As a result of one-stage purification by affinity chromatography, a protein preparation of 90% purity was obtained. The recombinant enzyme catalyzed the isomerization of glucose to fructose and exhibited maximum activity (0.8 U/mg) at 45 degrees C and pH 6.8. The enzyme required Mg2+ ions as a cofactor. When Mg2+ and Co2+ ions were simultaneously present in the reaction medium, the enzyme activity increased by 15-20%. Complete replacement of Mg2+ with Co2+ decreased the enzyme activity. In the presence of Ca2+ at concentrations comparable to the concentration of Mg2+, the enzyme was not inhibited, although published data reported inhibition of similar enzymes by Ca2+. The recombinant enzyme exhibited a very low thermostability: it underwent a slow inactivation when incubated at 45 degrees C and was completely inactivated after incubation at 65 degrees C for 1 h.

  20. Cyclophilins of a novel subfamily interact with SNW/SKIP coregulator in Dictyostelium discoideum and Schizosaccharomyces pombe.

    PubMed

    Skruzný, M; Ambrozková, M; Fuková, I; Martínková, K; Blahůsková, A; Hamplová, L; Půta, F; Folk, P

    2001-10-31

    We screened the Dictyostelium discoideum two-hybrid cDNA library with the SNW/SKIP transcription coregulator SnwA and identified a novel cyclophilin CypE. Independently, the Schizosaccharomyces pombe cDNA library was screened with the SnwA ortholog Snw1 and the ortholog of CypE (named Cyp2) was found. Both cyclophilins bind the respective SNW protein in their autologous systems. The interaction was localized to the N-terminal part of SnwA as well as of Snw1. CypE was confirmed in vitro to be a cyclosporin A-sensitive peptidyl-prolyl cis-trans isomerase. Remarkably, both SNW proteins bind the cyclophilins in a cyclosporin A independent manner, possibly serving as adaptors for these novel isomerases. These results are the first characterization of the members of a novel cyclophilin subfamily, which includes the human CGI-124/PPIL1 protein.

  1. Depletion of cyclophilins B and C leads to dysregulation of endoplasmic reticulum redox homeostasis.

    PubMed

    Stocki, Pawel; Chapman, Daniel C; Beach, Lori A; Williams, David B

    2014-08-15

    Protein folding within the endoplasmic reticulum is assisted by molecular chaperones and folding catalysts that include members of the protein-disulfide isomerase and peptidyl-prolyl isomerase families. In this report, we examined the contributions of the cyclophilin subset of peptidyl-prolyl isomerases to protein folding and identified cyclophilin C as an endoplasmic reticulum (ER) cyclophilin in addition to cyclophilin B. Using albumin and transferrin as models of cis-proline-containing proteins in human hepatoma cells, we found that combined knockdown of cyclophilins B and C delayed transferrin secretion but surprisingly resulted in more efficient oxidative folding and secretion of albumin. Examination of the oxidation status of ER protein-disulfide isomerase family members revealed a shift to a more oxidized state. This was accompanied by a >5-fold elevation in the ratio of oxidized to total glutathione. This "hyperoxidation" phenotype could be duplicated by incubating cells with the cyclophilin inhibitor cyclosporine A, a treatment that triggered efficient ER depletion of cyclophilins B and C by inducing their secretion to the medium. To identify the pathway responsible for ER hyperoxidation, we individually depleted several enzymes that are known or suspected to deliver oxidizing equivalents to the ER: Ero1αβ, VKOR, PRDX4, or QSOX1. Remarkably, none of these enzymes contributed to the elevated oxidized to total glutathione ratio induced by cyclosporine A treatment. These findings establish cyclophilin C as an ER cyclophilin, demonstrate the novel involvement of cyclophilins B and C in ER redox homeostasis, and suggest the existence of an additional ER oxidative pathway that is modulated by ER cyclophilins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Depletion of Cyclophilins B and C Leads to Dysregulation of Endoplasmic Reticulum Redox Homeostasis*

    PubMed Central

    Stocki, Pawel; Chapman, Daniel C.; Beach, Lori A.; Williams, David B.

    2014-01-01

    Protein folding within the endoplasmic reticulum is assisted by molecular chaperones and folding catalysts that include members of the protein-disulfide isomerase and peptidyl-prolyl isomerase families. In this report, we examined the contributions of the cyclophilin subset of peptidyl-prolyl isomerases to protein folding and identified cyclophilin C as an endoplasmic reticulum (ER) cyclophilin in addition to cyclophilin B. Using albumin and transferrin as models of cis-proline-containing proteins in human hepatoma cells, we found that combined knockdown of cyclophilins B and C delayed transferrin secretion but surprisingly resulted in more efficient oxidative folding and secretion of albumin. Examination of the oxidation status of ER protein-disulfide isomerase family members revealed a shift to a more oxidized state. This was accompanied by a >5-fold elevation in the ratio of oxidized to total glutathione. This “hyperoxidation” phenotype could be duplicated by incubating cells with the cyclophilin inhibitor cyclosporine A, a treatment that triggered efficient ER depletion of cyclophilins B and C by inducing their secretion to the medium. To identify the pathway responsible for ER hyperoxidation, we individually depleted several enzymes that are known or suspected to deliver oxidizing equivalents to the ER: Ero1αβ, VKOR, PRDX4, or QSOX1. Remarkably, none of these enzymes contributed to the elevated oxidized to total glutathione ratio induced by cyclosporine A treatment. These findings establish cyclophilin C as an ER cyclophilin, demonstrate the novel involvement of cyclophilins B and C in ER redox homeostasis, and suggest the existence of an additional ER oxidative pathway that is modulated by ER cyclophilins. PMID:24990953

  3. ALS-linked protein disulfide isomerase variants cause motor dysfunction.

    PubMed

    Woehlbier, Ute; Colombo, Alicia; Saaranen, Mirva J; Pérez, Viviana; Ojeda, Jorge; Bustos, Fernando J; Andreu, Catherine I; Torres, Mauricio; Valenzuela, Vicente; Medinas, Danilo B; Rozas, Pablo; Vidal, Rene L; Lopez-Gonzalez, Rodrigo; Salameh, Johnny; Fernandez-Collemann, Sara; Muñoz, Natalia; Matus, Soledad; Armisen, Ricardo; Sagredo, Alfredo; Palma, Karina; Irrazabal, Thergiory; Almeida, Sandra; Gonzalez-Perez, Paloma; Campero, Mario; Gao, Fen-Biao; Henny, Pablo; van Zundert, Brigitte; Ruddock, Lloyd W; Concha, Miguel L; Henriquez, Juan P; Brown, Robert H; Hetz, Claudio

    2016-04-15

    Disturbance of endoplasmic reticulum (ER) proteostasis is a common feature of amyotrophic lateral sclerosis (ALS). Protein disulfide isomerases (PDIs) areERfoldases identified as possibleALSbiomarkers, as well as neuroprotective factors. However, no functional studies have addressed their impact on the disease process. Here, we functionally characterized fourALS-linked mutations recently identified in two majorPDIgenes,PDIA1 andPDIA3/ERp57. Phenotypic screening in zebrafish revealed that the expression of thesePDIvariants induce motor defects associated with a disruption of motoneuron connectivity. Similarly, the expression of mutantPDIs impaired dendritic outgrowth in motoneuron cell culture models. Cellular and biochemical studies identified distinct molecular defects underlying the pathogenicity of thesePDImutants. Finally, targetingERp57 in the nervous system led to severe motor dysfunction in mice associated with a loss of neuromuscular synapses. This study identifiesERproteostasis imbalance as a risk factor forALS, driving initial stages of the disease.

  4. Solution structure of 3-oxo-delta5-steroid isomerase.

    PubMed

    Wu, Z R; Ebrahimian, S; Zawrotny, M E; Thornburg, L D; Perez-Alvarado, G C; Brothers, P; Pollack, R M; Summers, M F

    1997-04-18

    The three-dimensional structure of the enzyme 3-oxo-delta5-steroid isomerase (E.C. 5.3.3.1), a 28-kilodalton symmetrical dimer, was solved by multidimensional heteronuclear magnetic resonance spectroscopy. The two independently folded monomers pack together by means of extensive hydrophobic and electrostatic interactions. Each monomer comprises three alpha helices and a six-strand mixed beta-pleated sheet arranged to form a deep hydrophobic cavity. Catalytically important residues Tyr14 (general acid) and Asp38 (general base) are located near the bottom of the cavity and positioned as expected from mechanistic hypotheses. An unexpected acid group (Asp99) is also located in the active site adjacent to Tyr14, and kinetic and binding studies of the Asp99 to Ala mutant demonstrate that Asp99 contributes to catalysis by stabilizing the intermediate.

  5. Human glucose phosphate isomerase: Exon mapping and gene structure

    SciTech Connect

    Xu, Weiming; Lee, Pauline; Beutler, E.

    1995-10-10

    The structure of the gene for human glucose phosphate isomerase (GPI) has been determined. Three GPI clones were isolated from a human genomic library by using a full-length GPI cDNA probe and were characterized. Oligonucleotides based on the known cDNA sequence were used as primers in amplification and sequence analyses. This led to the identification of the exon-intron junctions. By this approach, 18 exons and 17 introns have been identified. The exons range in size from 44 to 431 nucleotides. The intronic sequences surrounding the exons provide useful information for the identification of mutations that give rise to human GPI deficiency associated with chronic hemolytic anemia. 13 refs., 4 figs., 1 tab.

  6. Three phenotypes of glucosephosphate isomerase in sheep: improved staining recipe.

    PubMed

    Manwell, C; Baker, C M; Graydon, R J

    1985-01-01

    Contrary to results published recently, we observe three, rather than two, phenotypes for the enzyme glucosephosphate isomerase (EC 5.3.1.9) from sheep. The phenotypic electrophoretic patterns conform to the patterns observed for this dimeric enzyme in other species. Genotype frequencies in a flock of Southdowns do not deviate significantly from those predicted under the assumption of the Hardy-Weinberg equilibrium. A remarkable observation is that the electrophoretically distinct phenotypes of GPI are largely or entirely obliterated by the addition of 1-10 mmol/l MgCl2 to the electrophoretic buffers. Modification of the usual staining recipe for GPI result in greater resolution and shorter staining times.

  7. Mammalian peptide isomerase: platypus-type activity is present in mouse heart.

    PubMed

    Koh, Jennifer M S; Chow, Stephanie J P; Crossett, Ben; Kuchel, Philip W

    2010-06-01

    Male platypus (Ornithorhynchus anatinus) venom has a peptidyl aminoacyl L/D-isomerase (hereafter called peptide isomerase) that converts the second amino acid residue in from the N-terminus from the L- to the D-form, and vice versa. A reversed-phase high-performance liquid chromatography (RP-HPLC) assay has been developed to monitor the interconversion using synthetic hexapeptides derived from defensin-like peptide-2 (DLP-2) and DLP-4 as substrates. It was hypothesised that animals other than the platypus would have peptide isomerase with the same substrate specificity. Accordingly, eight mouse tissues were tested and heart was shown to have the activity. This is notable for being the first evidence of a peptide isomerase being present in a higher mammal and heralds finding the activity in man.

  8. Cumene peroxide and Fe(2+)-ascorbate-induced lipid peroxidation and effect of phosphoglucose isomerase.

    PubMed

    Agadjanyan, Z S; Dugin, S F; Dmitriev, L F

    2006-09-01

    Malondialdehyde (MDA) is one of cytotoxic aldehydes produced in cells as a result of lipid peroxidation and further MDA metabolism in cytoplasm is not known. In our experiments the liver fraction 10,000 g containing phosphoglucose isomerase and enzymes of the glyoxalase system was used and obtained experimental data shows that in this fraction there is an aggregate of reactions taking place both in membranes (lipid peroxidation) and outside membranes. MDA accumulation is relatively slow because MDA is a substrate of aldehyde isomerase (MDA <--> methylglyoxal). The well known enzyme phosphoglucose isomerase acts as an aldehyde isomerase (Michaelis constant for this enzyme Km = 133 +/- 8 microM). MDA conversion to methylglyoxal and further to neutral product D-lactate (with GSH as a cofactor) occurs in cytoplasm and D-lactate should be regarded as the end product of two different parametabolic reactions: lipid peroxidation or protein glycation.

  9. A single and two step isomerization process for d-tagatose and l-ribose bioproduction using l-arabinose isomerase and d-lyxose isomerase.

    PubMed

    Patel, Manisha J; Akhani, Rekha C; Patel, Arti T; Dedania, Samir R; Patel, Darshan H

    2017-02-01

    l-ribose and d-tagatose are biochemically synthesized using sugar isomerases. The l-arabinose isomerase gene from Shigella flexneri (Sf-AI) was cloned and expressed in Escherichia coli BL-21. Sf-AI was applied for the bioproduction of d-tagatose from d-galactose. l-ribose synthesis was performed by two step isomerization using Sf-AI and d-lyxose/ribose isomerase from Cohnella laevoribosii. The overall 22.3% and 25% conversion rate were observed for d-tagatose and l-ribose production from d-galactose and l-arabinose respectively. In the present manuscript, synthesis of rare sugars from naturally available sugars is discussed along with the biochemical characterization of Sf-AI and its efficiency.

  10. Functional analysis of type 1 isopentenyl diphosphate isomerase from Halobacterium sp. NRC-1.

    PubMed

    Hoshino, Takeshi; Eguchi, Tadashi

    2007-10-01

    Here we report the characterization of the type-1 isopentenyl diphosphate isomerase derived from Halobacterium sp. NRC-1. The expressed purified enzyme showed maximum isomerase activity in the presence of 1 M NaCl at 37 degrees C at pH 6.0. This type-1 enzyme appears to be the first for which the Co2+ ion is required for activity.

  11. Detection of platypus-type L/D-peptide isomerase activity in aqueous extracts of papaya fruit.

    PubMed

    Arakawa, Kensuke; Koh, Jennifer M S; Crossett, Ben; Torres, Allan M; Kuchel, Philip W

    2012-09-01

    Peptide isomerase catalyses the post-translational isomerisation of the L: - to the D: -form of an amino acid residue around the N/C-termini of substrate peptides. To date, some peptide isomerases have been found in a limited number of animal secretions and cells. We show here that papaya extracts have weak peptide isomerase activity. The activity was detected in each 30-100 kDa fraction of the flesh and the seed extracts of unripe and ripe papaya fruit. The definitive activity was confirmed in the ripe papaya extracts, but even then it was much less active than that of the other peptide isomerases previously reported. The activity was markedly inhibited by methanol, and partly so by amastatin and diethyl pyrocarbonate. This is the first report of peptide isomerase activity in a plant and suggests that perhaps every living organism may have some peptide isomerase activity.

  12. Giardial triosephosphate isomerase as possible target of the cytotoxic effect of omeprazole in Giardia lamblia.

    PubMed

    Reyes-Vivas, Horacio; de la Mora-de la Mora, Ignacio; Castillo-Villanueva, Adriana; Yépez-Mulia, Lilian; Hernández-Alcántara, Gloria; Figueroa-Salazar, Rosalia; García-Torres, Itzhel; Gómez-Manzo, Saúl; Méndez, Sara T; Vanoye-Carlo, América; Marcial-Quino, Jaime; Torres-Arroyo, Angélica; Oria-Hernández, Jesús; Gutiérrez-Castrellón, Pedro; Enríquez-Flores, Sergio; López-Velázquez, Gabriel

    2014-12-01

    Giardiasis is highly prevalent in the developing world, and treatment failures with the standard drugs are common. This work deals with the proposal of omeprazole as a novel antigiardial drug, focusing on a giardial glycolytic enzyme used to follow the cytotoxic effect at the molecular level. We used recombinant technology and enzyme inactivation to demonstrate the capacity of omeprazole to inactivate giardial triosephosphate isomerase, with no adverse effects on its human counterpart. To establish the specific target in the enzyme, we used single mutants of every cysteine residue in triosephosphate isomerase. The effect on cellular triosephosphate isomerase was evaluated by following the remnant enzyme activity on trophozoites treated with omeprazole. The interaction of omeprazole with giardial proteins was analyzed by fluorescence spectroscopy. The susceptibility to omeprazole of drug-susceptible and drug-resistant strains of Giardia lamblia was evaluated to demonstrate its potential as a novel antigiardial drug. Our results demonstrate that omeprazole inhibits giardial triosephosphate isomerase in a species-specific manner through interaction with cysteine at position 222. Omeprazole enters the cytoplasmic compartment of the trophozoites and inhibits cellular triosephosphate isomerase activity in a dose-dependent manner. Such inhibition takes place concomitantly with the cytotoxic effect caused by omeprazole on trophozoites. G. lamblia triosephosphate isomerase (GlTIM) is a cytoplasmic protein which can help analyses of how omeprazole works against the proteins of this parasite and in the effort to understand its mechanism of cytotoxicity. Our results demonstrate the mechanism of giardial triosephosphate isomerase inhibition by omeprazole and show that this drug is effective in vitro against drug-resistant and drug-susceptible strains of G. lamblia. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  13. Role of LRAT on the retinoid isomerase activity and membrane association of Rpe65.

    PubMed

    Jin, Minghao; Yuan, Quan; Li, Songhua; Travis, Gabriel H

    2007-07-20

    Absorption of a photon by a vertebrate opsin pigment induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the bleached opsin requires chemical re-isomerization of the chromophore via an enzyme pathway called the visual cycle. The retinoid isomerase in this pathway is Rpe65, a membrane-associated protein in the retinal pigment epithelium (RPE) with no predicted membrane-spanning segments. It has been suggested that Rpe65 is S-palmitoylated by lecithin:retinol acyl transferase (LRAT) on Cys(231), Cys(329), and Cys(330), and that this palmitoylation is required for isomerase activity and the association of Rpe65 with membranes. Here we show that the affinity of Rpe65 for membranes is similar in wild-type and lrat(-/-) mice. The isomerase activity of Rpe65 is also similar in both strains when all-trans-retinyl palmitate is used as substrate. With all-trans-retinol substrate, isomerase activity is present in wild-type but undetectable in RPE homogenates from lrat(-/-) mice. Substitution of Cys(231), Cys(329), and Cys(330) with Ser or Ala did not affect the affinity of Rpe65 for membranes. Further, these Cys residues are not palmitoylated in Rpe65 by mass spectrometric analysis. Global inhibition of protein palmitoylation by 2-bromopalmitate did not affect the solubility or isomerase activity of Rpe65. Finally, we show that soluble and membrane-associated Rpe65 possesses similar isomerase specific activities. These results indicate that LRAT is not required for isomerase activity beyond synthesis of retinyl-ester substrate, and that the association of Rpe65 with membranes is neither dependent upon LRAT nor the result of S-palmitoylation. The affinity of Rpe65 for membranes is probably an intrinsic feature of this protein.

  14. Giardial Triosephosphate Isomerase as Possible Target of the Cytotoxic Effect of Omeprazole in Giardia lamblia

    PubMed Central

    Reyes-Vivas, Horacio; de la Mora-de la Mora, Ignacio; Castillo-Villanueva, Adriana; Yépez-Mulia, Lilian; Hernández-Alcántara, Gloria; Figueroa-Salazar, Rosalia; García-Torres, Itzhel; Gómez-Manzo, Saúl; Méndez, Sara T.; Vanoye-Carlo, América; Marcial-Quino, Jaime; Torres-Arroyo, Angélica; Oria-Hernández, Jesús; Gutiérrez-Castrellón, Pedro; Enríquez-Flores, Sergio

    2014-01-01

    Giardiasis is highly prevalent in the developing world, and treatment failures with the standard drugs are common. This work deals with the proposal of omeprazole as a novel antigiardial drug, focusing on a giardial glycolytic enzyme used to follow the cytotoxic effect at the molecular level. We used recombinant technology and enzyme inactivation to demonstrate the capacity of omeprazole to inactivate giardial triosephosphate isomerase, with no adverse effects on its human counterpart. To establish the specific target in the enzyme, we used single mutants of every cysteine residue in triosephosphate isomerase. The effect on cellular triosephosphate isomerase was evaluated by following the remnant enzyme activity on trophozoites treated with omeprazole. The interaction of omeprazole with giardial proteins was analyzed by fluorescence spectroscopy. The susceptibility to omeprazole of drug-susceptible and drug-resistant strains of Giardia lamblia was evaluated to demonstrate its potential as a novel antigiardial drug. Our results demonstrate that omeprazole inhibits giardial triosephosphate isomerase in a species-specific manner through interaction with cysteine at position 222. Omeprazole enters the cytoplasmic compartment of the trophozoites and inhibits cellular triosephosphate isomerase activity in a dose-dependent manner. Such inhibition takes place concomitantly with the cytotoxic effect caused by omeprazole on trophozoites. G. lamblia triosephosphate isomerase (GlTIM) is a cytoplasmic protein which can help analyses of how omeprazole works against the proteins of this parasite and in the effort to understand its mechanism of cytotoxicity. Our results demonstrate the mechanism of giardial triosephosphate isomerase inhibition by omeprazole and show that this drug is effective in vitro against drug-resistant and drug-susceptible strains of G. lamblia. PMID:25223993

  15. Plant phosphomannose isomerase as a selectable marker for rice transformation

    PubMed Central

    Hu, Lei; Li, Hao; Qin, Ruiying; Xu, Rongfang; Li, Juan; Li, Li; Wei, Pengcheng; Yang, Jianbo

    2016-01-01

    The E. coli phosphomannose isomerase (EcPMI) gene is widely used as a selectable marker gene (SMG) in mannose (Man) selection-based plant transformation. Although some plant species exhibit significant PMI activity and active PMIs were even identified in Man-sensitive plants, whether plant PMIs can be used as SMGs remains unclear. In this study, we isolated four novel PMI genes from Chlorella variabilis and Oryza sativa. Their isoenzymatic activities were examined in vitro and compared with that of EcPMI. The active plant PMIs were separately constructed into binary vectors as SMGs and then transformed into rice via Agrobacterium. In both Indica and Japonica subspecies, our results indicated that the plant PMIs could select and produce transgenic plants in a pattern similar to that of EcPMI. The transgenic plants exhibited an accumulation of plant PMI transcripts and enhancement of the in vivo PMI activity. Furthermore, a gene of interest was successfully transformed into rice using the plant PMIs as SMGs. Thus, novel SMGs for Man selection were isolated from plants, and our analysis suggested that PMIs encoding active enzymes might be common in plants and could potentially be used as appropriate genetic elements in cisgenesis engineering. PMID:27174847

  16. Solubility and crystallization of xylose isomerase from Streptomyces rubiginosus

    NASA Astrophysics Data System (ADS)

    Vuolanto, Antti; Uotila, Sinikka; Leisola, Matti; Visuri, Kalevi

    2003-10-01

    We have studied the crystallization and crystal solubility of xylose isomerase (XI) from Streptomyces rubiginosus. In this paper, we show a rational approach for developing a large-scale crystallization process for XI. Firstly, we measured the crystal solubility in salt solutions with respect to salt concentration, temperature and pH. In ammonium sulfate the solubility of XI decreased logarithmically when increasing the salt concentration. Surprisingly, the XI crystals had a solubility minimum at low concentration of magnesium sulfate. The solubility of XI in 0.17 M magnesium sulfate was less than 0.5 g l -1. The solubility of XI increased logarithmically when increasing the temperature. We also found a solubility minimum around pH 7. This is far from the isoelectric point of XI (pH 3.95). Secondly, based on the solubility study, we developed a large-scale crystallization process for XI. In a simple and economical cooling crystallization of XI from 0.17 M magnesium sulfate solution, the recovery of crystalline active enzyme was over 95%. Moreover, we developed a process for production of uniform crystals and produced homogenous crystals with average crystal sizes between 12 and 360 μm.

  17. Catalytic mechanism of a retinoid isomerase essential for vertebrate vision

    DOE PAGES

    Kiser, Philip D.; Zhang, Jianye; Badiee, Mohsen; ...

    2015-04-20

    Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive because of uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for the treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligandmore » positioned in an adjacent pocket. With the geometry of the RPE65–substrate complex clarified, we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. Finally, these data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules.« less

  18. Catalytic mechanism of a retinoid isomerase essential for vertebrate vision

    SciTech Connect

    Kiser, Philip D.; Zhang, Jianye; Badiee, Mohsen; Li, Qingjiang; Shi, Wuxian; Sui, Xuewu; Golczak, Marcin; Tochtrop, Gregory P.; Palczewski, Krzysztof

    2015-04-20

    Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive because of uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for the treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligand positioned in an adjacent pocket. With the geometry of the RPE65–substrate complex clarified, we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. Finally, these data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules.

  19. Crystal Structure of Triosephosphate Isomerase from Trypanosoma cruzi in Hexane

    NASA Astrophysics Data System (ADS)

    Gao, Xiu-Gong; Maldonado, Ernesto; Perez-Montfort, Ruy; Garza-Ramos, Georgina; Tuena de Gomez-Puyou, Marietta; Gomez-Puyou, Armando; Rodriguez-Romero, Adela

    1999-08-01

    To gain insight into the mechanisms of enzyme catalysis in organic solvents, the x-ray structure of some monomeric enzymes in organic solvents was determined. However, it remained to be explored whether the structure of oligomeric proteins is also amenable to such analysis. The field acquired new perspectives when it was proposed that the x-ray structure of enzymes in nonaqueous media could reveal binding sites for organic solvents that in principle could represent the starting point for drug design. Here, a crystal of the dimeric enzyme triosephosphate isomerase from the pathogenic parasite Trypanosoma cruzi was soaked and diffracted in hexane and its structure solved at 2- angstrom resolution. Its overall structure and the dimer interface were not altered by hexane. However, there were differences in the orientation of the side chains of several amino acids, including that of the catalytic Glu-168 in one of the monomers. No hexane molecules were detected in the active site or in the dimer interface. However, three hexane molecules were identified on the surface of the protein at sites, which in the native crystal did not have water molecules. The number of water molecules in the hexane structure was higher than in the native crystal. Two hexanes localized at <4 angstrom from residues that form the dimer interface; they were in close proximity to a site that has been considered a potential target for drug design.

  20. Purification and characterization of corticosteroid side chain isomerase

    SciTech Connect

    Marandici, A.; Monder, C. )

    1990-02-06

    Corticosteroid side chain isomerase of rat liver catalyzes the interconversion of the ketol (20-oxo-21-ol) and (20-hydroxy-21-al) forms of the corticosteroid side chain. The enzyme has now been purified to apparent homogeneity from rat liver cytosol by sequential chromatography on anionic, hydroxylapatite, and gel filtration columns. Ketol-aldol isomerization is followed by measuring the exchange of tritium from 21-tritiated steroids with water. The native enzyme is a dimer of MW 44,000. The isoelectric point is 4.8 {plus minus} 0.1 pH units. The purified enzyme is stimulated by Co{sup 3+} or Ni{sup 2+}. The enzyme utilizes 11-deoxycorticosterone, corticosterone, and 17-deoxycortisol as substrate but not cortisol, tetrahydrocortisol, and prednisolone. Tritium-water exchange of (21S)-(21-{sup 3}H)DOC is a pseudo-first-order reaction; 21-{sup 3}H exchange from the 21R isomer proceeds with first-order kinetics only after a lag associated with its epimerization to the 21S form.

  1. Molecular simulations of solute transport in xylose isomerase crystals.

    PubMed

    Malek, Kourosh; Coppens, Marc-Olivier

    2008-02-07

    Cross-linked enzyme crystals (CLECs) enclose an extensive regular matrix of chiral solvent-filled nanopores, via which ions and solutes travel in and out. Several cross-linked enzyme crystals have recently been used for chiral separation and as biocatalysts. We studied the dynamics of solute transport in orthorhombic d-xylose isomerase (XI) crystals by means of Brownian dynamics (BD) and molecular dynamics (MD) simulations, which show how the protein residues influence the dynamics of solute molecules in confined regions inside the lattice. In the BD simulations, coarse-grained beads represent solutes of different sizes. The diffusion of S-phenylglycine molecules inside XI crystals is investigated by long-time MD simulations. The computed diffusion coefficients within a crystal are found to be orders of magnitude lower than in bulk water. The simulation results are compared to the recent experimental studies of diffusion and reaction inside XI crystals. The insights obtained from simulations allow us to understand the nature of solute-protein interactions and transport phenomena in CLECs, which is useful for the design of novel nanoporous biocatalysts and bioseparations based on CLECs.

  2. Catalytic mechanism of a retinoid isomerase essential for vertebrate vision.

    PubMed

    Kiser, Philip D; Zhang, Jianye; Badiee, Mohsen; Li, Qingjiang; Shi, Wuxian; Sui, Xuewu; Golczak, Marcin; Tochtrop, Gregory P; Palczewski, Krzysztof

    2015-06-01

    Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive because of uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for the treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligand positioned in an adjacent pocket. With the geometry of the RPE65-substrate complex clarified, we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. These data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules.

  3. Structural and functional characterization of Mycobacterium tuberculosis triosephosphate isomerase

    SciTech Connect

    Connor, Sean E.; Capodagli, Glenn C.; Deaton, Michelle K.; Pegan, Scott D.

    2012-04-18

    Tuberculosis (TB) is a major infectious disease that accounts for over 1.7 million deaths every year. Mycobacterium tuberculosis, the causative agent of tuberculosis, enters the human host by the inhalation of infectious aerosols. Additionally, one third of the world's population is likely to be infected with latent TB. The incidence of TB is on the rise owing in part to the emergence of multidrug-resistant strains. As a result, there is a growing need to focus on novel M. tuberculosis enzyme targets. M. tuberculosis triosephosphate isomerase (MtTPI) is an essential enzyme for gluconeogenetic pathways, making it a potential target for future therapeutics. In order to determine its structure, the X-ray crystal structure of MtTPI has been determined, as well as that of MtTPI bound with a reaction-intermediate analog. As a result, two forms of the active site were revealed. In conjunction with the kinetic parameters obtained for the MtTPI-facilitated conversion of dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde-3-phosphate (D-GAP), this provides a greater structural and biochemical understanding of this enzyme. Additionally, isothermal titration calorimetry was used to determine the binding constant for a reaction-intermediate analog bound to the active site of MtTPI.

  4. Microbial sucrose isomerases: producing organisms, genes and enzymes.

    PubMed

    Goulter, Ken C; Hashimi, Saeed M; Birch, Robert G

    2012-01-05

    Sucrose isomerase (SI) activity is used industrially for the conversion of sucrose into isomers, particularly isomaltulose or trehalulose, which have properties advantageous over sucrose for some food uses. All of the known microbial SIs are TIM barrel proteins that convert sucrose without need for any cofactors, with varying kinetics and product specificities. The current analysis was undertaken to bridge key gaps between the information in patents and scientific publications about the microbes and enzymes useful for sucrose isomer production. This analysis shows that microbial SIs can be considered in 5 structural classes with corresponding functional distinctions that broadly align with the taxonomic differences between producing organisms. The most widely used bacterial strain for industrial production of isomaltulose, widely referred to as "Protaminobacter rubrum" CBS 574.77, is identified as Serratia plymuthica. The strain producing the most structurally divergent SI, with a high product specificity for trehalulose, widely referred to as "Pseudomonas mesoacidophila" MX-45, is identified as Rhizobium sp. Each tested SI-producer is shown to have a single SI gene and enzyme, so the properties reported previously for the isolated proteins can reasonably be associated with the products of the genes subsequently cloned from the same isolates and SI classes. Some natural isolates with potent SI activity do not catabolize the isomer under usual production conditions. The results indicate that their industrial potential may be further enhanced by selection for variants that do not catabolize the sucrose substrate.

  5. Structure and inactivation of triosephosphate isomerase from Entamoeba histolytica.

    PubMed

    Rodríguez-Romero, Adela; Hernández-Santoyo, Alejandra; del Pozo Yauner, Luis; Kornhauser, Adrián; Fernández-Velasco, D Alejandro

    2002-09-27

    Triosephosphate isomerase (TIM) has been proposed as a target for drug design. TIMs from several parasites have a cysteine residue at the dimer interface, whose derivatization with thiol-specific reagents induces enzyme inactivation and aggregation. TIMs lacking this residue, such as human TIM, are less affected. TIM from Entamoeba histolytica (EhTIM) has the interface cysteine residue and presents more than ten insertions when compared with the enzyme from other pathogens. To gain further insight into the role that interface residues play in the stability and reactivity of these enzymes, we determined the high-resolution structure and characterized the effect of methylmethane thiosulfonate (MMTS) on the activity and conformational properties of EhTIM. The structure of this enzyme was determined at 1.5A resolution using molecular replacement, observing that the dimer is not symmetric. EhTIM is completely inactivated by MMTS, and dissociated into stable monomers that possess considerable secondary structure. Structural and spectroscopic analysis of EhTIM and comparison with TIMs from other pathogens reveal that conformational rearrangements of the interface after dissociation, as well as intramonomeric contacts formed by the inserted residues, may contribute to the unusual stability of the derivatized EhTIM monomer.

  6. Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties

    PubMed Central

    Fessart, Delphine; Domblides, Charlotte; Avril, Tony; Eriksson, Leif A; Begueret, Hugues; Pineau, Raphael; Malrieux, Camille; Dugot-Senant, Nathalie; Lucchesi, Carlo; Chevet, Eric; Delom, Frederic

    2016-01-01

    The extracellular matrix (ECM) plays an instrumental role in determining the spatial orientation of epithelial polarity and the formation of lumens in glandular tissues during morphogenesis. Here, we show that the Endoplasmic Reticulum (ER)-resident protein anterior gradient-2 (AGR2), a soluble protein-disulfide isomerase involved in ER protein folding and quality control, is secreted and interacts with the ECM. Extracellular AGR2 (eAGR2) is a microenvironmental regulator of epithelial tissue architecture, which plays a role in the preneoplastic phenotype and contributes to epithelial tumorigenicity. Indeed, eAGR2, is secreted as a functionally active protein independently of its thioredoxin-like domain (CXXS) and of its ER-retention domain (KTEL), and is sufficient, by itself, to promote the acquisition of invasive and metastatic features. Therefore, we conclude that eAGR2 plays an extracellular role independent of its ER function and we elucidate this gain-of-function as a novel and unexpected critical ECM microenvironmental pro-oncogenic regulator of epithelial morphogenesis and tumorigenesis. DOI: http://dx.doi.org/10.7554/eLife.13887.001 PMID:27240165

  7. Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties.

    PubMed

    Fessart, Delphine; Domblides, Charlotte; Avril, Tony; Eriksson, Leif A; Begueret, Hugues; Pineau, Raphael; Malrieux, Camille; Dugot-Senant, Nathalie; Lucchesi, Carlo; Chevet, Eric; Delom, Frederic

    2016-05-30

    The extracellular matrix (ECM) plays an instrumental role in determining the spatial orientation of epithelial polarity and the formation of lumens in glandular tissues during morphogenesis. Here, we show that the Endoplasmic Reticulum (ER)-resident protein anterior gradient-2 (AGR2), a soluble protein-disulfide isomerase involved in ER protein folding and quality control, is secreted and interacts with the ECM. Extracellular AGR2 (eAGR2) is a microenvironmental regulator of epithelial tissue architecture, which plays a role in the preneoplastic phenotype and contributes to epithelial tumorigenicity. Indeed, eAGR2, is secreted as a functionally active protein independently of its thioredoxin-like domain (CXXS) and of its ER-retention domain (KTEL), and is sufficient, by itself, to promote the acquisition of invasive and metastatic features. Therefore, we conclude that eAGR2 plays an extracellular role independent of its ER function and we elucidate this gain-of-function as a novel and unexpected critical ECM microenvironmental pro-oncogenic regulator of epithelial morphogenesis and tumorigenesis.

  8. Genetic and functional aspects of linoleate isomerase in Lactobacillus acidophilus.

    PubMed

    Macouzet, Martin; Robert, Normand; Lee, Byong H

    2010-08-01

    While the remarkable health effects of conjugated linoleic acid (CLA) catalyzed from alpha-linoleic acid by the enzyme linoleate isomerase (LI, EC 5.2.1.5) are well recognized, how widely this biochemical activity is present and the mechanisms of its regulation in lactic acid bacteria are unknown. Although certain strains of Lactobacillus acidophilus can enrich CLA in fermented dairy products, it is unknown if other strains share this capacity. Due to its immense economic importance, this work aimed to investigate genetic aspects of CLA production in L. acidophilus for the first time. The genomic DNA from industrial and type strains of L. acidophilus were subjected to PCR and immunoblot analyses using the putative LI gene of L. reuteri ATCC 55739 as probe. The CLA production ability was estimated by gas chromatography of the biomass extracts. The presumptive LI gene from L. acidophilus ATCC 832 was isolated and sequenced. The resulting sequence shared 71% identity with that of L. reuteri and at least 99% with reported sequences from other L. acidophilus strains. All the strains accumulated detectable levels of CLA and tested positive by PCR and immunoblotting. However, no apparent correlation was observed between the yields and the hybridization patterns. The results suggest that LI activity might be common among L. acidophilus and related species and provide a new tool for screening potential CLA producers.

  9. Possible involvement of peptidylprolyl isomerase Pin1 in rheumatoid arthritis.

    PubMed

    Nagaoka, Akiko; Takizawa, Naohiro; Takeuchi, Ryohei; Inaba, Yutaka; Saito, Izumi; Nagashima, Yoji; Saito, Tomoyuki; Aoki, Ichiro

    2011-02-01

    The peptidylprolyl isomerase Pin1 is over-expressed in some human diseases including malignancies and chronic inflammatory diseases, this suggests that it contributes to the constitutive activation of certain intracellular signaling pathways that promote cell proliferation and cell invasion. Here, we investigate the possible role of Pin1 in rheumatoid arthritis (RA). Pin1 expression was immunohistochemically analyzed in synovial tissue (ST) obtained from patients with RA and osteoarthritis (OA). To investigate the correlation between Pin1 and motility and proliferation of synovial cells, Pin1 localization was immunohistochemically compared with matrix metalloproteinase (MMP)-1, MMP-3, and proliferating cell nuclear antigen (PCNA). Double immunofluorescent staining for Pin1 and p65 was performed to determine whether Pin1 is involved in nuclear factor κB (NF-κB) activation in RA-ST. Results showed Pin1 expression was significantly higher in RA-ST than in OA-ST. The expression of MMP-1, MMP-3, and PCNA was also significantly elevated in RA-ST. Double immunofluorescent staining revealed colocalization of Pin1 and p65 in the nuclei of RA-ST. These results suggest that Pin1 may be involved in the pathogenesis of RA binding with p65 to activate the proteins MMP-1, MMP-3, and PCNA. Therefore, Pin1 may play a pivotal role in the pathogenesis of RA. © 2010 The Authors. Pathology International © 2010 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd.

  10. Specificity and kinetics of triose phosphate isomerase from chicken muscle

    PubMed Central

    Putman, Sylvia J.; Coulson, A. F. W.; Farley, I. R. T.; Riddleston, B.; Knowles, J. R.

    1972-01-01

    The isolation of crystalline triose phosphate isomerase from chicken breast muscle is described. The values of kcat. and Km for the reaction in each direction were determined from experiments over wide substrate-concentration ranges, and the reactions were shown to obey simple Michaelis–Menten kinetics. With d-glyceraldehyde 3-phosphate as substrate, kcat. is 2.56×105min−1 and Km is 0.47mm; with dihydroxyacetone phosphate as substrate, kcat. is 2.59×104min−1 and Km is 0.97mm. The enzyme-catalysed exchange of the methyl hydrogen atoms of the `virtual substrate' monohydroxyacetone phosphate with solvent 2H2O or 3H2O was shown. This exchange is about 104-fold slower than the corresponding exchange of the C-3 hydrogen of dihydroxyacetone phosphate. The other deoxy substrate, 3-hydroxypropionaldehyde phosphate, was synthesized, but is too unstable in aqueous solution for analogous proton-exchange reactions to be studied. PMID:4643318

  11. Expression and Localization of Plant Protein Disulfide Isomerase.

    PubMed Central

    Shorrosh, B. S.; Subramaniam, J.; Schubert, K. R.; Dixon, R. A.

    1993-01-01

    A cDNA clone encoding a putative protein disulfide isomerase (PDI, EC 5.3.4.1) from alfalfa (Medicago sativa L.) was expressed in Escherichia coli cells, and an antiserum was raised against the expressed PDI-active protein. The antiserum recognized a protein of approximately 60 kD in extracts from alfalfa, soybean, and tobacco roots and stems. Levels of this protein remained relatively constant on exposure of alfalfa cell suspension cultures to the protein glycosylation inhibitor tunicamycin, whereas a slightly lower molecular mass form, also detected by the antiserum, was induced by this treatment. A lower molecular mass form of PDI was also observed in roots of alfalfa seedlings during the first 5 weeks after germination. PDI levels increased in developing soybean seeds up to 17 d after fertilization and then declined. Tissue print immunoblots revealed highest levels of PDI protein in the cambial tissues of soybean stems and petioles and in epidermal, subepidermal, cortical, and pith tissues of stems of alfalfa and tobacco. Immunogold electron microscopy confirmed the localization of PDI to the endoplasmic reticulum in soybean root nodules. PMID:12231974

  12. The flexibility and dynamics of protein disulfide isomerase

    PubMed Central

    Wells, Stephen A.; Emilio Jimenez‐Roldan, J.; Bhattacharyya, Moitrayee; Vishweshwara, Saraswathi; Freedman, Robert B.

    2016-01-01

    ABSTRACT We have studied the mobility of the multidomain folding catalyst, protein disulfide isomerase (PDI), by a coarse‐graining approach based on flexibility. We analyze our simulations of yeast PDI (yPDI) using measures of backbone movement, relative positions and orientations of domains, and distances between functional sites. We find that there is interdomain flexibility at every interdomain junction but these show very different characteristics. The extent of interdomain flexibility is such that yPDI's two active sites can approach much more closely than is found in crystal structures—and indeed hinge motion to bring these sites into proximity is the lowest energy normal mode of motion of the protein. The flexibility predicted for yPDI (based on one structure) includes the other known conformation of yPDI and is consistent with (i) the mobility observed experimentally for mammalian PDI and (ii) molecular dynamics. We also observe intradomain flexibility and clear differences between the domains in their propensity for internal motion. Our results suggest that PDI flexibility enables it to interact with many different partner molecules of widely different sizes and shapes, and highlights considerable similarities of yPDI and mammalian PDI. Proteins 2016; 84:1776–1785. © 2016 Wiley Periodicals, Inc. PMID:27616289

  13. Crystal structure of triosephosphate isomerase from Trypanosoma cruzi in hexane

    PubMed Central

    Gao, Xiu-Gong; Maldonado, Ernesto; Pérez-Montfort, Ruy; Garza-Ramos, Georgina; de Gómez-Puyou, Marietta Tuena; Gómez-Puyou, Armando; Rodríguez-Romero, Adela

    1999-01-01

    To gain insight into the mechanisms of enzyme catalysis in organic solvents, the x-ray structure of some monomeric enzymes in organic solvents was determined. However, it remained to be explored whether the structure of oligomeric proteins is also amenable to such analysis. The field acquired new perspectives when it was proposed that the x-ray structure of enzymes in nonaqueous media could reveal binding sites for organic solvents that in principle could represent the starting point for drug design. Here, a crystal of the dimeric enzyme triosephosphate isomerase from the pathogenic parasite Trypanosoma cruzi was soaked and diffracted in hexane and its structure solved at 2-Å resolution. Its overall structure and the dimer interface were not altered by hexane. However, there were differences in the orientation of the side chains of several amino acids, including that of the catalytic Glu-168 in one of the monomers. No hexane molecules were detected in the active site or in the dimer interface. However, three hexane molecules were identified on the surface of the protein at sites, which in the native crystal did not have water molecules. The number of water molecules in the hexane structure was higher than in the native crystal. Two hexanes localized at <4 Å from residues that form the dimer interface; they were in close proximity to a site that has been considered a potential target for drug design. PMID:10468562

  14. Immobilization of Recombinant Glucose Isomerase for Efficient Production of High Fructose Corn Syrup.

    PubMed

    Jin, Li-Qun; Xu, Qi; Liu, Zhi-Qiang; Jia, Dong-Xu; Liao, Cheng-Jun; Chen, De-Shui; Zheng, Yu-Guo

    2017-03-11

    Glucose isomerase is the important enzyme for the production of high fructose corn syrup (HFCS). One-step production of HFCS containing more than 55% fructose (HFCS-55) is receiving much attention for its industrial applications. In this work, the Escherichia coli harboring glucose isomerase mutant TEGI-W139F/V186T was immobilized for efficient production of HFCS-55. The immobilization conditions were optimized, and the maximum enzyme activity recovery of 92% was obtained. The immobilized glucose isomerase showed higher pH, temperature, and operational stabilities with a K m value of 272 mM and maximum reaction rate of 23.8 mM min(-1). The fructose concentration still retained above 55% after the immobilized glucose isomerase was reused for 10 cycles, and more than 85% of its initial activity was reserved even after 15 recycles of usage at temperature of 90 °C. The results highlighted the immobilized glucose isomerase as a potential biocatalyst for HFCS-55 production.

  15. Characterization of divergent pseudo-sucrose isomerase from Azotobacter vinelandii: Deciphering the absence of sucrose isomerase activity.

    PubMed

    Jung, Jong-Hyun; Kim, Min-Ji; Jeong, Woo-Soo; Seo, Dong-Ho; Ha, Suk-Jin; Kim, Young Wan; Park, Cheon-Seok

    2017-01-29

    Among members of the glycoside hydrolase (GH) family, sucrose isomerase (SIase) and oligo-1,6-glucosidase (O16G) are evolutionarily closely related even though their activities show different specificities. A gene (Avin_08330) encoding a putative SIase (AZOG: Azotobacterglucocosidase) from the nitrogen-fixing bacterium Azotobacter vinelandii is a type of pseudo-SIase harboring the "RLDRD" motif, a SIase-specific region in 329-333. However, neither sucrose isomerization nor hydrolysis activities were observed in recombinant AZOG (rAZOG). The rAZOG showed similar substrate specificity to Bacillus O16G as it catalyzes the hydrolysis of isomaltulose and isomaltose, which contain α-1,6-glycosidic linkages. Interestingly, rAZOG could generate isomaltose from the small substrate methyl-α-glucoside (MαG) via intermolecular transglycosylation. In addition, sucrose isomers isomaltulose and trehalulose were produced when 250 mM fructose was added to the MαG reaction mixture. The conserved regions I and II of AZOG are shared with many O16Gs, while regions III and IV are very similar to those of SIases. Strikingly, a shuffled AZOG, in which the N-terminal region of SIase containing conserved regions I and II was exchanged with the original enzyme, exhibited a production of sucrose isomers. This study demonstrates an evolutionary relationship between SIase and O16G and suggests some of the main regions that determine the specificity of SIase and O16G. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. 40 CFR 174.527 - Phosphomannose isomerase in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Phosphomannose isomerase in all plants... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.527 Phosphomannose isomerase in all plants...

  17. 40 CFR 174.527 - Phosphomannose isomerase in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Phosphomannose isomerase in all plants... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.527 Phosphomannose isomerase in all plants...

  18. Functional expression of a bacterial xylose isomerase in Saccharomyces cerevisiae.

    PubMed

    Brat, Dawid; Boles, Eckhard; Wiedemann, Beate

    2009-04-01

    In industrial fermentation processes, the yeast Saccharomyces cerevisiae is commonly used for ethanol production. However, it lacks the ability to ferment pentose sugars like d-xylose and l-arabinose. Heterologous expression of a xylose isomerase (XI) would enable yeast cells to metabolize xylose. However, many attempts to express a prokaryotic XI with high activity in S. cerevisiae have failed so far. We have screened nucleic acid databases for sequences encoding putative XIs and finally were able to clone and successfully express a highly active new kind of XI from the anaerobic bacterium Clostridium phytofermentans in S. cerevisiae. Heterologous expression of this enzyme confers on the yeast cells the ability to metabolize d-xylose and to use it as the sole carbon and energy source. The new enzyme has low sequence similarities to the XIs from Piromyces sp. strain E2 and Thermus thermophilus, which were the only two XIs previously functionally expressed in S. cerevisiae. The activity and kinetic parameters of the new enzyme are comparable to those of the Piromyces XI. Importantly, the new enzyme is far less inhibited by xylitol, which accrues as a side product during xylose fermentation. Furthermore, expression of the gene could be improved by adapting its codon usage to that of the highly expressed glycolytic genes of S. cerevisiae. Expression of the bacterial XI in an industrially employed yeast strain enabled it to grow on xylose and to ferment xylose to ethanol. Thus, our findings provide an excellent starting point for further improvement of xylose fermentation in industrial yeast strains.

  19. Evolutionary genomics of Colias Phosphoglucose Isomerase (PGI) introns.

    PubMed

    Wang, Baiqing; Mason Depasse, J; Watt, Ward B

    2012-02-01

    Little is known of intron sequences' variation in cases where eukaryotic gene coding regions undergo strong balancing selection. Phosphoglucose isomerase, PGI, of Colias butterflies offers such a case. Its 11 introns include many point mutations, insertions, and deletions. This variation changes with intron position and length, and may leave little evidence of homology within introns except for their first and last few basepairs. Intron position is conserved between PGIs of Colias and the silkmoth, but no intron sequence homology remains. % GC content and length are functional properties of introns which can affect whole-gene transcription; we find a relationship between these properties which may indicate selection on transcription speed. Intragenic recombination is active in these introns, as in coding sequences. The small extent of linkage disequilibrium (LD) in the introns decays over a few hundred basepairs. Subsequences of Colias introns match subsequences of other introns, untranslated regions of cDNAs, and insect-related transposons and pathogens, showing that a diverse pool of sequence fragments is the source of intron contents via turnover due to deletion, recombination, and transposition. Like Colias PGI's coding sequences, the introns evolve reticulately with little phylogenetic signal. Exceptions are coding-region allele clades defined by multiple amino acid variants in strong LD, whose introns are closely related but less so than their exons. Similarity of GC content between introns and flanking exons, lack of small introns despite mutational bias toward deletion, and findings already mentioned suggest constraining selection on introns, possibly balancing transcription performance against advantages of higher recombination rate conferred by intron length.

  20. Identification of fibrillogenic regions in human triosephosphate isomerase

    PubMed Central

    Carcamo-Noriega, Edson N.

    2016-01-01

    Background. Amyloid secondary structure relies on the intermolecular assembly of polypeptide chains through main-chain interaction. According to this, all proteins have the potential to form amyloid structure, nevertheless, in nature only few proteins aggregate into toxic or functional amyloids. Structural characteristics differ greatly among amyloid proteins reported, so it has been difficult to link the fibrillogenic propensity with structural topology. However, there are ubiquitous topologies not represented in the amyloidome that could be considered as amyloid-resistant attributable to structural features, such is the case of TIM barrel topology. Methods. This work was aimed to study the fibrillogenic propensity of human triosephosphate isomerase (HsTPI) as a model of TIM barrels. In order to do so, aggregation of HsTPI was evaluated under native-like and destabilizing conditions. Fibrillogenic regions were identified by bioinformatics approaches, protein fragmentation and peptide aggregation. Results. We identified four fibrillogenic regions in the HsTPI corresponding to the β3, β6, β7 y α8 of the TIM barrel. From these, the β3-strand region (residues 59–66) was highly fibrillogenic. In aggregation assays, HsTPI under native-like conditions led to amorphous assemblies while under partially denaturing conditions (urea 3.2 M) formed more structured aggregates. This slightly structured aggregates exhibited residual cross-β structure, as demonstrated by the recognition of the WO1 antibody and ATR-FTIR analysis. Discussion. Despite the fibrillogenic regions present in HsTPI, the enzyme maintained under native-favoring conditions displayed low fibrillogenic propensity. This amyloid-resistance can be attributed to the three-dimensional arrangement of the protein, where β-strands, susceptible to aggregation, are protected in the core of the molecule. Destabilization of the protein structure may expose inner regions promoting β-aggregation, as well as the

  1. Oxidation of kinetically trapped thiols by protein disulfide isomerase.

    PubMed

    Walker, K W; Gilbert, H F

    1995-10-17

    The formation of a stabilized structure during oxidative protein folding can severely retard disulfide formation if the structure must be disrupted to gain access to buried cysteines. These kinetic traps can slow protein folding and disulfide bond formation to the extent that unassisted folding is too slow to be kinetically competent in the cell. Protein disulfide isomerase (PDI) facilitates the oxidation of a kinetically trapped state of RTEM-1 beta-lactamase in which two cysteines that form the single disulfide bond in the native protein are buried and approximately 500-fold less reactive than exposed cysteines. Under second-order conditions, PDI-dependent oxidation of reduced, folded beta-lactamase is 500-fold faster than GSSG-dependent oxidation. The rate difference observed between PDI and GSSG can be accounted for by the 520-fold higher kinetic reactivity of PDI as an oxidant. Noncovalent interactions between PDI (35 microM) and beta-lactamase increase the reactivity or unfolding of beta-lactamase in the steady-state by less than 3-fold. At high concentrations of PDI or alkylating agents, the reaction of beta-lactamase cysteines approaches a constant rate, limited by the spontaneous unfolding of the protein (kunfold = 0.024 +/- 0.005 min-1). PDI does not substantially increase the rate of beta-lactamase unfolding; however, once beta-lactamase spontaneously unfolds, PDI at concentrations greater than 44 +/- 4 microM, oxidizes the unfolded substrate before it can refold (kfold = 1.5 +/- 0.2 min-1).(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Enzymes do what is expected (chalcone isomerase versus chorismate mutase).

    PubMed

    Hur, Sun; Bruice, Thomas C

    2003-02-12

    Madicago sativa chalcone isomerase (CI) catalyzes the isomerization of chalcone to flavanone, whereas E. coli chorismate mutase (CM) catalyzes the pericyclic rearrangement of chorismate to prephenate. Covalent intermediates are not formed in either of the enzyme-catalyzed reactions, K(M) and k(cat) are virtually the same for both enzymes, and the rate constants (k(o)) for the noncatalyzed reactions in water are also the same. This kinetic identity of both the enzymatic and the nonenzymatic reactions is not shared by a similarity in driving forces. The efficiency (DeltaG(o)() - DeltaG(cat)()) for the CI mechanism involves transition-state stabilization through general-acid catalysis and freeing of three water molecules trapped in the E.S species. The contribution to lowering DeltaG(cat)() by an increase in near attack conformer (NAC) formation in E.S as compared to S in water is not so important. In the CM reaction, the standard free energy for NAC formation in water is 8.4 kcal/mol as compared to 0.6 kcal/mol in E.S. Because the value of (DeltaG(o)() - DeltaG(cat)()) is 9 kcal/mol, the greater percentage of NACs accounts for approximately 90% of the kinetic advantage of the CM reaction. There is no discernible transition-state stabilization in the CM reaction. These results are discussed. In anthropomorphic terms, each enzyme has had to do what it must to have a biologically relevant rate of reaction.

  3. Protein disulfide isomerase is essential for viability in Saccharomyces cerevisiae.

    PubMed

    Farquhar, R; Honey, N; Murant, S J; Bossier, P; Schultz, L; Montgomery, D; Ellis, R W; Freedman, R B; Tuite, M F

    1991-12-01

    Protein disulfide isomerase (PDI) is an enzyme involved in the catalysis of disulfide bond formation in secretory and cell-surface proteins. Using an oligodeoxyribonucleotide designed to detect the conserved 'thioredoxin-like' active site of vertebrate PDIs, we have isolated a gene encoding PDI from the lower eukaryote, Saccharomyces cerevisiae. The nucleotide sequence and deduced open reading frame of the cloned gene predict a 530-amino-acid (aa) protein of Mr 59,082 and a pI of 4.1, physical properties characteristic of mammalian PDIs. Furthermore, the aa sequence shows 30-32% identity with mammalian and avian PDI sequences and has a very similar overall organisation, namely the presence of two approx. 100-aa segments, each of which is repeated, with the most significant homologies to mammalian and avian PDIs being in the regions (a, a') that contain the conserved 'thioredoxin-like' active site. The N-terminal region has the characteristics of a cleavable secretory signal sequence and the C-terminal four aa (-His-Asp-Glu-Leu) are consistent with the protein being a component of the S. cerevisiae endoplasmic reticulum. Transformants carrying multiple copies of this gene (designated PDI1) have tenfold higher levels of PDI activity and overproduce a protein of the predicted Mr. The PDI1 gene is unique in the yeast genome and encodes a single 1.8-kb transcript that is not found in stationary phase cells. Disruption of the PDI1 gene is haplo-lethal indicating that the product of this gene is essential for viability.

  4. Purification, characterization and catalytic properties of human sterol 8-isomerase.

    PubMed Central

    Nes, W David; Zhou, Wenxu; Dennis, Allen L; Li, Haoxia; Jia, Zhonghua; Keith, Richard A; Piser, Timothy M; Furlong, Stephen T

    2002-01-01

    CHO 2, encoding human sterol 8-isomerase (hSI), was introduced into plasmids pYX213 or pET23a. The resulting native protein was overexpressed in erg 2 yeast cells and purified to apparent homogeneity. The enzyme exhibited a K (m) of 50 microM and a turnover number of 0.423 s(-1) for zymosterol, an isoelectric point of 7.70, a native molecular mass of 107000 Da and was tetrameric. The structural features of zymosterol provided optimal substrate acceptability. Biomimetic studies of acid-catalysed isomerization of zymosterol resulted in formation of cholest-8(14)-enol, whereas the enzyme-generated product was a Delta(7)-sterol, suggesting absolute stereochemical control of the reaction by hSI. Using (2)H(2)O and either zymosterol or cholesta-7,24-dienol as substrates, the reversibility of the reaction was confirmed by GC-MS of the deuterated products. The positional specific incorporation of deuterium at C-9alpha was established by a combination of (1)H- and (13)C-NMR analyses of the enzyme-generated cholesta-7,24-dienol. Kinetic analyses indicated the reaction equilibrium ( K (eq)=14; DeltaG(o')=-6.5 kJ/mol) for double-bond isomerization favoured the forward direction, Delta(8) to Delta(7). Treatment of hSI with different high-energy intermediate analogues produced the following dissociation constants ( K (i)): emopamil (2 microM)=tamoxifen (1 microM)=tridemorph (1 microM)<25-azacholesterol (21 microM)

  5. Molecular identification, immunolocalization, and characterization of Clonorchis sinensis triosephosphate isomerase.

    PubMed

    Zhou, Juanjuan; Liao, Hua; Li, Shan; Zhou, Chenhui; Huang, Yan; Li, Xuerong; Liang, Chi; Yu, Xinbing

    2015-08-01

    Clonorchis sinensis triosephosphate isomerase (CsTIM) is a key regulatory enzyme of glycolysis and gluconeogenesis, which catalyzes the interconversion of glyceraldehyde 3-phosphate to dihydroxyacetone phosphate. In this study, the biochemical characterizations of CsTIM have been examined. A full-length complementary DNA (cDNA; Cs105350) sequence encoding CsTIM was obtained from our C. sinensis cDNA library. The open reading frame of CsTIM contains 759 bp which encodes 252 amino acids. The amino acid sequence of CsTIM shares 60-65% identity with other species. Western blot analysis displayed that recombinant CsTIM (rCsTIM) can be probed by anti-rCsTIM rat serum and anti-C. sinensis excretory/secretory products (anti-CsESPs) rat serum. Quantitative reverse transcription (RT)-PCR and western blotting analysis revealed that CsTIM messenger RNA (mRNA) and protein were differentially expressed in development cycle stages of the parasite, including adult worm, metacercaria, excysted metacercaria, and egg. In addition, immunolocalization assay showed that CsTIM was located in the seminal vesicle, eggs, and testicle. Moreover, rCsTIM exhibited active enzyme activity in catalytic reactions. The Michaelis constant (K m) of rCsTIM was 0.33 mM, when using glyceraldehyde 3-phosphate as the substrate. The optimal temperature and pH of CsTIM were 37 °C and 7.5-9.5, respectively. Collectively, these results suggest that CsTIM is an important protein involved in glycometabolism, and CsTIM possibly take part in many biological functions in the growth and development of C. sinensis.

  6. High production of D-tagatose, a potential sugar substitute, using immobilized L-arabinose isomerase.

    PubMed

    Kim, P; Yoon, S H; Roh, H J; Choi, J H

    2001-01-01

    An L-arabinose isomerase of Escherichia coli was immobilized using covalent binding to agarose to produce D-tagatose, a bulking sweetener that can be economically used as a sugar substitute. The immobilized L-arabinose isomerase stably produced an average of 7.5 g-tagatose/L.day for 7 days with a productivity exceeding that of the free enzyme (0.47 vs 0.30 mg/U.day). Using a scaled-up immobilized enzyme system, 99.9 g-tagatose/L was produced from galactose with 20% equilibrium in 48 h. The process was repeated two more times with production of 104.1 and 103.5 g-tagatose/L. D-Tagatose production using an immobilized L-arabinose isomerase has a high potential for commercial application.

  7. Directed evolution of xylose isomerase for improved xylose catabolism and fermentation in the yeast Saccharomyces cerevisiae.

    PubMed

    Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S

    2012-08-01

    The heterologous expression of a highly functional xylose isomerase pathway in Saccharomyces cerevisiae would have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways in S. cerevisiae suffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of the Piromyces sp. xylose isomerase (encoded by xylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing a gre3 knockout and tal1 and XKS1 overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.

  8. Purification and characterization of thermostable glucose isomerase from Clostridium thermosulfurogenes and Thermoanaerobacter strain B6A.

    PubMed Central

    Lee, C Y; Zeikus, J G

    1991-01-01

    Glucose isomerases produced by Thermoanaerobacter strain B6A and Clostridium thermosulfurogenes strain 4B were purified 10-11-fold to homogeneity and their physicochemical and catalytic properties were determined. Both purified enzymes displayed very similar properties (native Mr 200,000, tetrameric subunit composition, and apparent pH optima 7.0-7.5). The enzymes were stable at pH 5.5-12.0, and maintained more than 90% activity after incubation at high temperature (85 degrees C) for 1 h in the presence of metal ions. The N-terminal amino acid sequences of both thermostable glucose isomerases were Met-Asn-Lys-Tyr-Phe-Glu-Asn and were not similar to that of the thermolabile Bacillus subtilis enzyme. The glucose isomerase from C. thermosulfurogenes and Thermoanaerobacter displayed pI values of 4.9 and 4.8, and their kcat. and Km values for D-glucose at 65 degrees C were 1040 and 1260 min-1 and 140 and 120 mM respectively. Both enzymes displayed higher kcat. and lower Km values for D-xylose than for D-glucose. The C. thermosulfurogenes enzyme required Co2+ or Mg2+ for thermal stability and glucose isomerase activity, and Mn2+ or these metals for xylose isomerase activity. Crystals of C. thermosulfurogenes glucose isomerase were formed at room temperature by the hanging-drop method using 16-18% poly(ethylene glycol) (PEG) 4000 in 0.1 M-citrate buffer. Images Fig. 1. Fig. 5. PMID:1996956

  9. Method for the assay of glucose isomerase activity in complex fermentation mixtures

    SciTech Connect

    Boguslawski, G.; Bertch, S.W.

    1980-10-01

    A method for the determination of glucose isomerase activity is described. The method employs D-sorbitol dehydrogenase for conversion of fructose, formed in the glucose isomerase reaction, to sorbitol, with the concomitant oxidation of reduced nicotinamide adenine dinucleotide. The assay technique is simple, sensitive, and accurate. The few interferences by some sugars and components of a complex fermentation medium are easily corrected for. The method compares favorably with such alternative procedures as the cysteine--H/sub 2/SO/sub 4/ or hydrochloric acid methods of fructose determination.

  10. Interruption of the phosphoglucose isomerase gene results in glucose auxotrophy in Mycobacterium smegmatis.

    PubMed Central

    Tuckman, D; Donnelly, R J; Zhao, F X; Jacobs, W R; Connell, N D

    1997-01-01

    Two glycerol utilization mutants of Mycobacterium smegmatis that were unable to utilize most carbon sources except glucose were isolated. Supplementation of these media with small amounts of glucose restored growth in the mutants; these strains are therefore glucose auxotrophs. The mutant phenotype is complemented by the gene encoding phosphoglucose isomerase (pgi), and direct measurement of enzyme activities in the mutants suggests that this gene product is absent in the auxotrophic strains. Mapping of the mutant allele by Southern analysis demonstrates the presence of a 1-kb deletion extending into the coding sequence of pgi. The possible roles of phosphoglucose isomerase in mycobacterial cell wall synthesis and metabolic regulation are discussed. PMID:9098072

  11. Interruption of the phosphoglucose isomerase gene results in glucose auxotrophy in Mycobacterium smegmatis.

    PubMed

    Tuckman, D; Donnelly, R J; Zhao, F X; Jacobs, W R; Connell, N D

    1997-04-01

    Two glycerol utilization mutants of Mycobacterium smegmatis that were unable to utilize most carbon sources except glucose were isolated. Supplementation of these media with small amounts of glucose restored growth in the mutants; these strains are therefore glucose auxotrophs. The mutant phenotype is complemented by the gene encoding phosphoglucose isomerase (pgi), and direct measurement of enzyme activities in the mutants suggests that this gene product is absent in the auxotrophic strains. Mapping of the mutant allele by Southern analysis demonstrates the presence of a 1-kb deletion extending into the coding sequence of pgi. The possible roles of phosphoglucose isomerase in mycobacterial cell wall synthesis and metabolic regulation are discussed.

  12. The ULTRACURVATA2 Gene of Arabidopsis Encodes an FK506-Binding Protein Involved in Auxin and Brassinosteroid Signaling1

    PubMed Central

    Pérez-Pérez, José Manuel; Ponce, María Rosa; Micol, José Luis

    2004-01-01

    The dwarf ucu (ultracurvata) mutants of Arabidopsis display vegetative leaves that are spirally rolled downwards and show reduced expansion along the longitudinal axis. We have previously determined that the UCU1 gene encodes a SHAGGY/GSK3-like kinase that participates in the signaling pathways of auxins and brassinosteroids. Here, we describe four recessive alleles of the UCU2 gene, whose homozygotes display helical rotation of several organs in addition to other phenotypic traits shared with ucu1 mutants. Following a map-based strategy, we identified the UCU2 gene, which was found to encode a peptidyl-prolyl cis/trans-isomerase of the FK506-binding protein family, whose homologs in metazoans are involved in cell signaling and protein trafficking. Physiological and double mutant analyses suggest that UCU2 is required for growth and development and participates in auxin and brassinosteroid signaling. PMID:14730066

  13. Androgen receptor serine 81 mediates Pin1 interaction and activity

    PubMed Central

    La Montagna, Raffaele; Caligiuri, Isabella; Maranta, Pasquale; Lucchetti, Chiara; Esposito, Luca; Paggi, Marco G.; Toffoli, Giuseppe; Rizzolio, Flavio; Giordano, Antonio

    2012-01-01

    Hormone-dependent tumors are characterized by deregulated activity of specific steroid receptors, allowing aberrant expression of many genes involved in cancer initiation, progression and metastasis. In prostate cancer, the androgen receptor (AR) protein has pivotal functions, and over the years it has been the target of different drugs. AR is a nuclear receptor whose activity is regulated by a phosphorylation mechanism controlled by hormone and growth factors. Following phosphorylation, AR interacts with many cofactors that closely control its function. Among such cofactors, Pin1 is a peptidyl-prolyl isomerase that is involved in the control of protein phosphorylation and has a prognostic value in prostate cancer. In the present study, we demonstrate that ARSer81 is involved in the interaction with Pin1, and that this interaction is important for the transcriptional activity of AR. Since Pin1 expression positively correlates with tumor grade, our results suggest that Pin1 can participate in this process by modulating AR function. PMID:22894932

  14. Cyclophilin A: a key player for human disease

    PubMed Central

    Nigro, P; Pompilio, G; Capogrossi, M C

    2013-01-01

    Cyclophilin A (CyPA) is a ubiquitously distributed protein belonging to the immunophilin family. CyPA has peptidyl prolyl cis-trans isomerase (PPIase) activity, which regulates protein folding and trafficking. Although CyPA was initially believed to function primarily as an intracellular protein, recent studies have revealed that it can be secreted by cells in response to inflammatory stimuli. Current research in animal models and humans has provided compelling evidences supporting the critical function of CyPA in several human diseases. This review discusses recently available data about CyPA in cardiovascular diseases, viral infections, neurodegeneration, cancer, rheumatoid arthritis, sepsis, asthma, periodontitis and aging. It is believed that further elucidations of the role of CyPA will provide a better understanding of the molecular mechanisms underlying these diseases and will help develop novel pharmacological therapies. PMID:24176846

  15. Cyclophilin and Viruses: Cyclophilin as a Cofactor for Viral Infection and Possible Anti-Viral Target

    PubMed Central

    Watashi, Koichi; Shimotohno, Kunitada

    2007-01-01

    Cyclophilin (CyP) is a peptidyl prolyl cis/trans isomerase, catalyzing the cis-trans isomerization of proline residues in proteins. CyP plays key roles in several different aspects of cellular physiology including the immune response, transcription, mitochondrial function, cell death, and chemotaxis. In addition to these cellular events, a number of reports demonstrated that CyP plays a critical role in the life cycle of viruses, especially human immunodeficiency virus (HIV) and hepatitis C virus (HCV). These two viruses are significant causes of morbidity and mortality worldwide, but current therapies are often insufficient. CyP may provide a novel therapeutic target for the management and/or cure of these diseases, in particular HCV. PMID:21901058

  16. The Arabidopsis Cyclophilin Gene Family1

    PubMed Central

    Romano, Patrick G.N.; Horton, Peter; Gray, Julie E.

    2004-01-01

    Database searching has allowed the identification of a number of previously unreported single and multidomain isoform members of the Arabidopsis cyclophilin gene family. In addition to the cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, the latter contain a variety of other domains with characterized functions. Transcriptional analysis showed they are expressed throughout the plant, and different isoforms are present in all parts of the cell including the cytosol, nucleus, mitochondria, secretory pathway, and chloroplast. The abundance and diversity of cyclophilin isoforms suggests that, like their animal counterparts, plant cyclophilins are likely to be important proteins involved in a wide variety of cellular processes. As well as fulfilling the basic role of protein folding, they may also play important roles in mRNA processing, protein degradation, and signal transduction and thus may be crucial during both development and stress responsiveness. PMID:15051864

  17. Fragment-based discovery of a new family of non-peptidic small-molecule cyclophilin inhibitors with potent antiviral activities

    PubMed Central

    Ahmed-Belkacem, Abdelhakim; Colliandre, Lionel; Ahnou, Nazim; Nevers, Quentin; Gelin, Muriel; Bessin, Yannick; Brillet, Rozenn; Cala, Olivier; Douguet, Dominique; Bourguet, William; Krimm, Isabelle; Pawlotsky, Jean-Michel; Guichou, Jean- François

    2016-01-01

    Cyclophilins are peptidyl-prolyl cis/trans isomerases (PPIase) that catalyse the interconversion of the peptide bond at proline residues. Several cyclophilins play a pivotal role in the life cycle of a number of viruses. The existing cyclophilin inhibitors, all derived from cyclosporine A or sanglifehrin A, have disadvantages, including their size, potential for side effects unrelated to cyclophilin inhibition and drug–drug interactions, unclear antiviral spectrum and manufacturing issues. Here we use a fragment-based drug discovery approach using nucleic magnetic resonance, X-ray crystallography and structure-based compound optimization to generate a new family of non-peptidic, small-molecule cyclophilin inhibitors with potent in vitro PPIase inhibitory activity and antiviral activity against hepatitis C virus, human immunodeficiency virus and coronaviruses. This family of compounds has the potential for broad-spectrum, high-barrier-to-resistance treatment of viral infections. PMID:27652979

  18. Catalysis of cis/trans isomerization in native HIV-1 capsid by human cyclophilin A

    PubMed Central

    Bosco, Daryl A.; Eisenmesser, Elan Z.; Pochapsky, Susan; Sundquist, Wesley I.; Kern, Dorothee

    2002-01-01

    Packaging of cyclophilin A (CypA) into HIV-1 virions is essential for efficient replication; however, the reason for this is unknown. Incorporation is mediated through binding to the Gly-89–Pro-90 peptide bond of the N-terminal domain of HIV-1 capsid (CAN). Despite the fact that CypA is a peptidyl-prolyl cis/trans isomerase, catalytic activity on CAN has not been observed previously. We show here, using NMR exchange spectroscopy, that CypA does not only bind to CAN but also catalyzes efficiently the cis/trans isomerization of the Gly-89–Pro-90 peptide bond. In addition, conformational changes in CAN distal to the CypA binding loop are observed on CypA binding and catalysis. The results provide experimental evidence for efficient CypA catalysis on a natively folded and biologically relevant protein substrate. PMID:11929983

  19. Long-term inhibition of cyclophilin D results in intracellular translocation of calcein AM from mitochondria to lysosomes.

    PubMed

    Shinohe, Daisuke; Kobayashi, Asuka; Gotoh, Marina; Tanaka, Kotaro; Ohta, Yoshihiro

    2017-01-01

    Cyclophilin D is a peptidyl-prolyl cis-trans isomerase localized in the mitochondrial matrix. Although its effects on mitochondrial characteristics have been well studied, its relation to the uptake of molecules by mitochondria remains unknown. Here, we demonstrated the effects of cyclophilin D on the intracellular translocation of calcein AM. Following addition of calcein AM to control cells or cells overexpressing wild-type cyclophilin D, calcein fluorescence was observed in mitochondria. However, long-term inhibition of cyclophilin D in these cells altered the localization of calcein fluorescence from mitochondria to lysosomes without changing mitochondrial esterase activity. In addition, depletion of glucose from the medium recovered calcein localization from lysosomes to mitochondria. This is the first demonstration of the effects of cyclophilin D on the intracellular translocation of molecules other than proteins and suggests that cyclophilin D may modify mitochondrial features by inducing the translocation of molecules to the mitochondria through the mechanism associated with cellular energy metabolism.

  20. Cyclophilins as modulators of viral replication.

    PubMed

    Frausto, Stephen D; Lee, Emily; Tang, Hengli

    2013-07-11

    Cyclophilins are peptidyl-prolyl cis/trans isomerases important in the proper folding of certain proteins. Mounting evidence supports varied roles of cyclophilins, either positive or negative, in the life cycles of diverse viruses, but the nature and mechanisms of these roles are yet to be defined. The potential for cyclophilins to serve as a drug target for antiviral therapy is evidenced by the success of non-immunosuppressive cyclophilin inhibitors (CPIs), including Alisporivir, in clinical trials targeting hepatitis C virus infection. In addition, as cyclophilins are implicated in the predisposition to, or severity of, various diseases, the ability to specifically and effectively modulate their function will prove increasingly useful for disease intervention. In this review, we will summarize the evidence of cyclophilins as key mediators of viral infection and prospective drug targets.

  1. The cyclophilins.

    PubMed

    Wang, Ping; Heitman, Joseph

    2005-01-01

    Cyclophilins (Enzyme Commission (EC) number 5.1.2.8) belong to a group of proteins that have peptidyl-prolyl cis-trans isomerase activity; such proteins are collectively known as immunophilins and also include the FK-506-binding proteins and the parvulins. Cyclophilins are found in all cells of all organisms studied, in both prokaryotes and eukaryotes; humans have a total of 16 cyclophilin proteins, Arabidopsis up to 29 and Saccharomyces 8. The first member of the cyclophilins to be identified in mammals, cyclophilin A, is the major cellular target for, and thus mediates the actions of, the immunosuppressive drug cyclosporin A. Cyclophilin A forms a ternary complex with cyclosporin A and the calcium-calmodulin-activated serine/threonine-specific protein phosphatase calcineurin; formation of this complex prevents calcineurin from regulating cytokine gene transcription. Recent studies have implicated a diverse array of additional cellular functions for cyclophilins, including roles as chaperones and in cell signaling.

  2. Cyclophilin function in Cancer; lessons from virus replication.

    PubMed

    Lavin, Paul T M; Mc Gee, Margaret M

    2015-01-01

    Cyclophilins belong to a group of proteins that possess peptidyl prolyl isomerase activity and catalyse the cis-trans conversion of proline peptide bonds. Cyclophilin members play important roles in protein folding and as molecular chaperones, in addition to a well-established role as host factors required for completion of the virus life cycle. Members of the cyclophilin family are overexpressed in a range of human malignancies including hepatocellular cancer, pancreatic cancer, nonsmall cell lung cancer, gastric cancer, colorectal cancer and glioblastoma multiforme, however, their precise role in tumourigenesis remains unclear. In recent years, mounting evidence supports a role for prolyl isomerisation during mammalian cell division; a process with striking similarity to plasma membrane remodelling during virus replication. Here, we summarise our current understanding of the role of cyclophilins in cancer. We review the function of cyclophilins during mammalian cell division and during HIV-1 infection, and highlight common processes involving members of the ESCRT and Rab GTPase families.

  3. Structural, biochemical, and in vivo characterization of the first virally encoded cyclophilin from the Mimivirus.

    PubMed

    Thai, Vu; Renesto, Patricia; Fowler, C Andrew; Brown, Darin J; Davis, Tara; Gu, Wanjun; Pollock, David D; Kern, Dorothee; Raoult, Didier; Eisenmesser, Elan Z

    2008-04-18

    Although multiple viruses utilize host cell cyclophilins, including severe acute respiratory syndrome (SARS) and human immunodeficiency virus type-1(HIV-1), their role in infection is poorly understood. To help elucidate these roles, we have characterized the first virally encoded cyclophilin (mimicyp) derived from the largest virus discovered to date (the Mimivirus) that is also a causative agent of pneumonia in humans. Mimicyp adopts a typical cyclophilin-fold, yet it also forms trimers unlike any previously characterized homologue. Strikingly, immunofluorescence assays reveal that mimicyp localizes to the surface of the mature virion, as recently proposed for several viruses that recruit host cell cyclophilins such as SARS and HIV-1. Additionally mimicyp lacks peptidyl-prolyl isomerase activity in contrast to human cyclophilins. Thus, this study suggests that cyclophilins, whether recruited from host cells (i.e. HIV-1 and SARS) or virally encoded (i.e. Mimivirus), are localized on viral surfaces for at least a subset of viruses.

  4. The cyclophilins

    PubMed Central

    Wang, Ping; Heitman, Joseph

    2005-01-01

    Summary: Cyclophilins (Enzyme Commission (EC) number 5.1.2.8) belong to a group of proteins that have peptidyl-prolyl cis-trans isomerase activity; such proteins are collectively known as immunophilins and also include the FK-506-binding proteins and the parvulins. Cyclophilins are found in all cells of all organisms studied, in both prokaryotes and eukaryotes; humans have a total of 16 cyclophilin proteins, Arabidopsis up to 29 and Saccharomyces 8. The first member of the cyclophilins to be identified in mammals, cyclophilin A, is the major cellular target for, and thus mediates the actions of, the immunosuppressive drug cyclosporin A. Cyclophilin A forms a ternary complex with cyclosporin A and the calcium-calmodulin-activated serine/threonine-specific protein phosphatase calcineurin; formation of this complex prevents calcineurin from regulating cytokine gene transcription. Recent studies have implicated a diverse array of additional cellular functions for cyclophilins, including roles as chaperones and in cell signaling. PMID:15998457

  5. Solution characterization of the extracellular region of CD147 and its interaction with its enzyme ligand cyclophilin-A

    SciTech Connect

    Schlegel, Jennifer; Redzic, Jasmina S.; Porter, Christopher; Yurchenko, Vyacheslav; Bukrinsky, Michael; Labeikovsky, Wladimir; Armstrong, Geoffrey S.; Zhang, Fengli; Isern, Nancy G.; Degregori, James; Hodges, Robert; Eisenmesser, Elan Z.

    2009-08-21

    The CD147 receptor plays an integral role in numerous diseases by stimulating the expression of several protein families and serving as the receptor for extracellular cyclophilins, however, neither CD147 nor its interactions with its cyclophilin ligands have been well characterized in solution. CD147 is a unique protein in that it can function both at the cell membrane and after being released from cells where it continues to retain activity. Thus, the CD147 receptor functions through at least two mechanisms that include both cyclophilin-independent and cyclophilin-dependent modes of action. In regard to CD147 cyclophilin-independent activity, CD147 homophilic interactions are thought to underlie its activity. In regard to CD147 cyclophilin-dependent activity, cyclophilin/CD147 interactions may represent a novel means of signaling since cyclophilins are also peptidyl-prolyl isomerases.

  6. Current implications of cyclophilins in human cancers.

    PubMed

    Lee, Jinhwa; Kim, Sung Soo

    2010-07-19

    Cyclophilins (Cyps), the intracellular receptor for immunosuppressant cyclosporine A (CsA), play important cellular roles through activities of peptidyl-prolyl cis-trans isomerase (PPIase) and chaperones. Cyps are structurally conserved and found in both prokaryotic and eukaryotic organisms, including humans which contain 16 Cyp isoforms. Although human Cyps were identified about 25 years ago, their physiological and pathological roles have only been the focus of attention recently because of their possible involvement in diseases and ailments such as HIV infection, hepatitis B and C viral infection, atherosclerosis, ER stress-related diseases and neurodegenerative diseases, etc. There are reports for upregulated Cyps in many human cancers and there are also strong correlations found between Cyps overexpression and malignant transformation. This review discusses the important and diverse roles of Cyps overexpression in human cancers. Understanding biological functions of Cyps will eventually lead to improved strategies for cancer treatment and prevention.

  7. Isoform-specific inhibition of cyclophilins.

    PubMed

    Daum, Sebastian; Schumann, Michael; Mathea, Sebastian; Aumüller, Tobias; Balsley, Molly A; Constant, Stephanie L; de Lacroix, Boris Féaux; Kruska, Fabian; Braun, Manfred; Schiene-Fischer, Cordelia

    2009-07-07

    Cyclophilins belong to the enzyme class of peptidyl prolyl cis-trans isomerases which catalyze the cis-trans isomerization of prolyl bonds in peptides and proteins in different folding states. Cyclophilins have been shown to be involved in a multitude of cellular functions like cell growth, proliferation, and motility. Among the 20 human cyclophilin isoenzymes, the two most abundant members of the cyclophilin family, CypA and CypB, exhibit specific cellular functions in several inflammatory diseases, cancer development, and HCV replication. A small-molecule inhibitor on the basis of aryl 1-indanylketones has now been shown to discriminate between CypA and CypB in vitro. CypA binding of this inhibitor has been characterized by fluorescence anisotropy- and isothermal titration calorimetry-based cyclosporin competition assays. Inhibition of CypA- but not CypB-mediated chemotaxis of mouse CD4(+) T cells by the inhibitor provided biological proof of discrimination in vivo.

  8. Structural, Biochemical, and in Vivo Characterization of the First Virally Encoded Cyclophilin from the Mimivirus

    PubMed Central

    Thai, Vu; Renesto, Patricia; Fowler, C. Andrew; Brown, Darin J.; Davis, Tara; Gu, Wanjun; Pollock, David D.; Kern, Dorothee; Raoult, Didier; Eisenmesser, Elan Z.

    2010-01-01

    Although multiple viruses utilize host cell cyclophilins, including severe acute respiratory syndrome (SARS) and human immunodeficiency virus type-1(HIV-1), their role in infection is poorly understood. To help elucidate these roles, we have characterized the first virally encoded cyclophilin (mimicyp) derived from the largest virus discovered to date (the Mimivirus) that is also a causative agent of pneumonia in humans. Mimicyp adopts a typical cyclophilin-fold, yet it also forms trimers unlike any previously characterized homologue. Strikingly, immunofluorescence assays reveal that mimicyp localizes to the surface of the mature virion, as recently proposed for several viruses that recruit host cell cyclophilins such as SARS and HIV-1. Additionally mimicyp lacks peptidyl-prolyl isomerase activity in contrast to human cyclophilins. Thus, this study suggests that cyclophilins, whether recruited from host cells (i.e. HIV-1 and SARS) or virally encoded (i.e. Mimivirus), are localized on viral surfaces for at least a subset of viruses. PMID:18342330

  9. Cyclophilin A Associates with Enterovirus-71 Virus Capsid and Plays an Essential Role in Viral Infection as an Uncoating Regulator

    PubMed Central

    Huang, Jiaoyan; Yan, Wenzhong; Wang, Jinglan; Su, Dan; Ni, Cheng; Li, Jian; Rao, Zihe; Liu, Lei; Lou, Zhiyong

    2014-01-01

    Viruses utilize host factors for their efficient proliferation. By evaluating the inhibitory effects of compounds in our library, we identified inhibitors of cyclophilin A (CypA), a known immunosuppressor with peptidyl-prolyl cis-trans isomerase activity, can significantly attenuate EV71 proliferation. We demonstrated that CypA played an essential role in EV71 entry and that the RNA interference-mediated reduction of endogenous CypA expression led to decreased EV71 multiplication. We further revealed that CypA directly interacted with and modified the conformation of H-I loop of the VP1 protein in EV71 capsid, and thus regulated the uncoating process of EV71 entry step in a pH-dependent manner. Our results aid in the understanding of how host factors influence EV71 life cycle and provide new potential targets for developing antiviral agents against EV71 infection. PMID:25275585

  10. Treating hepatitis C: can you teach old dogs new tricks?

    PubMed

    Rice, Charles M; You, Shihyun

    2005-12-01

    Viruses depend on host-derived factors for their efficient genome replication. Here, we demonstrate that a cellular peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin B (CyPB), is critical for the efficient replication of the hepatitis C virus genome. CyPB interacted with the HCV RNA polymerase NS5B to directly stimulate its RNA binding activity. Both the RNA interference (RNAi)-mediated reduction of endogenous CyPB expression and the induced loss of NS5B binding to CyPB decreased the levels of HCV replication. Thus, CyPB functions as a stimulatory regulator of NS5B in HCV replication machinery. This regulation mechanism for viral replication identifies CyPB as a target for antiviral therapeutic strategies.

  11. Protein aggregation activates erratic stress response in dietary restricted yeast cells

    PubMed Central

    Bhadra, Ankan Kumar; Das, Eshita; Roy, Ipsita

    2016-01-01

    Chronic stress and prolonged activation of defence pathways have deleterious consequences for the cell. Dietary restriction is believed to be beneficial as it induces the cellular stress response machinery. We report here that although the phenomenon is beneficial in a wild-type cell, dietary restriction leads to an inconsistent response in a cell that is already under proteotoxicity-induced stress. Using a yeast model of Huntington’s disease, we show that contrary to expectation, aggregation of mutant huntingtin is exacerbated and activation of the unfolded protein response pathway is dampened under dietary restriction. Global proteomic analysis shows that when exposed to a single stress, either protein aggregation or dietary restriction, the expression of foldases like peptidyl-prolyl isomerase, is strongly upregulated. However, under combinatorial stress, this lead is lost, which results in enhanced protein aggregation and reduced cell survival. Successful designing of aggregation-targeted therapeutics will need to take additional stressors into account. PMID:27633120

  12. Interaction with Ppil3 leads to the cytoplasmic localization of Apoptin in tumor cells

    SciTech Connect

    Huo Dehua; Yi Lina; Yang Jine

    2008-07-18

    Apoptin, a small protein encoded by chicken anemia virus (CAV), induces cell death specifically in cancer cells. In normal cells, Apoptin remains in the cytoplasm; whereas in cancerous cells, it migrates into the nucleus and kills the cell. Cellular localization appears to be crucial. Through a yeast two-hybrid screen, we identified human Peptidyl-prolyl isomerase-like 3 (Ppil3) as one of the Apoptin-associated proteins. Ppil3 could bind Apoptin directly, and held Apoptin in cytoplasm even in tumor cells. We then demonstrated that the nuclearcytoplasmic distribution of Apoptin is related to the expression level of intrinsic Ppil3. Moreover, extrinsic modifying of Ppil3 levels also resulted in nuclearcytoplasmic shuffling of Apoptin. The Apoptin P109A mutant, located between the putative nuclear localization and export signals, could significantly impair the function of Ppil3. Our results suggest a new direction for the localization mechanism study of Apoptin in cells.

  13. Novel inhibitors of the calcineurin/NFATc hub - alternatives to CsA and FK506?

    PubMed Central

    Sieber, Matthias; Baumgrass, Ria

    2009-01-01

    The drugs cyclosporine A (CsA) and tacrolimus (FK506) revolutionized organ transplantation. Both compounds are still widely used in the clinic as well as for basic research, even though they have dramatic side effects and modulate other pathways than calcineurin-NFATc, too. To answer the major open question - whether the adverse side effects are secondary to the actions of the drugs on the calcineurin-NFATc pathway - alternative inhibitors were developed. Ideal inhibitors should discriminate between the inhibition of (i) calcineurin and peptidyl-prolyl cis-trans isomerases (PPIases; the matchmaker proteins of CsA and FK506), (ii) calcineurin and the other Ser/Thr protein phosphatases, and (iii) NFATc and other transcription factors. In this review we summarize the current knowledge about novel inhibitors, synthesized or identified in the last decades, and focus on their mode of action, specificity, and biological effects. PMID:19860902

  14. Spliceosomal Immunophilins

    PubMed Central

    Mesa, Annia; Somarelli, Jason A.; Herrera, Rene J.

    2008-01-01

    The spliceosome is a dynamic, macromolecular complex, which removes non-protein-coding introns from pre-mRNA to form mature mRNA in a process known as splicing. This ribonucleoprotein assembly is comprised of five uridine-rich small nuclear RNAs (snRNAs) as well as over 300 proteins. In humans, several of the known splicing factors are members of the immunophilin superfamily. Immunophilins are peptidyl-prolyl cis-trans isomerases that catalyze the conversion of proteins from cis to trans at Xaa-Pro bonds. Our review of the data portrays a picture of this protein family as activators of spliceosomal proteins by way of folding and transport. PMID:18544344

  15. RNA-interference-mediated downregulation of Pin1 suppresses tumorigenicity of malignant melanoma A375 cells.

    PubMed

    Jin, J; Zhang, Y; Li, Y; Zhang, H; Li, H; Yuan, X; Li, X; Zhou, W; Xu, B; Zhang, C; Zhang, Z; Zhu, L; Chen, X

    2013-01-01

    The peptidyl-prolyl isomerase Pin1 is overexpressed in many human cancers, including melanoma. To investigate its possible role in oncogenesis of melanoma and as a therapeutic target, we suppressed Pin1 expression in the human melanoma cell line A375 by microRNA (miRNA) interference technology. Two stable clones with suppressed Pin1 were established by stable transfection of miRNA plasmid targeting Pin1 into A375 cells. Both clones showed reduced proliferation and invasion in vitro and suppressed tumorigenic potential in athymic mice. Furthermore, Pin1 inhibition also resulted in decreased phosphorylation of Akt and repressed expression of C-Jun N-terminal kinase and pro-matrix metalloproteinase 2, which were associated closely with the development of melanoma. These findings indicate that Pin1 plays an important role in the tumorigenesis of melanoma and might serve as a promising therapeutic target.

  16. Cyclophilin A: a key player for human disease.

    PubMed

    Nigro, P; Pompilio, G; Capogrossi, M C

    2013-10-31

    Cyclophilin A (CyPA) is a ubiquitously distributed protein belonging to the immunophilin family. CyPA has peptidyl prolyl cis-trans isomerase (PPIase) activity, which regulates protein folding and trafficking. Although CyPA was initially believed to function primarily as an intracellular protein, recent studies have revealed that it can be secreted by cells in response to inflammatory stimuli. Current research in animal models and humans has provided compelling evidences supporting the critical function of CyPA in several human diseases. This review discusses recently available data about CyPA in cardiovascular diseases, viral infections, neurodegeneration, cancer, rheumatoid arthritis, sepsis, asthma, periodontitis and aging. It is believed that further elucidations of the role of CyPA will provide a better understanding of the molecular mechanisms underlying these diseases and will help develop novel pharmacological therapies.

  17. Cyclophilin D in Mitochondrial Pathophysiology

    PubMed Central

    Giorgio, Valentina; Soriano, Maria Eugenia; Basso, Emy; Bisetto, Elena; Lippe, Giovanna; Forte, Michael A.; Bernardi, Paolo

    2010-01-01

    Cyclophilins are a family of peptidyl-prolyl cis-trans isomerases whose enzymatic activity can be inhibited by Cyclosporin A. Sixteen cyclophilins have been identified in humans, and cyclophilin D is a unique isoform that is imported into the mitochondrial matrix. Here we shall (i) review the best characterized functions of cyclophilin D in mitochondria, i.e. regulation of the permeability transition pore, an inner membrane channel that plays an important role in the execution of cell death; (ii) highlight new regulatory interactions that are emerging in the literature, including the modulation of the mitochondrial F1FO ATP synthase through an interaction with the lateral stalk of the enzyme complex; and (iii) discuss diseases where cyclophilin D plays a pathogenetic role that makes it a suitable target for pharmacologic intervention. PMID:20026006

  18. Functional aspects of extracellular cyclophilins.

    PubMed

    Hoffmann, Henrik; Schiene-Fischer, Cordelia

    2014-07-01

    The cyclophilin family of peptidyl prolyl cis/trans isomerases includes several isoforms found to be secreted in response to different stimuli, thus existing both in the interior and the exterior of cells. The extracellular fractions of the cyclophilins CypA and CypB are involved in the control of cell-cell communication. By binding to the cell membrane receptor CD147 and cell surface heparans they elicit a variety of intracellular signaling cascades involved in inflammatory processes. Increased levels of cyclophilins in inflammatory tissues and body fluids are considered as an inflammatory response to injury. Thus, the extracellular portion of cyclophilins probably plays an important role in human diseases associated with acute or chronic inflammation like rheumatoid arthritis, sepsis, asthma and cardiovascular diseases. Specific inhibition of the cyclophilins in the extracellular space may open an effective therapeutic approach for treating inflammatory diseases.

  19. A novel role for hGas7b in microtubular maintenance: possible implication in tau-associated pathology in Alzheimer disease.

    PubMed

    Akiyama, Hirotada; Gotoh, Aina; Shin, Ryong-Woon; Koga, Tomoe; Ohashi, Tsubasa; Sakamoto, Wataru; Harada, Akihiro; Arai, Hiroyuki; Sawa, Akira; Uchida, Chiyoko; Uchida, Takafumi

    2009-11-20

    Here, we report a novel role for hGas7b (human growth arrest specific protein 7b) in the regulation of microtubules. Using a bioinformatic approach, we studied the actin-binding protein hGas7b with a structural similarity to the WW domain of a peptidyl prolyl cis/trans isomerase, Pin1, that facilitates microtubule assembly. Thus, we have demonstrated that hGas7b binds Tau at the WW motif and that the hGas7b/Tau protein complex interacts with the microtubules, promoting tubulin polymerization. Tau, in turn, contributes to protein stability of hGas7b. Furthermore, we observed decreased levels of hGas7b in the brains from patients with Alzheimer disease. These results suggest an important role for hGas7b in microtubular maintenance and possible implication in Alzheimer disease.

  20. Chemotactic Activity of Cyclophilin A in the Skin Mucus of Yellow Catfish (Pelteobagrus fulvidraco) and Its Active Site for Chemotaxis

    PubMed Central

    Dawar, Farman Ullah; Tu, Jiagang; Xiong, Yang; Lan, Jiangfeng; Dong, Xing Xing; Liu, Xiaoling; Khattak, Muhammad Nasir Khan; Mei, Jie; Lin, Li

    2016-01-01

    Fish skin mucus is a dynamic barrier for invading pathogens with a variety of anti-microbial enzymes, including cyclophilin A (CypA), a multi-functional protein with peptidyl-prolyl cis/trans isomerase (PPIase) activity. Beside various other immunological functions, CypA induces leucocytes migration in vitro in teleost. In the current study, we have discovered several novel immune-relevant proteins in yellow catfish skin mucus by mass spectrometry (MS). The CypA present among them was further detected by Western blot. Moreover, the CypA present in the skin mucus displayed strong chemotactic activity for yellow catfish leucocytes. Interestingly, asparagine (like arginine in mammals) at position 69 was the critical site in yellow catfish CypA involved in leucocyte attraction. These novel efforts do not only highlight the enzymatic texture of skin mucus, but signify CypA to be targeted for anti-inflammatory therapeutics. PMID:27589721

  1. Transcriptional and epigenetic phenomena in peripheral blood cells of monozygotic twins discordant for alzheimer's disease, a case report.

    PubMed

    D'Addario, Claudio; Candia, Sussy Bastias; Arosio, Beatrice; Di Bartolomeo, Martina; Abbate, Carlo; Casè, Alessandra; Candeletti, Sanzio; Romualdi, Patrizia; Damanti, Sarah; Maccarrone, Mauro; Bergamaschini, Luigi; Mari, Daniela

    2017-01-15

    Target genes in Alzheimer's disease (AD) have been identified. In monozygotic twins discordant for AD we analysed the expression of selected genes, and their possible regulation by epigenetic mechanisms in peripheral blood mononuclear cells, possibly useful to discover biomarkers. Amyloid precursor protein, sirtuin 1 and peptidyl prolyl isomerase 1 gene expressions were highly up-regulated in the AD twin versus the healthy one. Consistently with sirtuin 1 role in controlling acetylation status, we observed a substantial reduction of the acetylation on histone 3 lysine 9, associated with gene transcription in the AD twin. Noteworthy in the AD twin we also observed an increased gene expression in two histone deacetylases (HDACs) isoforms: HDAC2 and HDAC9. A general DNA hypomethylation of all gene promoters studied was also observed in both twins. Our results unravel transcriptional and epigenetic differences potentially helpful to better understand environmental factors and phenotypic differences in monozygotic twins.

  2. Structural, Biochemical, and in Vivo Characterization of the First Virally Encoded Cyclophilin from the Mimivirus

    SciTech Connect

    Thai,V.; Renesto, P.; Fowler, C.; Brown, D.; Davis, T.; Gu, W.; Pollock, D.; Kern, D.; Raoult, D.; Eisenmesser, E.

    2008-01-01

    Although multiple viruses utilize host cell cyclophilins, including severe acute respiratory syndrome (SARS) and human immunodeficiency virus type-1(HIV-1), their role in infection is poorly understood. To help elucidate these roles, we have characterized the first virally encoded cyclophilin (mimicyp) derived from the largest virus discovered to date (the Mimivirus) that is also a causative agent of pneumonia in humans. Mimicyp adopts a typical cyclophilin-fold, yet it also forms trimers unlike any previously characterized homologue. Strikingly, immunofluorescence assays reveal that mimicyp localizes to the surface of the mature virion, as recently proposed for several viruses that recruit host cell cyclophilins such as SARS and HIV-1. Additionally mimicyp lacks peptidyl-prolyl isomerase activity in contrast to human cyclophilins. Thus, this study suggests that cyclophilins, whether recruited from host cells (ie HIV-1 and SARS) or virally encoded (ie Mimivirus), are localized on viral surfaces for at least a subset of viruses.

  3. Involvement of cyclophilin D in mitochondrial permeability transition induction in intact cells.

    PubMed

    Tazawa, Hidejiro; Fujita, Chisako; Machida, Kiyotaka; Osada, Hiroyuki; Ohta, Yoshihiro

    2009-01-01

    The mitochondrial permeability transition (MPT) is involved in both Ca(2+) signaling and cell death. The present study aimed to clarify the involvement of cyclophilin D, a peptidyl prolyl cis-trans isomerase (PPIase), in MPT induction in intact cells. To achieve this, we used C6 cells overexpressing wild-type or PPIase-deficient cyclophilin D, and measured the inner mitochondrial membrane permeability to calcein, a 623-Da hydrophilic fluorescent molecule, to evaluate MPT induction. In vector control cells, the percentage of MPT induction by ionomycin increased as the Ca(2+) concentration in the extracellular medium increased. This result indicates that the present method is valid for numerical evaluation of MPT induction. In C6 cells expressing the PPIase-deficient mutant, the percentage of MPT induction was significantly decreased compared with wild-type CypD-overexpressing cells or vector control cells. These results suggest that cyclophilin D is involved in MPT induction by Ca(2+) in intact cells.

  4. Functional analysis of the two cyclophilin isoforms of Sinorhizobium meliloti.

    PubMed

    Thomloudi, Eirini-Evangelia; Skagia, Aggeliki; Venieraki, Anastasia; Katinakis, Panagiotis; Dimou, Maria

    2017-02-01

    The nitrogen fixing Sinorhizobium meliloti possesses two genes, ppiA and ppiB, encoding two cyclophilin isoforms which belong to the superfamily of peptidyl prolyl cis/trans isomerases (PPIase, EC: 5.2.1.8). Here, we functionally characterize the two proteins and we demonstrate that both recombinant cyclophilins are able to isomerise the Suc-AAPF-pNA synthetic peptide but neither of them displays chaperone function in the citrate synthase thermal aggregation assay. Furthermore, we observe that the expression of both enzymes increases the viability of E. coli BL21 in the presence of abiotic stress conditions such as increased heat and salt concentration. Our results support and strengthen previous high-throughput studies implicating S. meliloti cyclophilins in various stress conditions.

  5. Identification of native Escherichia coli BL21 (DE3) proteins that bind to immobilized metal affinity chromatography under high imidazole conditions and use of 2D-DIGE to evaluate contamination pools with respect to recombinant protein expression level.

    PubMed

    Bartlow, Patrick; Uechi, Guy T; Cardamone, John J; Sultana, Tamanna; Fruchtl, McKinzie; Beitle, Robert R; Ataai, Mohammad M

    2011-08-01

    Immobilized metal affinity chromatography (IMAC) is a widely used purification tool for the production of active, soluble recombinant proteins. Escherichia coli proteins that routinely contaminate IMAC purifications have been characterized to date. The work presented here narrows that focus to the most problematic host proteins, those retaining nickel affinity under elevated imidazole conditions, using a single bind-and-elute step. Two-dimensional difference gel electrophoresis, a favored technique for resolving complex protein mixtures and evaluating their expression, here discerns variation in the soluble extract pools that are loaded in IMAC and the remaining contaminants with respect to varied levels of recombinant protein expression. Peptidyl-prolyl isomerase SlyD and catabolite activator protein (CAP) are here shown to be the most persistent contaminants and have greater prevalence at low target protein expression.

  6. Induction of chalcone isomerase in elicitor-treated bean cells. Comparison of rates of synthesis and appearance of immunodetectable enzyme.

    PubMed

    Robbins, M P; Dixon, R A

    1984-11-15

    Chalcone isomerase, an enzyme involved in the formation of flavonoid-derived compounds in plants, has been purified nearly 600-fold from cell suspension cultures of dwarf French bean (Phaseolus vulgaris L.). Chromatofocussing yielded a single form of the enzyme of apparent pI 5.0. This preparation was used to raise rabbit anti-(chalcone isomerase) serum. Changes in the rate of synthesis of chalcone isomerase have been investigated by indirect immunoprecipitation of enzyme labelled in vivo with [35S]methionine in elicitor-treated cultures of P. vulgaris. Elicitor, heat-released from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum, the causal agent of anthracnose disease of bean, causes increased synthesis of the isomerase, with maximum synthetic rate occurring 11-12 h after exposure to elicitor. Immune blotting studies indicate that the elicitor-mediated increase in extractable activity of the isomerase is associated with increased appearance of immunodetactable isomerase protein of Mr 27 000. However, the maximum level of immunodetectable isomerase was attained approximately 6 h earlier than maximum extractable activity. Furthermore, a 2.8-fold increase in enzyme activity above basal levels at 12 h after elicitor-treatment was associated with a corresponding 5.8-fold increase in immunodetectable enzyme. It is concluded that elicitor induces the synthesis of both active and inactive chalcone isomerase of Mr 27 000, and that some activation of inactive enzyme occurs during the elicitor-mediated increase in isomerase activity. The presence of a pool of inactive chalcone isomerase in bean cell cultures has recently been suggested on the basis of density labelling experiments utilising 2H from 2H2O [Dixon et al. (1983) Planta (Berl.) 159, 561-569].

  7. Xylose Isomerase Improves Growth and Ethanol Production Rates from Biomass Sugars for Both Saccharomyces Pastorianus and Saccharomyces Cerevisiae

    PubMed Central

    Miller, Kristen P.; Gowtham, Yogender Kumar; Henson, J. Michael; Harcum, Sarah W.

    2013-01-01

    The demand for biofuel ethanol made from clean, renewable nonfood sources is growing. Cellulosic biomass, such as switch grass (Panicum virgatum L.), is an alternative feedstock for ethanol production; however, cellulosic feedstock hydrolysates contain high levels of xylose, which needs to be converted to ethanol to meet economic feasibility. In this study, the effects of xylose isomerase on cell growth and ethanol production from biomass sugars representative of switch grass were investigated using low cell density cultures. The lager yeast species Saccharomyces pastorianus was grown with immobilized xylose isomerase in the fermentation step to determine the impact of the glucose and xylose concentrations on the ethanol production rates. Ethanol production rates were improved due to xylose isomerase; however, the positive effect was not due solely to the conversion of xylose to xylulose. Xylose isomerase also has glucose isomerase activity, so to better understand the impact of the xylose isomerase on S. pastorianus, growth and ethanol production were examined in cultures provided fructose as the sole carbon. It was observed that growth and ethanol production rates were higher for the fructose cultures with xylose isomerase even in the absence of xylose. To determine whether the positive effects of xylose isomerase extended to other yeast species, a side-by-side comparison of S. pastorianus and Saccharomyces cerevisiae was conducted. These comparisons demonstrated that the xylose isomerase increased ethanol productivity for both the yeast species by increasing the glucose consumption rate. These results suggest that xylose isomerase can contribute to improved ethanol productivity, even without significant xylose conversion. PMID:22866331

  8. Proteomic identification of abnormally expressed proteins in early-stage placenta derived from cloned cat embryos.

    PubMed

    Bang, Jae-Il; Lee, Hyo-Sang; Deb, Gautam Kumar; Ha, A-Na; Kwon, Young-Sang; Cho, Seong-Keun; Kim, Byeong-Woo; Cho, Kyu-Woan; Kong, Il-Keun

    2013-01-15

    It is unknown whether gene expression in cloned placenta during pre- and postimplantation is associated with early pregnancy failure in the cat. In this study, protein expression patterns were examined in early-stage (21-day-old) domestic cat placentas of fetuses derived from AI (CP; N = 4) and cloned embryo transfer (CEP; N = 2). Differentially expressed proteins were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrometry (MS). A total of 21 proteins were aberrantly expressed (P < 0.05) by >1.5-fold in CEP compared with CP. Compared with CP, 12 proteins were upregulated in CEP (peptidyl-prolyl cis-trans isomerase A, annexin A2, protein DJ-1, adenylate kinase isoenzyme 1, protein disulfide-isomerase A3, actin cytoplasmic 1, serum albumin, protein disulfide-isomerase A6, and triosephosphate isomerase), and nine proteins were downregulated (triosephosphate isomerase; heterogeneous nuclear ribonucleoprotein H; tropomyosin alpha-4; triosephosphate isomerase 1; 60 kDa heat shock protein, mitochondrial; serum albumin; calumenin; keratin type 1; and prohibitin). The identities of the differentially expressed proteins were validated by peptide mass fingerprinting using matrix-assisted laser desorption/ionization-TOF/TOF MS/MS. The abnormally expressed proteins identified in this study might be associated with impaired development and dysfunction of CEP during early pregnancy. Abnormal protein expression might also induce fetal loss and contribute to failure to maintain pregnancy to term.

  9. Diagnostic value of glucose-6-phosphate isomerase in rheumatoid arthritis.

    PubMed

    Fan, Lie Ying; Zong, Ming; Wang, Qiang; Yang, Lin; Sun, Li Shan; Ye, Qin; Ding, Yuan Yuan; Ma, Jian Wei

    2010-12-14

    Although glucose-6-phosphate isomerase (G6PI), anti-G6PI antibodies and G6PI-containing immune complexes (G6PI-CIC) have proved high expression in patients with rheumatoid arthritis (RA), comprehensive evaluation of the G6PI-derived markers, G6PI antigen, anti-G6PI Abs, G6PI-CIC and G6PI mRNA, in the diagnosis of RA remains necessary. We measured G6PI antigen, anti-G6PI Abs, C1q/G6PI-CIC as well as anti-cyclic citrullinated peptide antibodies (anti-CCP Abs) in serum and concomitantly synovial fluid (SF) by ELISA in RA, other rheumatic diseases and healthy controls. The G6PI mRNA expression in peripheral blood mononuclear cells (PBMCs) was assessed with real-time PCR. As compared with non-RA patients, RA patients had increased levels of G6PI antigen, anti-G6PI Abs, C1q/G6PI-CIC and G6PI mRNA expression in sera or PBMCs, and increased levels of G6PI and C1q/G6PI-CIC in SF. The serum G6PI levels in RA patients positively correlated with anti-G6PI Abs, C1q/G6PI-CIC, G6PI mRNA, anti-CCP Abs, RF, CRP and ESR, respectively. The area under curve analyses demonstrated that serum G6PI had the best discriminating power for RA and active RA followed by C1q/G6PI-CIC, anti-G6PI Abs and G6PI mRNA. The simultaneous use of serum G6PI and anti-CCP Abs assays in the form of either of them tested positive gave improved sensitivities of 88.1% for RA and 95.8% for active RA. Despite the elevated expression of all G6PI-derived markers in RA, the serum G6PI has the best discriminating power among the four G6PI-derived markers. The serum G6PI determination either alone or in combination with anti-CCP Abs improves the diagnosis of RA. Copyright © 2010 Elsevier B.V. All rights reserved.

  10. Prevalent overexpression of prolyl isomerase Pin1 in human cancers.

    PubMed

    Bao, Lere; Kimzey, Amy; Sauter, Guido; Sowadski, Janusz M; Lu, Kun Ping; Wang, Da-Gong

    2004-05-01

    Phosphorylation of proteins on serine or threonine residues preceding proline (pSer/Thr-Pro) is a major regulatory mechanism in cell proliferation and transformation. Interestingly, the pSer/Thr-Pro motifs in proteins exist in two distinct cis and trans conformations, whose conversion rate is normally reduced on phosphorylation, but is catalyzed specifically by the prolyl isomerase Pin1. Pin1 can catalytically induce conformational changes in proteins after phosphorylation, thereby having profound effects on catalytic activity, dephosphorylation, protein-protein interactions, subcellular location, and/or turnover of certain phosphorylated proteins. Recently, it has been shown that Pin1 is overexpressed in human breast cancer cell lines and cancer tissues and plays a critical role in the transformation of mammary epithelial cells by activating multiple oncogenic pathways. Furthermore, Pin1 expression is an excellent independent prognostic marker in prostate cancer. However, little is known about Pin1 expression in other human normal and cancerous tissues. In the present study, we quantified Pin1 expression in 2041 human tumor samples and 609 normal tissue samples as well as normal and transformed human cell lines. We found that Pin1 was usually expressed at very low levels in most normal tissues and its expression was normally associated with cell proliferation, with high Pin1 levels being found only in a few cell types. However, Pin1 was strikingly overexpressed in many different human cancers. Most tumors (38 of 60 tumor types) have Pin1 overexpression in more than 10% of the cases, as compared with the corresponding normal controls, which included prostate, lung, ovary, cervical, brain tumors, and melanoma. Consistent with these findings, Pin1 expression in human cancer cell lines was also higher than that in the normal cell lines examined. These results indicate that Pin1 overexpression is a prevalent and specific event in human cancers. Given previous findings

  11. Interactions of Arabidopsis RS domain containing cyclophilins with SR proteins and U1 and U11 small nuclear ribonucleoprotein-specific proteins suggest their involvement in pre-mRNA Splicing.

    PubMed

    Lorkovic, Zdravko J; Lopato, Sergiy; Pexa, Monika; Lehner, Reinhard; Barta, Andrea

    2004-08-06

    Ser/Arg (SR)-rich proteins are important splicing factors in both general and alternative splicing. By binding to specific sequences on pre-mRNA and interacting with other splicing factors via their RS domain they mediate different intraspliceosomal contacts, thereby helping in splice site selection and spliceosome assembly. While characterizing new members of this protein family in Arabidopsis, we have identified two proteins, termed CypRS64 and CypRS92, consisting of an N-terminal peptidyl-prolyl cis/trans isomerase domain and a C-terminal domain with many SR/SP dipeptides. Cyclophilins possess a peptidyl-prolyl cis/trans isomerase activity and are implicated in protein folding, assembly, and transport. CypRS64 interacts in vivo and in vitro with a subset of Arabidopsis SR proteins, including SRp30 and SRp34/SR1, two homologs of mammalian SF2/ASF, known to be important for 5' splice site recognition. In addition, both cyclophilins interact with U1-70K and U11-35K, which in turn are binding partners of SRp34/SR1. CypRS64 is a nucleoplasmic protein, but in most cells expressing CypRS64-GFP fusion it was also found in one to six round nuclear bodies. However, co-expression of CypRS64 with its binding partners resulted in re-localization of CypRS64 from the nuclear bodies to nuclear speckles, indicating functional interactions. These findings together with the observation that binding of SRp34/SR1 to CypRS64 is phosphorylation-dependent indicate an involvement of CypRS64 in nuclear pre-mRNA splicing, possibly by regulating phosphorylation/dephosphorylation of SR proteins and other spliceosomal components. Alternatively, binding of CypRS64 to proteins important for 5' splice site recognition suggests its involvement in the dynamics of spliceosome assembly.

  12. Cyclophilin-facilitated membrane translocation as pharmacological target to prevent intoxication of mammalian cells by binary clostridial actin ADP-ribosylated toxins.

    PubMed

    Ernst, Katharina; Langer, Simon; Kaiser, Eva; Osseforth, Christian; Michaelis, Jens; Popoff, Michel R; Schwan, Carsten; Aktories, Klaus; Kahlert, Viktoria; Malesevic, Miroslav; Schiene-Fischer, Cordelia; Barth, Holger

    2015-03-27

    Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin and Clostridium difficile CDT belong to the family of binary actin ADP-ribosylating toxins and are composed of a binding/translocation component and a separate enzyme component. The enzyme components ADP-ribosylate G-actin in the cytosol of target cells resulting in depolymerization of F-actin, cell rounding and cell death. The binding/translocation components bind to their cell receptors and form complexes with the respective enzyme components. After receptor-mediated endocytosis, the binding/translocation components form pores in membranes of acidified endosomes and the enzyme components translocate through these pores into the cytosol. This step is facilitated by the host cell chaperone heat shock protein 90 and peptidyl-prolyl cis/trans isomerases including cyclophilin A. Here, we demonstrate that a large isoform of cyclophilin A, the multi-domain enzyme cyclophilin 40 (Cyp40), binds to the enzyme components C2I, Ia and CDTa in vitro. Isothermal titration calorimetry revealed a direct binding to C2I with a calculated affinity of 101 nM and to Ia with an affinity of 1.01 μM. Closer investigation for the prototypic C2I revealed that binding to Cyp40 did not depend on its ADP-ribosyltransferase activity but was stronger for unfolded C2I. The interaction of C2I with Cyp40 was also demonstrated in lysates from C2-treated cells by pull-down. Treatment of cells with a non-immunosuppressive cyclosporine A derivative, which still binds to and inhibits the peptidyl-prolyl cis/trans isomerase activity of cyclophilins, protected cells from intoxication with C2, iota and CDT toxins, offering an attractive approach for development of novel therapeutic strategies against binary actin ADP-ribosylating toxins. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Solution structure of the parvulin-type PPIase domain of Staphylococcus aureus PrsA – Implications for the catalytic mechanism of parvulins

    PubMed Central

    Heikkinen, Outi; Seppala, Raili; Tossavainen, Helena; Heikkinen, Sami; Koskela, Harri; Permi, Perttu; Kilpeläinen, Ilkka

    2009-01-01

    Background Staphylococcus aureus is a Gram-positive pathogenic bacterium causing many kinds of infections from mild respiratory tract infections to life-threatening states as sepsis. Recent emergence of S. aureus strains resistant to numerous antibiotics has created a need for new antimicrobial agents and novel drug targets. S. aureus PrsA is a membrane associated extra-cytoplasmic lipoprotein which contains a parvulin-type peptidyl-prolyl cis-trans isomerase domain. PrsA is known to act as an essential folding factor for secreted proteins in Gram-positive bacteria and thus it is a potential target for antimicrobial drugs against S. aureus. Results We have solved a high-resolution solution structure of the parvulin-type peptidyl-prolyl cis-trans isomerase domain of S. aureus PrsA (PrsA-PPIase). The results of substrate peptide titrations pinpoint the active site and demonstrate the substrate preference of the enzyme. With detailed NMR spectroscopic investigation of the orientation and tautomeric state of the active site histidines we are able to give further insight into the structure of the catalytic site. NMR relaxation analysis gives information on the dynamic behaviour of PrsA-PPIase. Conclusion Detailed structural description of the S. aureus PrsA-PPIase lays the foundation for structure-based design of enzyme inhibitors. The structure resembles hPin1-type parvulins both structurally and regarding substrate preference. Even though a wealth of structural data is available on parvulins, the catalytic mechanism has yet to be resolved. The structure of S. aureus PrsA-PPIase and our findings on the role of the conserved active site histidines help in designing further experiments to solve the detailed catalytic mechanism. PMID:19309529

  14. Extracellular cyclophilin-A stimulates ERK1/2 phosphorylation in a cell-dependent manner but broadly stimulates nuclear factor kappa B

    PubMed Central

    2012-01-01

    Background Although the peptidyl-prolyl isomerase, cyclophilin-A (peptidyl-prolyl isomerase, PPIA), has been studied for decades in the context of its intracellular functions, its extracellular roles as a major contributor to both inflammation and multiple cancers have more recently emerged. A wide range of activities have been ascribed to extracellular PPIA that include induction of cytokine and matrix metalloproteinase (MMP) secretion, which potentially underlie its roles in inflammation and tumorigenesis. However, there have been conflicting reports as to which particular signaling events are under extracellular PPIA regulation, which may be due to either cell-dependent responses and/or the use of commercial preparations recently shown to be highly impure. Methods We have produced and validated the purity of recombinant PPIA in order to subject it to a comparative analysis between different cell types. Specifically, we have used a combination of multiple methods such as luciferase reporter screens, translocation assays, phosphorylation assays, and nuclear magnetic resonance to compare extracellular PPIA activities in several different cell lines that included epithelial and monocytic cells. Results Our findings have revealed that extracellular PPIA activity is cell type-dependent and that PPIA signals via multiple cellular receptors beyond the single transmembrane receptor previously identified, Extracellular Matrix MetalloPRoteinase Inducer (EMMPRIN). Finally, while our studies provide important insight into the cell-specific responses, they also indicate that there are consistent responses such as nuclear factor kappa B (NFκB) signaling induced in all cell lines tested. Conclusions We conclude that although extracellular PPIA activates several common pathways, it also targets different receptors in different cell types, resulting in a complex, integrated signaling network that is cell type-specific. PMID:22631225

  15. Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation.

    PubMed

    Kuyper, Marko; Hartog, Miranda M P; Toirkens, Maurice J; Almering, Marinka J H; Winkler, Aaron A; van Dijken, Johannes P; Pronk, Jack T

    2005-02-01

    After an extensive selection procedure, Saccharomyces cerevisiae strains that express the xylose isomerase gene from the fungus Piromyces sp. E2 can grow anaerobically on xylose with a mu(max) of 0.03 h(-1). In order to investigate whether reactions downstream of the isomerase control the rate of xylose consumption, we overexpressed structural genes for all enzymes involved in the conversion of xylulose to glycolytic intermediates, in a xylose-isomerase-expressing S. cerevisiae strain. The overexpressed enzymes were xylulokinase (EC 2.7.1.17), ribulose 5-phosphate isomerase (EC 5.3.1.6), ribulose 5-phosphate epimerase (EC 5.3.1.1), transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2). In addition, the GRE3 gene encoding aldose reductase was deleted to further minimise xylitol production. Surprisingly the resulting strain grew anaerobically on xylose in synthetic media with a mu(max) as high as 0.09 h(-1) without any non-defined mutagenesis or selection. During growth on xylose, xylulose formation was absent and xylitol production was negligible. The specific xylose consumption rate in anaerobic xylose cultures was 1.1 g xylose (g biomass)(-1) h(-1). Mixtures of glucose and xylose were sequentially but completely consumed by anaerobic batch cultures, with glucose as the preferred substrate.

  16. Positive selection sites in tertiary structure of Leguminosae chalcone isomerase 1.

    PubMed

    Wang, R K; Zhan, S F; Zhao, T J; Zhou, X L; Wang, C E

    2015-03-20

    Isoflavonoids and the related synthesis enzyme, chalcone isomerase 1 (CHI1), are unique in the Leguminosae, with diverse biological functions. Among the Leguminosae, the soybean is an important oil, protein crop, and model plant. In this study, we aimed to detect the generation pattern of Leguminosae CHI1. Genome-wide sequence analysis of CHI in 3 Leguminosae and 3 other closely related model plants was performed; the expression levels of soybean chalcone isomerases were also analyzed. By comparing positively selected sites and their protein structures, we retrieved the evolution patterns for Leguminosae CHI1. A total of 28 CHI and 7 FAP3 (CHI4) genes were identified and separated into 4 clades: CHI1, CHI2, CHI3, and FAP3. Soybean genes belonging to the same chalcone isomerase subfamily had similar expression patterns. CHI1, the unique chalcone isomerase subfamily in Leguminosae, showed signs of significant positive selection as well as special expression characteristics, indicating an accelerated evolution throughout its divergence. Eight sites were identified as undergoing positive selection with high confidence. When mapped onto the tertiary structure of CHI1, these 8 sites were observed surrounding the enzyme substrate only; some of them connected to the catalytic core of CHI. Thus, we inferred that the generation of Leguminosae CHI1 is dependent on the positively selected amino acids surrounding its catalytic substrate. In other words, the evolution of CHI1 was driven by specific selection or processing conditions within the substrate.

  17. Triosephosphate isomerase and filamin C share common epitopes as novel allergens of Procambarus clarkii

    USDA-ARS?s Scientific Manuscript database

    Triosephosphate isomerase (TIM) is a key enzyme in glycolysis and has been identified as an allergen in saltwater products. In this study, TIM with a molecular mass of 28 kDa was purified from the freshwater crayfish (Procambarus clarkii) muscle. A 90-kDa protein that showed IgG/IgE cross-reactivity...

  18. Identification of Triosephosphate Isomerase as a Novel Allergen in Octopus fangsiao

    USDA-ARS?s Scientific Manuscript database

    A 28 kDa-protein was purified from octopus (Octopus fangsiao) and identified to be triosephosphate isomerase (TIM). The purified TIM is a glycoprotein with 1.7% carbohydrates and the isoelectric point is 7.6. TIM aggregated after heating above 45 °C, and the secondary structure was altered in extre...

  19. Characterization of the highly efficient sucrose isomerase from Pantoea dispersa UQ68J and cloning of the sucrose isomerase gene.

    PubMed

    Wu, Luguang; Birch, Robert G

    2005-03-01

    Sucrose isomerase (SI) genes from Pantoea dispersa UQ68J, Klebsiella planticola UQ14S, and Erwinia rhapontici WAC2928 were cloned and expressed in Escherichia coli. The predicted products of the UQ14S and WAC2928 genes were similar to known SIs. The UQ68J SI differed substantially, and it showed the highest isomaltulose-producing efficiency in E. coli cells. The purified recombinant WAC2928 SI was unstable, whereas purified UQ68J and UQ14S SIs were very stable. UQ68J SI activity was optimal at pH 5 and 30 to 35 degrees C, and it produced a high ratio of isomaltulose to trehalulose (>22:1) across its pH and temperature ranges for activity (pH 4 to 7 and 20 to 50 degrees C). In contrast, UQ14S SI showed optimal activity at pH 6 and 35 degrees C and produced a lower ratio of isomaltulose to trehalulose (<8:1) across its pH and temperature ranges for activity. UQ68J SI had much higher catalytic efficiency; the Km was 39.9 mM, the Vmax was 638 U mg(-1), and the Kcat/Km was 1.79 x 10(4) M(-1) s(-1), compared to a Km of 76.0 mM, a Vmax of 423 U mg(-1), and a Kcat/Km of 0.62 x 10(4) M(-1) s(-1) for UQ14S SI. UQ68J SI also showed no apparent reverse reaction producing glucose, fructose, or trehalulose from isomaltulose. These properties of the P. dispersa UQ68J enzyme are exceptional among purified SIs, and they indicate likely differences in the mechanism at the enzyme active site. They may favor the production of isomaltulose as an inhibitor of competing microbes in high-sucrose environments, and they are likely to be highly beneficial for industrial production of isomaltulose.

  20. Characterization of the Highly Efficient Sucrose Isomerase from Pantoea dispersa UQ68J and Cloning of the Sucrose Isomerase Gene

    PubMed Central

    Wu, Luguang; Birch, Robert G.

    2005-01-01

    Sucrose isomerase (SI) genes from Pantoea dispersa UQ68J, Klebsiella planticola UQ14S, and Erwinia rhapontici WAC2928 were cloned and expressed in Escherichia coli. The predicted products of the UQ14S and WAC2928 genes were similar to known SIs. The UQ68J SI differed substantially, and it showed the highest isomaltulose-producing efficiency in E. coli cells. The purified recombinant WAC2928 SI was unstable, whereas purified UQ68J and UQ14S SIs were very stable. UQ68J SI activity was optimal at pH 5 and 30 to 35°C, and it produced a high ratio of isomaltulose to trehalulose (>22:1) across its pH and temperature ranges for activity (pH 4 to 7 and 20 to 50°C). In contrast, UQ14S SI showed optimal activity at pH 6 and 35°C and produced a lower ratio of isomaltulose to trehalulose (<8:1) across its pH and temperature ranges for activity. UQ68J SI had much higher catalytic efficiency; the Km was 39.9 mM, the Vmax was 638 U mg−1, and the Kcat/Km was 1.79 × 104 M−1 s−1, compared to a Km of 76.0 mM, a Vmax of 423 U mg−1, and a Kcat/Km of 0.62 × 104 M−1 s−1 for UQ14S SI. UQ68J SI also showed no apparent reverse reaction producing glucose, fructose, or trehalulose from isomaltulose. These properties of the P. dispersa UQ68J enzyme are exceptional among purified SIs, and they indicate likely differences in the mechanism at the enzyme active site. They may favor the production of isomaltulose as an inhibitor of competing microbes in high-sucrose environments, and they are likely to be highly beneficial for industrial production of isomaltulose. PMID:15746363

  1. Segmental movement: definition of the structural requirements for loop closure in catalysis by triosephosphate isomerase.

    PubMed

    Sampson, N S; Knowles, J R

    1992-09-15

    To determine what drives the closure of the active-site loop in the reaction catalyzed by triosephosphate isomerase, several residues involved in hydrogen bonding between the loop and the bulk of the protein have been altered. It was known from earlier work that the loop serves two functions: to stabilize the reaction intermediate (and the two transition states that flank it) and to prevent the loss of this unstable species into free solution. To discover what elements of the protein are necessary for proper closure of the loop, selective destabilization of the "open" and the "closed" forms of the enzyme with respect to one another has been attempted. The mutant Y164F isomerase has been prepared to evaluate the importance of the structure of the "open" form, and the mutant E129Q, Y208F, and S211A enzymes have allowed investigation of the "closed" form. The integrity of the loop itself has been destabilized by making the T172A isomerase. We have found that only those mutations that destabilize the "closed" form of the enzyme significantly perturb the catalytic properties of the isomerase. The second-order rate constants (kcat/Km) of the S211A and E129Q enzymes are reduced 30-fold, and that of the mutant Y208F enzyme is reduced 2000-fold, from the level of the wild-type enzyme. The dramatic drop in activity of the Y208F enzyme is accompanied by a 200-fold increase in the dissociation constant of the intermediate analogue phosphoglycolohydroxamate. The most important property of the mobile loop of triosephosphate isomerase lies, therefore, in the stability of the system when the active site contains ligand and the loop is closed.

  2. Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

    PubMed Central

    Bettiga, Maurizio; Hahn-Hägerdal, Bärbel; Gorwa-Grauslund, Marie F

    2008-01-01

    Background Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose. Results The specific aerobic arabinose growth rate was identical, 0.03 h-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells)-1 h-1 than the xylose isomerase strain, which only reached 0.03 h-1 and 0.02 g (g cells)-1h-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g-1 compared with 0.18 g g-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g-1 compared with 0.32 g g-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells)-1 h-1 compared with 0.01 g (g cells)-1 h-1 for the xylose reductase

  3. Analysis of the arabinose-5-phosphate isomerase of Bacteroides fragilis provides insight into regulation of single-domain arabinose phosphate isomerases.

    PubMed

    Cech, David; Wang, Pan Fen; Holler, Tod P; Woodard, Ronald W

    2014-08-01

    Arabinose-5-phosphate isomerases (APIs) catalyze the interconversion of d-ribulose-5-phosphate and D-arabinose-5-phosphate, the first step in the biosynthesis of 3-deoxy-D-manno-octulosonic acid (Kdo), an essential component of the lipopolysaccharide in Gram-negative bacteria. Classical APIs, such as Escherichia coli KdsD, contain a sugar isomerase domain and a tandem cystathionine beta-synthase domain. Despite substantial effort, little is known about structure-function relationships in these APIs. We recently reported an API containing only a sugar isomerase domain. This protein, c3406 from E. coli CFT073, has no known physiological function. In this study, we investigated a putative single-domain API from the anaerobic Gram-negative bacterium Bacteroides fragilis. This putative API (UniProt ID Q5LIW1) is the only protein encoded by the B. fragilis genome with significant identity to any known API, suggesting that it is responsible for lipopolysaccharide biosynthesis in B. fragilis. We tested this hypothesis by preparing recombinant Q5LIW1 protein (here referred to by the UniProt ID Q5LIW1), characterizing its API activity in vitro, and demonstrating that the gene encoding Q5LIW1 (GenBank ID YP_209877.1) was able to complement an API-deficient E. coli strain. We demonstrated that Q5LIW1 is inhibited by cytidine 5'-monophospho-3-deoxy-D-manno-2-octulosonic acid, the final product of the Kdo biosynthesis pathway, with a Ki of 1.91 μM. These results support the assertion that Q5LIW1 is the API that supports lipopolysaccharide biosynthesis in B. fragilis and is subject to feedback regulation by CMP-Kdo. The sugar isomerase domain of E. coli KdsD, lacking the two cystathionine beta-synthase domains, demonstrated API activity and was further characterized. These results suggest that Q5LIW1 may be a suitable system to study API structure-function relationships. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  4. Analysis of the Arabinose-5-Phosphate Isomerase of Bacteroides fragilis Provides Insight into Regulation of Single-Domain Arabinose Phosphate Isomerases

    PubMed Central

    Cech, David; Wang, Pan Fen; Holler, Tod P.

    2014-01-01

    Arabinose-5-phosphate isomerases (APIs) catalyze the interconversion of d-ribulose-5-phosphate and d-arabinose-5-phosphate, the first step in the biosynthesis of 3-deoxy-d-manno-octulosonic acid (Kdo), an essential component of the lipopolysaccharide in Gram-negative bacteria. Classical APIs, such as Escherichia coli KdsD, contain a sugar isomerase domain and a tandem cystathionine beta-synthase domain. Despite substantial effort, little is known about structure-function relationships in these APIs. We recently reported an API containing only a sugar isomerase domain. This protein, c3406 from E. coli CFT073, has no known physiological function. In this study, we investigated a putative single-domain API from the anaerobic Gram-negative bacterium Bacteroides fragilis. This putative API (UniProt ID Q5LIW1) is the only protein encoded by the B. fragilis genome with significant identity to any known API, suggesting that it is responsible for lipopolysaccharide biosynthesis in B. fragilis. We tested this hypothesis by preparing recombinant Q5LIW1 protein (here referred to by the UniProt ID Q5LIW1), characterizing its API activity in vitro, and demonstrating that the gene encoding Q5LIW1 (GenBank ID YP_209877.1) was able to complement an API-deficient E. coli strain. We demonstrated that Q5LIW1 is inhibited by cytidine 5′-monophospho-3-deoxy-d-manno-2-octulosonic acid, the final product of the Kdo biosynthesis pathway, with a Ki of 1.91 μM. These results support the assertion that Q5LIW1 is the API that supports lipopolysaccharide biosynthesis in B. fragilis and is subject to feedback regulation by CMP-Kdo. The sugar isomerase domain of E. coli KdsD, lacking the two cystathionine beta-synthase domains, demonstrated API activity and was further characterized. These results suggest that Q5LIW1 may be a suitable system to study API structure-function relationships. PMID:24891442

  5. Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase that increases the production rate of D-tagatose.

    PubMed

    Kim, H-J; Kim, J-H; Oh, H-J; Oh, D-K

    2006-07-01

    Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase used to increase the production rate of D-tagatose. A mutated gene was obtained by an error-prone polymerase chain reaction using L-arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in Escherichia coli. The expressed mutated L-arabinose isomerase exhibited the change of three amino acids (Met322-->Val, Ser393-->Thr, and Val408-->Ala), compared with the wild-type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerization activity at pH 8.0, 65 degrees C, and 1.0 mM Co2+, while the wild-type enzyme had a maximum activity at pH 8.0, 60 degrees C, and 1.0-mM Mn2+. The mutated L-arabinose isomerase exhibited increases in D-galactose isomerization activity, optimum temperature, catalytic efficiency (kcat/Km) for D-galactose, and the production rate of D-tagatose from D-galactose. The mutated L-arabinose isomerase from G. stearothermophilus is valuable for the commercial production of D-tagatose. This work contributes knowledge on the characterization of a mutated L-arabinose isomerase, and allows an increased production rate for D-tagatose from D-galactose using the mutated enzyme.

  6. The Hsp90-binding peptidylprolyl isomerase FKBP52 potentiates glucocorticoid signaling in vivo.

    PubMed

    Riggs, Daniel L; Roberts, Patricia J; Chirillo, Samantha C; Cheung-Flynn, Joyce; Prapapanich, Viravan; Ratajczak, Thomas; Gaber, Richard; Picard, Didier; Smith, David F

    2003-03-03

    Hsp90 is required for the normal activity of steroid receptors, and in steroid receptor complexes it is typically bound to one of the immunophilin-related co-chaperones: the peptidylprolyl isomerases FKBP51, FKBP52 or CyP40, or the protein phosphatase PP5. The physiological roles of the immunophilins in regulating steroid receptor function have not been well defined, and so we examined in vivo the influences of immunophilins on hormone-dependent gene activation in the Saccharomyces cerevisiae model for glucocorticoid receptor (GR) function. FKBP52 selectively potentiates hormone-dependent reporter gene activation by as much as 20-fold at limiting hormone concentrations, and this potentiation is readily blocked by co-expression of the closely related FKBP51. The mechanism for potentiation is an increase in GR hormone-binding affinity that requires both the Hsp90-binding ability and the prolyl isomerase activity of FKBP52.

  7. The Hsp90-binding peptidylprolyl isomerase FKBP52 potentiates glucocorticoid signaling in vivo

    PubMed Central

    Riggs, Daniel L.; Roberts, Patricia J.; Chirillo, Samantha C.; Cheung-Flynn, Joyce; Prapapanich, Viravan; Ratajczak, Thomas; Gaber, Richard; Picard, Didier; Smith, David F.

    2003-01-01

    Hsp90 is required for the normal activity of steroid receptors, and in steroid receptor complexes it is typically bound to one of the immunophilin-related co-chaperones: the peptidylprolyl isomerases FKBP51, FKBP52 or CyP40, or the protein phosphatase PP5. The physiological roles of the immunophilins in regulating steroid receptor function have not been well defined, and so we examined in vivo the influences of immunophilins on hormone-dependent gene activation in the Saccharomyces cerevisiae model for glucocorticoid receptor (GR) function. FKBP52 selectively potentiates hormone-dependent reporter gene activation by as much as 20-fold at limiting hormone concentrations, and this potentiation is readily blocked by co-expression of the closely related FKBP51. The mechanism for potentiation is an increase in GR hormone-binding affinity that requires both the Hsp90-binding ability and the prolyl isomerase activity of FKBP52. PMID:12606580

  8. Expression, purification, crystallization and preliminary X-ray diffraction analysis of Bifidobacterium adolescentis xylose isomerase

    PubMed Central

    dos Reis, Caio Vinicius; Bernardes, Amanda; Polikarpov, Igor

    2013-01-01

    Xylose isomerase (EC 5.3.1.5) is a key enzyme in xylose metabolism which is industrially important for the transformation of glucose and xylose into fructose and xylulose, respectively. The Bifidobacterium adolescentis xylA gene (NC_008618.1) encoding xylose isomerase (XI) was cloned and the enzyme was overexpressed in Escherichia coli. Purified recombinant XI was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 3350 as the precipitating agent. A complete native data set was collected to 1.7 Å resolution using a synchrotron-radiation source. The crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 88.78, b = 123.98, c = 78.63 Å. PMID:23695585

  9. Purification of Δ(5)-3-ketosteroid isomerase from Digitalis lanata.

    PubMed

    Meitinger, Nadine; Geiger, Daniel; Augusto, Thierry W; Maia de Pádua, Rodrigo; Kreis, Wolfgang

    2015-01-01

    The isomerization of 5-pregnene-3,20-dione into 4-pregnene-3,20-dione was investigated to shed further light on cardenolide biosynthesis and to characterize the enzymes involved in cardenolide formation. It was shown that the Δ(5)-3-ketosteroid isomerase of Digitalis lanata, which catalyzes this isomerization, is an individual enzyme and not, as previously thought, associated with Δ(5)-3β-hydroxysteroid dehydrogenase. The enzyme was purified by fractionated ammonium sulfate precipitation, hydrophobic interaction chromatography and gel filtration. The purification protocol resulted in a 68.1-fold enriched specific enzyme activity with a yield of 2.2%. After an additional chromatofocusing step the 3KSI activity appeared as a single protein band at 17kDa in SDS-PAGE. Plant 3KSI displayed similar properties to microbial 3-ketosteroid isomerases.

  10. Bacterial L-arabinose isomerases: industrial application for D-tagatose production.

    PubMed

    Boudebbouze, Samira; Maguin, Emmanuelle; Rhimi, Moez

    2011-12-01

    D-tagatose is a natural monosaccharide with a low caloric value and has an anti-hyperglycemiant effect. This hexose has potential applications both in pharmaceutical and agro-food industries. However, the use of D-tagatose remains limited by its production cost. Many production procedures including chemical and biological processes were developed and patented. The most profitable production way is based on the use of L-arabinose isomerase which allows the manufacture of D-tagatose with an attractive rate. Future developments are focused on the generation of L-arabinose isomerases having biochemical properties satisfying the industrial applications. This report provides a brief review of the most recent patents that have been published relating to this area.

  11. Mutations inducing an active-site aperture in Rhizobium sp. sucrose isomerase confer hydrolytic activity.

    PubMed

    Lipski, Alexandra; Watzlawick, Hildegard; Ravaud, Stéphanie; Robert, Xavier; Rhimi, Moez; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

    2013-02-01

    Sucrose isomerase is an enzyme that catalyzes the production of sucrose isomers of high biotechnological and pharmaceutical interest. Owing to the complexity of the chemical synthesis of these isomers, isomaltulose and trehalulose, enzymatic conversion remains the preferred method for obtaining these products. Depending on the microbial source, the ratio of the sucrose-isomer products varies significantly. In studies aimed at understanding and explaining the underlying molecular mechanisms of these reactions, mutations obtained using a random-mutagenesis approach displayed a major hydrolytic activity. Two of these variants, R284C and F164L, of sucrose isomerase from Rhizobium sp. were therefore crystallized and their crystal structures were determined. The three-dimensional structures of these mutants allowed the identification of the molecular determinants that favour hydrolytic activity compared with transferase activity. Substantial conformational changes resulting in an active-site opening were observed, as were changes in the pattern of water molecules bordering the active-site region.

  12. A Quasi-Laue Neutron Crystallographic Study of D-Xylose Isomerase

    NASA Technical Reports Server (NTRS)

    Meilleur, Flora; Snell, Edward H.; vanderWoerd, Mark; Judge, Russell A.; Myles, Dean A. A.

    2006-01-01

    Hydrogen atom location and hydrogen bonding interaction determination are often critical to explain enzymatic mechanism. Whilst it is difficult to determine the position of hydrogen atoms using X-ray crystallography even with subatomic (less than 1.0 Angstrom) resolution data available, neutron crystallography provides an experimental tool to directly localise hydrogeddeuteriwn atoms in biological macromolecules at resolution of 1.5-2.0 Angstroms. Linearisation and isomerisation of xylose at the active site of D-xylose isomerase rely upon a complex hydrogen transfer. Neutron quasi-Laue data were collected on Streptomyces rubiginosus D-xylose isomerase crystal using the LADI instrument at ILL with the objective to provide insight into the enzymatic mechanism (Myles et al. 1998). The neutron structure unambiguously reveals the protonation state of His 53 in the active site, identifying the model for the enzymatic pathway.

  13. TXNDC5, a newly discovered disulfide isomerase with a key role in cell physiology and pathology.

    PubMed

    Horna-Terrón, Elena; Pradilla-Dieste, Alberto; Sánchez-de-Diego, Cristina; Osada, Jesús

    2014-12-17

    Thioredoxin domain-containing 5 (TXNDC5) is a member of the protein disulfide isomerase family, acting as a chaperone of endoplasmic reticulum under not fully characterized conditions As a result, TXNDC5 interacts with many cell proteins, contributing to their proper folding and correct formation of disulfide bonds through its thioredoxin domains. Moreover, it can also work as an electron transfer reaction, recovering the functional isoform of other protein disulfide isomerases, replacing reduced glutathione in its role. Finally, it also acts as a cellular adapter, interacting with the N-terminal domain of adiponectin receptor. As can be inferred from all these functions, TXNDC5 plays an important role in cell physiology; therefore, dysregulation of its expression is associated with oxidative stress, cell ageing and a large range of pathologies such as arthritis, cancer, diabetes, neurodegenerative diseases, vitiligo and virus infections. Its implication in all these important diseases has made TXNDC5 a susceptible biomarker or even a potential pharmacological target.

  14. Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118.

    PubMed

    Lobley, Carina M C; Aller, Pierre; Douangamath, Alice; Reddivari, Yamini; Bumann, Mario; Bird, Louise E; Nettleship, Joanne E; Brandao-Neto, Jose; Owens, Raymond J; O'Toole, Paul W; Walsh, Martin A

    2012-12-01

    The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Å resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and β D-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography.

  15. Crystallization and preliminary X-ray characterization of phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv

    PubMed Central

    Mathur, Divya; Anand, Kanchan; Mathur, Deepika; Jagadish, Nirmala; Suri, Anil; Garg, Lalit C.

    2007-01-01

    Phosphoglucose isomerase is a ubiquitous enzyme that catalyzes the isomerization of d-glucopyranose-6-phosphate to d-fructofuranose-6-phosphate. The present investigation reports the expression, purification, crystallization and preliminary crystallographic studies of the phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv, which shares 46% sequence identity with that of its human host. The recombinant protein, which was prepared using an Escherichia coli expression system, was crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.8 Å and belonged to the orthorhombic space group I212121, with unit-cell parameters a = 109.0, b = 119.8, c = 138.9 Å. PMID:17401215

  16. A protein disulfide isomerase gene fusion expression system that increases the extracellular productivity of Bacillus brevis.

    PubMed

    Kajino, T; Ohto, C; Muramatsu, M; Obata, S; Udaka, S; Yamada, Y; Takahashi, H

    2000-02-01

    We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.

  17. Catalysis of Protein Folding by Protein Disulfide Isomerase and Small-Molecule Mimics

    PubMed Central

    KERSTEEN, ELIZABETH A.; RAINES, RONALD T.

    2010-01-01

    Protein disulfide isomerase (PDI) catalyzes the formation of native disulfide pairings in secretory proteins. The ability of PDI to act as a disulfide isomerase makes it an essential enzyme in eukaryotes. PDI also fulfills other important roles. Recent studies have emphasized the importance of PDI as an oxidant in the endoplasmic reticulum. Intriguing questions remain regarding how PDI is able to catalyze both isomerization and oxidation in vivo. Studies of PDI and its homologs have led to the development of small-molecule folding catalysts that are able to accelerate disulfide isomerization in vitro and in vivo. PDI will continue to provide both an inspiration for the design of such artificial foldases and a benchmark with which to gauge the success of those designs. Here, we review current understanding of the chemistry and biology of PDI, its homologs, and small molecules that mimic its catalytic activity. PMID:13678529

  18. A Quasi-Laue Neutron Crystallographic Study of D-Xylose Isomerase

    NASA Technical Reports Server (NTRS)

    Meilleur, Flora; Snell, Edward H.; vanderWoerd, Mark; Judge, Russell A.; Myles, Dean A. A.

    2006-01-01

    Hydrogen atom location and hydrogen bonding interaction determination are often critical to explain enzymatic mechanism. Whilst it is difficult to determine the position of hydrogen atoms using X-ray crystallography even with subatomic (less than 1.0 Angstrom) resolution data available, neutron crystallography provides an experimental tool to directly localise hydrogeddeuteriwn atoms in biological macromolecules at resolution of 1.5-2.0 Angstroms. Linearisation and isomerisation of xylose at the active site of D-xylose isomerase rely upon a complex hydrogen transfer. Neutron quasi-Laue data were collected on Streptomyces rubiginosus D-xylose isomerase crystal using the LADI instrument at ILL with the objective to provide insight into the enzymatic mechanism (Myles et al. 1998). The neutron structure unambiguously reveals the protonation state of His 53 in the active site, identifying the model for the enzymatic pathway.

  19. Chalcone Isomerase from Eubacterium ramulus Catalyzes the Ring Contraction of Flavanonols.

    PubMed

    Braune, Annett; Engst, Wolfram; Elsinghorst, Paul W; Furtmann, Norbert; Bajorath, Jürgen; Gütschow, Michael; Blaut, Michael

    2016-11-01

    The enzyme catalyzing the ring-contracting conversion of the flavanonol taxifolin to the auronol alphitonin in the course of flavonoid degradation by the human intestinal anaerobe Eubacterium ramulus was purified and characterized. It stereospecifically catalyzed the isomerization of (+)-taxifolin but not that of (-)-taxifolin. The Km for (+)-taxifolin was 6.4 ± 0.8 μM, and the Vmax was 108 ± 4 μmol min(-1) (mg protein)(-1) The enzyme also isomerized (+)-dihydrokaempferol, another flavanonol, to maesopsin. Inspection of the encoding gene revealed its complete identity to that of the gene encoding chalcone isomerase (CHI) from E. ramulus Based on the reported X-ray crystal structure of CHI (M. Gall et al., Angew Chem Int Ed 53:1439-1442, 2014, http://dx.doi.org/10.1002/anie.201306952), docking experiments suggest the substrate binding mode of flavanonols and their stereospecific conversion. Mutation of the active-site histidine (His33) to alanine led to a complete loss of flavanonol isomerization by CHI, which indicates that His33 is also essential for this activity. His33 is proposed to mediate the stereospecific abstraction of a proton from the hydroxymethylene carbon of the flavanonol C-ring followed by ring opening and recyclization. A flavanonol-isomerizing enzyme was also identified in the flavonoid-converting bacterium Flavonifractor plautii based on its 50% sequence identity to the CHI from E. ramulus IMPORTANCE: Chalcone isomerase was known to be involved in flavone/flavanone conversion by the human intestinal bacterium E. ramulus Here we demonstrate that this enzyme moreover catalyzes a key step in the breakdown of flavonols/flavanonols. Thus, a single isomerase plays a dual role in the bacterial conversion of dietary bioactive flavonoids. The identification of a corresponding enzyme in the human intestinal bacterium F. plautii suggests a more widespread occurrence of this isomerase in flavonoid-degrading bacteria.

  20. BIOPHYSICS. Comment on "Extreme electric fields power catalysis in the active site of ketosteroid isomerase".

    PubMed

    Chen, Deliang; Savidge, Tor

    2015-08-28

    Fried et al. (Reports, 19 December 2014, p. 1510) demonstrate electric field-dependent acceleration of biological catalysis using ketosteroid isomerase as a prototypic example. These findings were not extended to aqueous solution because water by itself has field fluctuations that are too large and fast to provide a catalytic effect. Given physiological context, when water electrostatic interactions are considered, electric fields play a less important role in the catalysis.

  1. BIOPHYSICS. Comment on "Extreme electric fields power catalysis in the active site of ketosteroid isomerase".

    PubMed

    Natarajan, Aditya; Yabukarski, Filip; Lamba, Vandana; Schwans, Jason P; Sunden, Fanny; Herschlag, Daniel

    2015-08-28

    Fried et al. (Reports, 19 December 2014, p. 1510) demonstrated a strong correlation between reaction rate and the carbonyl stretching frequency of a product analog bound to ketosteroid isomerase oxyanion hole mutants and concluded that the active-site electric field provides 70% of catalysis. Alternative comparisons suggest a smaller contribution, relative to the corresponding solution reaction, and highlight the importance of atomic-level descriptions.

  2. Role of isopentenyl-diphosphate isomerase in heterologous cyanobacterial (Synechocystis) isoprene production.

    PubMed

    Chaves, Julie E; Romero, Paloma Rueda; Kirst, Henning; Melis, Anastasios

    2016-12-01

    Heterologous production of isoprene (C5H8) hydrocarbons in cyanobacteria, emanating from sunlight, CO2, and water, is now attracting increasing attention. The concept entails application of an isoprene synthase transgene from terrestrial plants, heterologously expressed in cyanobacteria, aiming to reprogram carbon flux in the terpenoid biosynthetic pathway toward formation and spontaneous release of this volatile chemical from the cell and liquid culture. However, flux manipulations and carbon-partitioning reactions between isoprene (the product) and native terpenoid biosynthesis for cellular needs are not yet optimized for isoprene yield. The primary reactant for isoprene biosynthesis is dimethylallyl diphosphate (DMAPP), whereas both DMAPP and its isopentenyl diphosphate (IPP) isomer are needed for cellular terpenoid biosynthesis. The present work addressed the function of an isopentenyl diphosphate (IPP) isomerase in cyanobacteria and its role in carbon partitioning between IPP and DMAPP, both of which serve, in variable ratios, as reactants for the synthesis of different cellular terpenoids. The work was approached upon the heterologous expression in Synechocystis of the "isopentenyl diphosphate isomerase" gene (FNI) from Streptococcus pneumoniae, using isoprene production as a "reporter process" for substrate partitioning between DMAPP and IPP. It is shown that transgenic expression of the FNI gene in Synechocystis resulted in a 250 % increase in the "reporter isoprene" rate and yield, suggesting that the FNI isomerase shifted the endogenous DMAPP-IPP steady-state pool size toward DMAPP, thereby enhancing rates and yield of isoprene production. The work provides insight into the significance and functional role of the IPP isomerase in these photosynthetic microorganisms.

  3. Synthesis of conjugated linoleic acid by the linoleate isomerase complex in food-derived lactobacilli.

    PubMed

    Yang, B; Chen, H; Gu, Z; Tian, F; Ross, R P; Stanton, C; Chen, Y Q; Chen, W; Zhang, H

    2014-08-01

    To assess strains of lactobacilli for their capacity to produce functional fatty acid-conjugated linoleic acid. To assess the linoleate isomerase for CLA production in the most efficient CLA producer. In this study, strains of food-derived lactobacilli were cultured in media with linoleic acid and CLA production was assessed. Most of the selected strains produced CLA at different levels, with Lactobacillus plantarum ZS2058 being the most efficient CLA producer converting over 50% of linoleic acid to c9, t11-CLA and t9, t11-CLA. Some intermediates 10-hydroxy-cis-12-octadecenoic acid, 10-oxo-cis-12-octadecenoic acid and 10-oxo-trans-11-octadecenoic acid were determined via GC-MS. The genes coding the multicomponent linoleate isomerase containing myosin-cross-reactive antigen, short-chain dehydrogenase/oxidoreductase and acetoacetate decarboxylase for CLA production in Lact. plantarum ZS2058 were cloned and expressed in Escherichia coli. With the mixture of recombinant E. coli, c9, t11-CLA and three kinds of intermediates were produced from linoleic acid, which were in line with those in the lactobacilli. The ability for CLA production by lactobacilli exhibited variation. Lactobacillus plantarum and Lact. bulgaricus were the most efficient producers in the selected strains. Lact. plantarum ZS2058 converted linoleic acid to CLAs with 10-hydroxy-cis-12-octadecenoic acid, 10-oxo-cis-12-octadecenoic acid and 10-oxo-trans-11-octadecenoic acid as intermediates. The multiple-step reactions for CLA production catalysed by multicomponent linoleate isomerase in Lact. plantarum ZS2058 were confirmed successfully. Multicomponent linoleate isomerase provides important results for the illustration of the mechanism for CLA production in lactic acid bacteria. Food-derived lactobacilli with CLA production ability offers novel opportunities for functional foods development. © 2014 The Society for Applied Microbiology.

  4. Synthesis of conjugated linoleic acid by the linoleate isomerase complex in food-derived lactobacilli

    PubMed Central

    Yang, B.; Chen, H.; Gu, Z.; Tian, F.; Ross, R. P.; Stanton, C.; Chen, Y. Q.; Chen, W.; Zhang, H.

    2015-01-01

    Aims To assess strains of lactobacilli for their capacity to produce functional fatty acid-conjugated linoleic acid. To assess the linoleate isomerase for CLA production in the most efficient CLA producer. Methods and Results In this study, strains of food-derived lactobacilli were cultured in media with linoleic acid and CLA production was assessed. Most of the selected strains produced CLA at different levels, with Lactobacillus plantarum ZS2058 being the most efficient CLA producer converting over 50% of linoleic acid to c9, t11-CLA and t9, t11-CLA. Some intermediates 10-hydroxy-cis-12-octadecenoic acid, 10-oxo-cis-12-octadecenoic acid and 10-oxo-trans-11-octadecenoic acid were determined via GC-MS. The genes coding the multicomponent linoleate isomerase containing myosin-cross-reactive antigen, short-chain dehydrogenase/oxidoreductase and acetoacetate decarboxylase for CLA production in Lact. plantarum ZS2058 were cloned and expressed in Escherichia coli. With the mixture of recombinant E. coli, c9, t11-CLA and three kinds of intermediates were produced from linoleic acid, which were in line with those in the lactobacilli. Conclusions The ability for CLA production by lactobacilli exhibited variation. Lactobacillus plantarum and Lact. bulgaricus were the most efficient producers in the selected strains. Lact. plantarum ZS2058 converted linoleic acid to CLAs with 10-hydroxy-cis-12-octadecenoic acid, 10-oxo-cis-12-octadecenoic acid and 10-oxo-trans-11-octadecenoic acid as intermediates. The multiple-step reactions for CLA production catalysed by multicomponent linoleate isomerase in Lact. plantarum ZS2058 were confirmed successfully. Significance and Impact of the study Multicomponent linoleate isomerase provides important results for the illustration of the mechanism for CLA production in lactic acid bacteria. Food-derived lactobacilli with CLA production ability offers novel opportunities for functional foods development. PMID:24750362

  5. Substrate specificity of a recombinant D-lyxose isomerase from Providencia stuartii for monosaccharides.

    PubMed

    Kwon, Hyun-Jung; Yeom, Soo-Jin; Park, Chang-Su; Oh, Deok-Kun

    2010-07-01

    The specific activity and catalytic efficiency (k(cat)/K(m)) of the recombinant putative protein from Providencia stuartii was the highest for D-lyxose among the aldose substrates, indicating that it is a D-lyxose isomerase. Gel filtration analysis suggested that the native enzyme is a dimer with a molecular mass of 44 kDa. The maximal activity for D-lyxose isomerization was observed at pH 7.5 and 45 degrees C in the presence of 1 mM Mn(2+). The enzyme exhibited high isomerization activity for aldose substrates with the C2 and C3 hydroxyl groups in the left-hand configuration, such as D-lyxose, D-mannose, L-ribose, D-talose, and L-allose (listed in decreasing order of activity). The enzyme exhibited the highest activity for D-xylulose among all pentoses and hexoses. Thus, D-lyxose was produced at 288 g/l from 500 g/l D-xylulose by D-lyxose isomerase at pH 7.5 and 45 degrees C for 2 h, with a conversion yield of 58% and a volumetric productivity of 144 g l(-1) h(-1). The observed k(cat)/K(m) (920 mM(-1) s(-1)) of P. stuartiid-lyxose isomerase for D-xylulose is higher than any of the k(cat)/K(m) values previously reported for sugar and sugar phosphate isomerases with monosaccharide substrates. These results suggest that the enzyme will be useful as an industrial producer of D-lyxose.

  6. Structure of the Noncatalytic Domains and Global Fold of the Protein Disulfide Isomerase ERp72

    SciTech Connect

    Kozlov, G.; Määttänen, P; Schrag, J; Hura, G; Gabrielli, L; Cygler, M; Thomas, D; Gehring, K

    2009-01-01

    Protein disulfide isomerases are a family of proteins that catalyze the oxidation and isomerization of disulfide bonds in newly synthesized proteins in the endoplasmic reticulum. The family includes general enzymes such as PDI that recognize unfolded proteins, and others that are selective for specific classes of proteins. Here, we report the X-ray crystal structure of central non-catalytic domains of a specific isomerase, ERp72 (also called CaBP2 and protein disulfide-isomerase A4) from Rattus norvegicus. The structure reveals strong similarity to ERp57, a PDI-family member that interacts with the lectin-like chaperones calnexin and calreticulin but, unexpectedly, ERp72 does not interact with calnexin as shown by isothermal titration calorimetry and nuclear magnetic resonance (NMR) spectroscopy. Small-angle X-ray scattering (SAXS) of ERp72 was used to develop models of the full-length protein using both rigid body refinement and ab initio simulated annealing of dummy atoms. The two methods show excellent agreement and define the relative positions of the five thioredoxin-like domains of ERp72 and potential substrate or chaperone binding sites.

  7. Biochemical characterization of phosphoglucose isomerase and genetic variants from mouse and Drosophila melanogaster.

    PubMed

    Charles, D; Lee, C Y

    1980-01-16

    A simple and unique procedure was developed to purify phosphoglucose isomerase variants from the whole mouse body extracts and Drosophila homogenate. It involved the use of an 8-(6-aminohexyl)-amino-ATP-Sepharose column followed by a preparative isoelectric focusing. In each case, the enzyme in the homogenate was adsorbed by ionic interaction on the ATP-Sepharose column. Substantial purification was achieved by the affinity elution with the substrate-glucose-6-phosphate. Mouse and Drosophila phosphoglucose isomerase as well as the corresponding variants were shown to be dimers of similar molecular weight and to exhibit similar kinetic properties. The isoelectric points for the variants from DBA/2J and C57BL/6J mice were determined to be 8.4 and 8.7 respectively, while they were 6.8 and 6.3 respectively for Drosophila and 4/4 variants. Differential thermal stability was observed for the two mouse variants but not for the Drosophila ones. Amino acid composition analysis was performed for both mouse and Drosophila enzymes. Rabbit antisera for mouse (DBA/2J) and Drosophila (2/2) enzymes were raised. Within each species, complete immunological identity was observed between the variants. The antisera were used to characterize the null mutants of phosphoglucose isomerase identified in the mouse and Drosophila populations. By rocket immunoelectrophoresis, the null allele of the naturally occurring heterozygous null variant of Drosophila was shown to express no cross-reacting materials (CRM).

  8. Distinct sucrose isomerases catalyze trehalulose synthesis in whiteflies, Bemisia argentifolii, and Erwinia rhapontici.

    PubMed

    Salvucci, Michael E

    2003-06-01

    Isomaltulose [alpha-D-glucopyranosyl-(1,6)-D-fructofuranose] and trehalulose [alpha-D-glucopyranosyl-(1,1)-D-fructofuranose] are commercially valuable sucrose-substitutes that are produced in several microorganisms by the palI gene product, a sucrose isomerase. Trehalulose also occurs in the silverleaf whitefly, Bemisia argentifoli, as the major carbohydrate in the insect's honeydew. To determine if the enzyme that synthesizes trehalulose in whiteflies was similar to the well-characterized sucrose isomerase from microbial sources, the properties of the enzymes from whiteflies and the bacterium, Erwinia rhapontici, were compared. Partial purification of both enzymes showed that the enzyme from whiteflies was a 116 kD membrane-associated polypeptide, in contrast to the enzyme from E. rhapontici, which was soluble and 66 kD. The enzyme from E. rhapontici converted sucrose to isomaltulose and trehalulose in a 5:1 ratio, whereas the enzyme from whiteflies produced only trehalulose. Unlike the E. rhapontici enzyme, the whitefly enzyme did not convert isomaltulose to trehalulose, but both enzymes catalyzed the transfer of fructose to trehalulose using sucrose as the glucosyl donor. The results indicate that trehalulose synthase from whiteflies is structurally and functionally distinct from the sucrose isomerases described in bacteria. The whitefly enzyme is the first reported case of an enzyme that converts sucrose to exclusively trehalulose.

  9. Crystal structures of human IPP isomerase: new insights into the catalytic mechanism.

    PubMed

    Zhang, Cheng; Liu, Lin; Xu, Hang; Wei, Zhiyi; Wang, Yanli; Lin, Yajing; Gong, Weimin

    2007-03-09

    Type I isopentenyl diphosphate (IPP): dimethylally diphosphate (DMAPP) isomerase is an essential enzyme in human isoprenoid biosynthetic pathway. It catalyzes isomerization of the carbon-carbon double bonds in IPP and DMAPP, which are the basic building blocks for the subsequent biosynthesis. We have determined two crystal structures of human IPP isomerase I (hIPPI) under different crystallization conditions. High similarity between structures of human and Escherichia coli IPP isomerases proves the conserved catalytic mechanism. Unexpectedly, one of the hIPPI structures contains a natural substrate analog ethanol amine pyrophosphate (EAPP). Based on this structure, a water molecule is proposed to be the direct proton donor for IPP and different conformations of IPP and DMAPP bound in the enzyme are also proposed. In addition, structures of human IPPI show a flexible N-terminal alpha-helix covering the active pocket and blocking the entrance, which is absent in E. coli IPPI. Besides, the active site conformation is not the same in the two hIPPI structures. Such difference leads to a hypothesis that substrate binding induces conformational change in the active site. The inhibition mechanism of high Mn(2+) concentrations is also discussed.

  10. Actinobacteria cyclophilins: phylogenetic relationships and description of new class- and order-specific paralogues.

    PubMed

    Manteca, Angel; Pelaez, Ana I; Zardoya, Rafael; Sanchez, Jesus

    2006-12-01

    Cyclophilins are folding helper enzymes belonging to the class of peptidyl-prolyl cis-trans isomerases (PPIases; EC 5.2.1.8) that catalyze the cis-trans isomerization of peptidyl-prolyl bonds in proteins. They are ubiquitous proteins present in almost all living organisms analyzed to date, with extremely rare exceptions. Few cyclophilins have been described in Actinobacteria, except for three reported in the genus Streptomyces and another one in Mycobacterium tuberculosis. In this study, we performed a complete phylogenetic analysis of all Actinobacteria cyclophilins available in sequence databases and new Streptomyces cyclophilin genes sequenced in our laboratory. Phylogenetic analyses of cyclophilins recovered six highly supported groups of paralogy. Streptomyces appears as the bacteria having the highest cyclophilin diversity, harboring proteins from four groups. The first group was named "A" and is made up of highly conserved cytosolic proteins of approximately 18 kDa present in all Actinobacteria. The second group, "B," includes cytosolic proteins widely distributed throughout the genus Streptomyces and closely related to eukaryotic cyclophilins. The third group, "M" cyclophilins, consists of high molecular mass cyclophilins ( approximately 30 kDa) that contain putative membrane binding domains and would constitute the only membrane cyclophilins described to date in bacteria. The fourth group, named "C" cyclophilins, is made up of proteins of approximately 18 kDa that are orthologous to Gram-negative proteobacteria cyclophilins. Ancestral character reconstruction under parsimony was used to identify shared-derived (and likely functionally important) amino acid residues of each paralogue. Southern and Western blot experiments were performed to determine the taxonomic distribution of the different cyclophilins in Actinobacteria.

  11. Mutations in FKBP14 Cause a Variant of Ehlers-Danlos Syndrome with Progressive Kyphoscoliosis, Myopathy, and Hearing Loss

    PubMed Central

    Baumann, Matthias; Giunta, Cecilia; Krabichler, Birgit; Rüschendorf, Franz; Zoppi, Nicoletta; Colombi, Marina; Bittner, Reginald E.; Quijano-Roy, Susana; Muntoni, Francesco; Cirak, Sebahattin; Schreiber, Gudrun; Zou, Yaqun; Hu, Ying; Romero, Norma Beatriz; Carlier, Robert Yves; Amberger, Albert; Deutschmann, Andrea; Straub, Volker; Rohrbach, Marianne; Steinmann, Beat; Rostásy, Kevin; Karall, Daniela; Bönnemann, Carsten G.; Zschocke, Johannes; Fauth, Christine

    2012-01-01

    We report on an autosomal-recessive variant of Ehlers-Danlos syndrome (EDS) characterized by severe muscle hypotonia at birth, progressive scoliosis, joint hypermobility, hyperelastic skin, myopathy, sensorineural hearing impairment, and normal pyridinoline excretion in urine. Clinically, the disorder shares many features with the kyphoscoliotic type of EDS (EDS VIA) and Ullrich congenital muscular dystrophy. Linkage analysis in a large Tyrolean kindred identified a homozygous frameshift mutation in FKBP14 in two affected individuals. Based on the cardinal clinical characteristics of the disorder, four additional individuals originating from different European countries were identified who carried either homozygous or compound heterozygous mutations in FKBP14. FKBP14 belongs to the family of FK506-binding peptidyl-prolyl cis-trans isomerases (PPIases). ER-resident FKBPs have been suggested to act as folding catalysts by accelerating cis-trans isomerization of peptidyl-prolyl bonds and to act occasionally also as chaperones. We demonstrate that FKBP14 is localized in the endoplasmic reticulum (ER) and that deficiency of FKBP14 leads to enlarged ER cisterns in dermal fibroblasts in vivo. Furthermore, indirect immunofluorescence of FKBP14-deficient fibroblasts indicated an altered assembly of the extracellular matrix in vitro. These findings suggest that a disturbance of protein folding in the ER affecting one or more components of the extracellular matrix might cause the generalized connective tissue involvement in this disorder. FKBP14 mutation analysis should be considered in all individuals with apparent kyphoscoliotic type of EDS and normal urinary pyridinoline excretion, in particular in conjunction with sensorineural hearing impairment. PMID:22265013

  12. Mutations in FKBP14 cause a variant of Ehlers-Danlos syndrome with progressive kyphoscoliosis, myopathy, and hearing loss.

    PubMed

    Baumann, Matthias; Giunta, Cecilia; Krabichler, Birgit; Rüschendorf, Franz; Zoppi, Nicoletta; Colombi, Marina; Bittner, Reginald E; Quijano-Roy, Susana; Muntoni, Francesco; Cirak, Sebahattin; Schreiber, Gudrun; Zou, Yaqun; Hu, Ying; Romero, Norma Beatriz; Carlier, Robert Yves; Amberger, Albert; Deutschmann, Andrea; Straub, Volker; Rohrbach, Marianne; Steinmann, Beat; Rostásy, Kevin; Karall, Daniela; Bönnemann, Carsten G; Zschocke, Johannes; Fauth, Christine

    2012-02-10

    We report on an autosomal-recessive variant of Ehlers-Danlos syndrome (EDS) characterized by severe muscle hypotonia at birth, progressive scoliosis, joint hypermobility, hyperelastic skin, myopathy, sensorineural hearing impairment, and normal pyridinoline excretion in urine. Clinically, the disorder shares many features with the kyphoscoliotic type of EDS (EDS VIA) and Ullrich congenital muscular dystrophy. Linkage analysis in a large Tyrolean kindred identified a homozygous frameshift mutation in FKBP14 in two affected individuals. Based on the cardinal clinical characteristics of the disorder, four additional individuals originating from different European countries were identified who carried either homozygous or compound heterozygous mutations in FKBP14. FKBP14 belongs to the family of FK506-binding peptidyl-prolyl cis-trans isomerases (PPIases). ER-resident FKBPs have been suggested to act as folding catalysts by accelerating cis-trans isomerization of peptidyl-prolyl bonds and to act occasionally also as chaperones. We demonstrate that FKBP14 is localized in the endoplasmic reticulum (ER) and that deficiency of FKBP14 leads to enlarged ER cisterns in dermal fibroblasts in vivo. Furthermore, indirect immunofluorescence of FKBP14-deficient fibroblasts indicated an altered assembly of the extracellular matrix in vitro. These findings suggest that a disturbance of protein folding in the ER affecting one or more components of the extracellular matrix might cause the generalized connective tissue involvement in this disorder. FKBP14 mutation analysis should be considered in all individuals with apparent kyphoscoliotic type of EDS and normal urinary pyridinoline excretion, in particular in conjunction with sensorineural hearing impairment.

  13. Irreversible inhibition of delta 5-3-oxosteroid isomerase by 2-substituted progesterones.

    PubMed Central

    Penning, T M

    1985-01-01

    2 alpha-Cyanoprogesterone (I) and 2-hydroxymethyleneprogesterone (II) were synthesized and screened as irreversible active-site-directed inhibitors of the delta 5-3-oxosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni. Both compounds were found to inhibit the purified bacterial enzyme in a time-dependent manner. In either case the inactivated enzyme could be dialysed without return of activity, indicating that a stable covalent bond had formed between the inhibitor and the enzyme. Inactivation mediated by compounds (I) and (II) followed pseudo-first-order kinetics, and at higher inhibitor concentrations saturation was observed. The competitive inhibitor 17 beta-oestradiol offered protection against the inactivation mediated by both compounds, and initial-rate studies indicated that compounds (I) and (II) can also act as competitive inhibitors yielding Ki values identical with those generated during inactivation experiments. 2 alpha-Cyanoprogesterone (I) and 2-hydroxymethyleneprogesterone (II) thus appear to be active-site-directed. To compare the reactivity of these 2-substituted progesterones with other irreversible inhibitors of the isomerase, 3 beta-spiro-oxiranyl-5 alpha-pregnan-20 beta-ol (III) was synthesized as the C21 analogue of 3 beta-spiro-oxiranyl-5 alpha-androstan-17 beta-ol, which is a potent inactivator of the isomerase [Pollack, Kayser & Bevins (1979) Biochem. Biophys. Res. Commun. 91, 783-790]. Comparison of the bimolecular rate constants for inactivation (k+3/Ki) mediated by compounds (I)-(III) indicated the following order of reactivity: (III) greater than (II) greater than (I). 2-Mercaptoethanol offers complete protection against the inactivation of the isomerase mediated by 2 alpha-cyanoprogesterone (I). Under the conditions of inactivation compound (I) appears to be completely stable, and no evidence could be obtained for enolate ion formation in the presence or absence of enzyme. It is suggested that cyanoprogesterone inactivates

  14. The secreted L-arabinose isomerase displays anti-hyperglycemic effects in mice.

    PubMed

    Rhimi, Moez; Bermudez-Humaran, Luis G; Huang, Yuan; Boudebbouze, Samira; Gaci, Nadia; Garnier, Alexandrine; Gratadoux, Jean-Jacques; Mkaouar, Héla; Langella, Philippe; Maguin, Emmanuelle

    2015-12-21

    The L-arabinose isomerase is an intracellular enzyme which converts L-arabinose into L-ribulose in living systems and D-galactose into D-tagatose in industrial processes and at industrial scales. D-tagatose is a natural ketohexose with potential uses in pharmaceutical and food industries. The D-galactose isomerization reaction is thermodynamically equilibrated, and leads to secondary subproducts at high pH. Therefore, an attractive L-arabinose isomerase should be thermoactive and acidotolerant with high catalytic efficiency. While many reports focused on the set out of a low cost process for the industrial production of D-tagatose, these procedures remain costly. When compared to intracellular enzymes, the production of extracellular ones constitutes an interesting strategy to increase the suitability of the biocatalysts. The L-arabinose isomerase (L-AI) from Lactobacillus sakei was expressed in Lactococcus lactis in fusion with the signal peptide of usp45 (SP(Usp45)). The L-AI protein and activity were detected only in the supernatant of the induced cultures of the recombinant L. lactis demonstrating the secretion in the medium of the intracellular L. sakei L-AI in an active form. Moreover, we showed an improvement in the enzyme secretion using either (1) L. lactis strains deficient for their two major proteases, ClpP and HtrA, or (2) an enhancer of protein secretion in L. lactis fused to the recombinant L-AI with the SP(Usp45). Th L-AI enzyme secreted by the recombinant L. lactis strains or produced intracellularly in E. coli, showed the same functional properties than the native enzyme. Furthermore, when mice are fed with the L. lactis strain secreting the L-AI and galactose, tagatose was produced in vivo and reduced the glycemia index. We report for the first time the secretion of the intracellular L-arabinose isomerase in the supernatant of food grade L. lactis cultures with hardly display other secreted proteins. The secreted L-AI originated from the food

  15. Triosephosphate isomerase: removal of a putatively electrophilic histidine residue results in a subtle change in catalytic mechanism

    SciTech Connect

    Nickbarg, E.B.; Davenport, R.C.; Petsko, G.A.; Knowles, J.R.

    1988-08-09

    An important active-site residue in the glycolytic enzyme triosephosphate isomerase is His-95, which appears to act as an electrophilic component in catalyzing the enolization of the substrates. With the techniques of site-directed mutagenesis, His-95 has been replaced by Gln in the isomerase from Saccharomyces cerevisiae. The mutant isomerase has been expressed in Escherichia coli strain DF502 and purified to homogeneity. The specific catalytic activity of the mutant enzyme is less than that of wild type by a factor of nearly 400. The mutant enzyme can be resolved from the wild-type isomerase on nondenaturing isoelectric focusing gels, and an isomerase activity stain shows that the observed catalytic activity indeed derives from the mutant protein. The mutant enzyme shows the same stereospecificity of proton transfer as the wild type. Tritium exchange experiments similar to those used to define the free energy profile for the wild-type yeast isomerase, together with a new method of analysis involving /sup 14/C and /sup 3/H doubly labeled substrates, have been used to investigate the energetics of the mutant enzyme catalyzed reaction. The deuterium kinetic isotope effects observed with the mutant isomerase using (1(R)-/sup 2/H)dihydroxyacetone phosphate and (2-/sup 2/H)glyceraldehyde 3-phosphate are 2.15 +/- 0.04 and 2.4 +/- 0.1, respectively. These results lead to the conclusion that substitution of Gln for His-95 so impairs the ability of the enzyme to stabilize the reaction intermediate that there is a change in the pathways of proton transfer mediated by the mutant enzyme.

  16. Bioproduction of D-Tagatose from D-Galactose Using Phosphoglucose Isomerase from Pseudomonas aeruginosa PAO1.

    PubMed

    Patel, Manisha J; Patel, Arti T; Akhani, Rekha; Dedania, Samir; Patel, Darshan H

    2016-07-01

    Pseudomonas aeruginosa PAO1 phosphoglucose isomerase was purified as an active soluble form by a single-step purification using Ni-NTA chromatography that showed homogeneity on SDS-PAGE with molecular mass ∼62 kDa. The optimum temperature and pH for the maximum isomerization activity with D-galactose were 60 °C and 7.0, respectively. Generally, sugar phosphate isomerases show metal-independent activity but PA-PGI exhibited metal-dependent isomerization activity with aldosugars and optimally catalyzed the D-galactose isomerization in the presence of 1.0 mM MnCl2. The apparent Km and Vmax for D-galactose under standardized conditions were calculated to be 1029 mM (±31.30 with S.E.) and 5.95 U/mg (±0.9 with S.E.), respectively. Equilibrium reached after 180 min with production of 567.51 μM D-tagatose from 1000 mM of D-galactose. Though, the bioconversion ratio is low but it can be increased by immobilization and enzyme engineering. Although various L-arabinose isomerases have been characterized for bioproduction of D-tagatose, P. aeruginosa glucose phosphate isomerase is distinguished from the other L-arabinose isomerases by its optimal temperature (60 °C) for D-tagatose production being mesophilic bacteria, making it an alternate choice for bulk production.

  17. Xylose isomerase from polycentric fungus Orpinomyces: gene sequencing, cloning, and expression in Saccharomyces cerevisiae for bioconversion of xylose to ethanol.

    PubMed

    Madhavan, Anjali; Tamalampudi, Sriappareddy; Ushida, Kazunari; Kanai, Daisuke; Katahira, Satoshi; Srivastava, Aradhana; Fukuda, Hideki; Bisaria, Virendra S; Kondo, Akihiko

    2009-04-01

    The cDNA sequence of the gene for xylose isomerase from the rumen fungus Orpinomyces was elucidated by rapid amplification of cDNA ends. The 1,314-nucleotide gene was cloned and expressed constitutively in Saccharomyces cerevisiae. The deduced polypeptide sequence encoded a protein of 437 amino acids which showed the highest similarity to the family II xylose isomerases. Further, characterization revealed that the recombinant enzyme was a homodimer with a subunit of molecular mass 49 kDa. Cell extract of the recombinant strain exhibited high specific xylose isomerase activity. The pH optimum of the enzyme was 7.5, while the low temperature optimum at 37 degrees C was the property that differed significantly from the majority of the reported thermophilic xylose isomerases. In addition to the xylose isomerase gene, the overexpression of the S. cerevisiae endogenous xylulokinase gene and the Pichia stipitis SUT1 gene for sugar transporter in the recombinant yeast facilitated the efficient production of ethanol from xylose.

  18. Characterization of a F280N variant of L-arabinose isomerase from Geobacillus thermodenitrificans identified as a D-galactose isomerase.

    PubMed

    Kim, Baek-Joong; Hong, Seung-Hye; Shin, Kyung-Chul; Jo, Ye-Seul; Oh, Deok-Kun

    2014-11-01

    The double-site variant (C450S-N475K) L-arabinose isomerase (L-AI) from Geobacillus thermodenitrificans catalyzes the isomerization of D-galactose to D-tagatose, a functional sweetener. Using a substrate-docking homology model, the residues near to D-galactose O6 were identified as Met186, Phe280, and Ile371. Several variants obtained by site-directed mutagenesis of these three residues were analyzed, and a triple-site (F280N) variant enzyme exhibited the highest activity for D-galactose isomerization. The k cat/K m of the triple-site variant enzyme for D-galactose was 2.1-fold higher than for L-arabinose, whereas the k cat/K m of the double-site variant enzyme for L-arabinose was 43.9-fold higher than for D-galactose. These results suggest that the triple-site variant enzyme is a D-galactose isomerase. The conversion rate of D-galactose to D-tagatose by the triple-site variant enzyme was approximately 3-fold higher than that of the double-site variant enzyme for 30 min. However, the conversion yields of L-arabinose to L-ribulose by the triple-site and double-site variant enzymes were 10.6 and 16.0 % after 20 min, respectively. The triple-site variant enzyme exhibited increased specific activity, turnover number, catalytic efficiency, and conversion rate for D-galactose isomerization compared to the double-site variant enzyme. Therefore, the amino acid at position 280 determines the substrate specificity for D-galactose and L-arabinose, and the triple-site variant enzyme has the potential to produce D-tagatose on an industrial scale.

  19. Truncation of a Protein Disulfide Isomerase, PDIL2-1, Delays Embryo Sac Maturation and Disrupts Pollen Tube Guidance in Arabidopsis thaliana

    USDA-ARS?s Scientific Manuscript database

    Pollen tubes navigate through different female tissues and deliver the sperm to the embryo sac for fertilization. Protein disulfide isomerases play important roles in the maturation of secreted or plasma membrane proteins. Here we show that truncated versions of a protein disulfide isomerase (PDI), ...

  20. Structural characterization and comparison of the large subunits of IPM isomerase and homoaconitase from Methanococcus jannaschii.

    PubMed

    Lee, Eun Hye; Lee, Kitaik; Hwang, Kwang Yeon

    2014-04-01

    The aconitase family of proteins includes three classes of hydro-lyase enzymes: aconitases, homoaconitases and isopropylmalate (IPM) isomerases. They have a common Fe-S cluster-binding site and catalyze the isomerization of specific substrates by sequential dehydration and hydration. The archaeon Methanococcus jannaschii contains two aconitase family proteins, IPM isomerase and homoaconitase, which have 50% sequence identity. These two enzymes are heterodimeric proteins composed of large and small subunits encoded by separate genes. Although structures have been reported for the small subunits of the two enzymes, the first structures of oxidized and reduced forms of the large subunit of IPM isomerase (ox-MJ0499 and red-MJ0499, respectively) from M. jannaschii are reported here at 1.8 and 2.7 Å resolution, respectively, together with the structure of the large subunit of homoaconitase (MJ1003) at 2.5 Å resolution. The structures of both proteins have unbound Fe-S clusters and contain a fourth cysteine in the active site. The active site of MJ1003 is homologous to that of aconitase, whereas MJ0499 has significant structural distortion at the active site compared with aconitase. In addition, significant large conformational changes were observed in the active site of red-MJ0499 when compared with ox-MJ0499. The active sites of the two proteins adopt two different states before changing to the Fe-S cluster-bound `activated' state observed in aconitase. MJ1003 has an `open' active site, which forms an active pocket for the cluster, while ox-MJ0499 has a `closed' active site, with four cysteines in disulfide bonds. These data will be helpful in understanding the biochemical mechanism of clustering of the Fe-S protein family.

  1. Studies on linoleic acid 8R-dioxygenase and hydroperoxide isomerase of the fungus Gaeumannomyces graminis.

    PubMed

    Su, C; Brodowsky, I D; Oliw, E H

    1995-01-01

    Linoleic acid is sequentially converted to 7S,8S-dihydroxy-9Z,12Z-octadecadienoic acid by the 8R-dioxygenase and hydroperoxide isomerase of the fungus Gaeumannomyces graminis, which is a common pathogen of wheat. The objective of this study was to separate and characterize the two enzyme activities. The isomerase activity was found mainly in the microsomal fraction of the mycelia and the 8R-dioxygenase in the cytosol. The 8R-dioxygenase could be partially purified by ammonium sulfate precipitation, gel filtration, ion exchange chromatography or isoelectric focusing. The 8R-dioxygenase was unstable during purification, but it could be stabilized by glutathione, glutathione peroxidase and ethylenediaminetetraacetic acid. Several protease inhibitors reduced the enzyme activity. Gel filtration with Sephacryl S-300 showed that most 8R-dioxygenase activity was eluted with the front with little retention. Isoelectric focusing in the presence of ethylene glycol (20%) indicated an isoelectric point of pl 6.1-6.3. The enzyme was retained on strong anion exchange columns at pH 7.4 and could be eluted with 0.3-0.5 M NaCl. Incubation of the enzyme with 0.1 mM linoleic acid led to partial inactivation, which may indicate product inhibition. Paracetamol and the lipoxygenase inhibitor ICI 230,487 at 30 microM inhibited the 8R-dioxygenase by 44 and 58%, respectively. 8R-hydroperoxy-9Z,12Z-octadecadienoic acid was isolated from incubations of linoleic acid with the partially purified enzyme or with the cytosol in the presence of p-hydroxymercuribenzoate. The hydroperoxide was rapidly converted by the hydroperoxide isomerase in the microsomal fractions to 7S,8S-dihydroxy-9Z,12Z-octadecadienoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Inhibition of RPE65 Retinol Isomerase Activity by Inhibitors of Lipid Metabolism*

    PubMed Central

    Eroglu, Abdulkerim; Gentleman, Susan; Poliakov, Eugenia; Redmond, T. Michael

    2016-01-01

    RPE65 is the isomerase catalyzing conversion of all-trans-retinyl ester (atRE) into 11-cis-retinol in the retinal visual cycle. Crystal structures of RPE65 and site-directed mutagenesis reveal aspects of its catalytic mechanism, especially retinyl moiety isomerization, but other aspects remain to be determined. To investigate potential interactions between RPE65 and lipid metabolism enzymes, HEK293-F cells were transfected with expression vectors for visual cycle proteins and co-transfected with either fatty acyl:CoA ligases (ACSLs) 1, 3, or 6 or the SLC27A family fatty acyl-CoA synthase FATP2/SLCA27A2 to test their effect on isomerase activity. These experiments showed that RPE65 activity was reduced by co-expression of ACSLs or FATP2. Surprisingly, however, in attempting to relieve the ACSL-mediated inhibition, we discovered that triacsin C, an inhibitor of ACSLs, also potently inhibited RPE65 isomerase activity in cellulo. We found triacsin C to be a competitive inhibitor of RPE65 (IC50 = 500 nm). We confirmed that triacsin C competes directly with atRE by incubating membranes prepared from chicken RPE65-transfected cells with liposomes containing 0–1 μm atRE. Other inhibitors of ACSLs had modest inhibitory effects compared with triascin C. In conclusion, we have identified an inhibitor of ACSLs as a potent inhibitor of RPE65 that competes with the atRE substrate of RPE65 for binding. Triacsin C, with an alkenyl chain resembling but not identical to either acyl or retinyl chains, may compete with binding of the acyl moiety of atRE via the alkenyl moiety. Its inhibitory effect, however, may reside in its nitrosohydrazone/triazene moiety. PMID:26719343

  3. Glucose(xylose) isomerase production by Streptomyces sp. CH7 grown on agricultural residues.

    PubMed

    Chanitnun, Kankiya; Pinphanichakarn, Pairoh

    2012-07-01

    Streptomyces sp. CH7 was found to efficiently produce glucose(xylose) isomerase when grown on either xylan or agricultural residues. This strain produced a glucose(xylose) isomerase activity of roughly 1.8 U/mg of protein when it was grown in medium containing 1% xylose as a carbon source. Maximal enzymatic activities of about 5 and 3 U/mg were obtained when 1% xylan and 2.5% corn husks were used, respectively. The enzyme was purified from a mycelial extract to 16-fold purity with only two consecutive column chromatography steps using Macro-prep DEAE and Sephacryl-300, respectively. The approximate molecular weight of the purified enzyme is 170 kDa, and it has four identical subunits of 43.6 kDa as estimated by SDS-PAGE. Its K m values for glucose and xylose were found to be 258.96 and 82.77 mM, respectively, and its V max values are 32.42 and 63.64 μM/min/mg, respectively. The purified enzyme is optimally active at 85°C and pH 7.0. It is stable at pH 5.5-8.5 and at temperatures up to 60°C after 30 min. These findings indicate that glucose(xylose) isomerase from Streptomyces sp. CH7 has the potential for industrial applications, especially for high-fructose syrup production and bioethanol fermentation from hemicellulosic hydrolysates by Saccharomyces cerevisiae.

  4. Triosephosphate isomerase: energetics of the reaction catalyzed by the yeast enzyme expressed in Escherichia coli

    SciTech Connect

    Nickbarg, E.B.; Knowles, J.R.

    1988-08-09

    Triosephosphate isomerase from bakers' yeast, expressed in Escherichia coli strain DF502(p12), has been purified to homogeneity. The kinetics of the reaction in each direction have been determined at pH 7.5 and 30 degrees C. Deuterium substitution at the C-2 position of substrate (R)-glyceraldehyde phosphate and at the 1-pro-R position of substrate dihydroxyacetone phosphate results in kinetic isotope effects on kcat of 1.6 and 3.4, respectively. The extent of transfer of tritium from (1(R)-TH)dihydroxyacetone phosphate to product (R)-glyceraldehyde phosphate during the catalyzed reaction is only 3% after 66% conversion to product, indicating that the enzymic base that mediates proton transfer is in rapid exchange with solvent protons. When the isomerase-catalyzed reaction is run in tritiated water in each direction, radioactivity is incorporated both into the remaining substrate and into the product. In the exchange-conversion experiment with dihydroxyacetone phosphate as substrate, the specific radioactivity of remaining dihydroxyacetone phosphate rises as a function of the extent of reaction with a slope of about 0.3, while the specific radioactivity of the products is 54% that of the solvent. In the reverse direction with (R)-glyceraldehyde phosphate as substrate, the specific radioactivity of the product formed is only 11% that of the solvent, while the radioactivity incorporated into the remaining substrate (R)-glyceraldehyde phosphate also rises as a function of the extent of reaction with a slope of 0.3. These results have been analyzed according to the protocol described earlier to yield the free energy profile of the reaction catalyzed by the yeast isomerase.

  5. Overexpression, purification, crystallization and preliminary X-ray crystal analysis of Bacillus pallidus d-arabinose isomerase

    PubMed Central

    Takeda, Kosei; Yoshida, Hiromi; Takada, Goro; Izumori, Ken; Kamitori, Shigehiro

    2008-01-01

    d-Arabinose isomerase catalyzes the isomerization of d-arabinose to d-ribulose. Bacillus pallidus d-arabinose isomerase has broad substrate specificity and can catalyze the isomerization of d-arabinose, l-fucose, l-xylose, l-galactose and d-­altrose. Recombinant B. pallidus d-arabinose isomerase was overexpressed, purified and crystallized. A crystal of the enzyme was obtained by the sitting-drop method at room temperature and belonged to the orthorhombic space group P21212, with unit-cell parameters a = 144.9, b = 127.9, c = 109.5 Å. Diffraction data were collected to 2.3 Å resolution. PMID:18931442

  6. Crystal structure and putative mechanism of 3-methylitaconate-delta-isomerase from Eubacterium barkeri.

    PubMed

    Velarde, Milko; Macieira, Sofia; Hilberg, Markus; Bröker, Gerd; Tu, Shang-Min; Golding, Bernard T; Pierik, Antonio J; Buckel, Wolfgang; Messerschmidt, Albrecht

    2009-08-21

    3-Methylitaconate-Delta-isomerase (Mii) participates in the nicotinate fermentation pathway of the anaerobic soil bacterium Eubacterium barkeri (order Clostridiales) by catalyzing the reversible conversion of (R)-3-methylitaconate (2-methylene-3-methylsuccinate) to 2,3-dimethylmaleate. The enzyme is also able to catalyze the isomerization of itaconate (methylenesuccinate) to citraconate (methylmaleate) with ca 10-fold higher K(m) but > 1000-fold lower k(cat). The gene mii from E. barkeri was cloned and expressed in Escherichia coli. The protein produced with a C-terminal Strep-tag exhibited the same specific activity as the wild-type enzyme. The crystal structure of Mii from E. barkeri has been solved at a resolution of 2.70 A. The asymmetric unit of the P2(1)2(1)2(1) unit cell with parameters a = 53.1 A, b = 142.3 A, and c = 228.4 A contains four molecules of Mii. The enzyme belongs to a group of isomerases with a common structural feature, the so-called diaminopimelate epimerase fold. The monomer of 380 amino acid residues has two topologically similar domains exhibiting an alpha/beta-fold. The active site is situated in a cleft between these domains. The four Mii molecules are arranged as a tetramer with 222 symmetry for the N-terminal domains. The C-terminal domains have different relative positions with respect to the N-terminal domains resulting in a closed conformation for molecule A and two distinct open conformations for molecules B and D. The C-terminal domain of molecule C is disordered. The Mii active site contains the putative catalytic residues Lys62 and Cys96, for which mechanistic roles are proposed based on a docking experiment of the Mii substrate complex. The active sites of Mii and the closely related PrpF, most likely a methylaconitate Delta-isomerase, have been compared. The overall architecture including the active-site Lys62, Cys96, His300, and Ser17 (Mii numbering) is similar. This positioning of (R)-3-methylitaconate allows Cys96 (as

  7. Characterization of an L-arabinose isomerase from Bacillus thermoglucosidasius for D-tagatose production.

    PubMed

    Seo, Myung-Ji

    2013-01-01

    L-Arabinose isomerase from Bacillus thermoglucosidasius KCTC 1828 (BTAI) was expressed in Escherichia coli. The optimal temperature and pH for the activity of the purified BTAI were 40 °C and pH 7.0. The Mn(2+) ion was an activator of BTAI activity. The kinetic parameters of BTAI for D-galactose were a K(m) of 175 mM and a k(cat)/K(m) of 2.8 mM(-1)min(-1). The conversion ratio by BTAI to D-tagatose reached 45.6% at 40 °C.

  8. Crystal Structure and Substrate Specificity of D-Galactose-6-Phosphate Isomerase Complexed with Substrates

    PubMed Central

    Lee, Jung-Kul; Pan, Cheol-Ho

    2013-01-01

    D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26), which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD), catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi). Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays. PMID:24015281

  9. Inhibition of hexose transport by glucose in a glucose-6-phosphate isomerase mutant of Saccharomyces cerevisiae.

    PubMed

    Alonso, A; Pascual, C; Romay, C; Herrera, L; Kotyk, A

    1989-01-01

    The rate of hexose transport was approximately 60% lower for both the high- and the low-affinity components of hexose uptake when a glucose-6-phosphate isomerase mutant of Saccharomyces cerevisiae was preincubated with glucose, as compared with preincubation with water. Similarly the Jmax value of the high-affinity system of the mutant was 25-35% of the corresponding Jmax value for normal cells incubated with glucose. Accumulation of glucose 6-phosphate or of some other metabolite, such as fructose 6-phosphate or trehalose, may be responsible for this striking inhibition.

  10. A coleopteran triosephosphate isomerase: X-ray structure and phylogenetic impact of insect sequences.

    PubMed

    Knobeloch, D; Schmidt, A; Scheerer, P; Krauss, N; Wessner, H; Scholz, Ch; Küttner, G; von Rintelen, T; Wessel, A; Höhne, W

    2010-02-01

    A coleopteran triosephosphate isomerase (TIM) from Tenebrio molitor (yellow mealworm beetle) was recombinantly expressed in Escherichia coli and characterized with respect to thermal stability, kinetic parameters and oligomeric state. The enzyme was successfully crystallized and the structure determined by X-ray analysis to 2.0 A resolution. This is the first example of an invertebrate TIM. We compare structural features with known structures of TIMs from microorganisms, plants and vertebrates, and discuss the utility of the Tenebrio TIM sequence, together with several newly sequenced insect TIMs, for molecular phylogenetic analysis.

  11. Chemical modification of chalcone isomerase by mercurials and tetrathionate. Evidence for a single cysteine residue in the active site

    SciTech Connect

    Bednar, R.A.; Fried, W.B.; Lock, Y.W.; Pramanik, B. )

    1989-08-25

    Chalcone isomerase from soybean is inactivated by stoichiometric amounts of p-mercuribenzoate or HgCl{sub 2}. Spectral titration of the enzyme with p-mercuribenzoate indicates that a single thiol group is modified. Treatment of modified enzyme with KCN or thiols results in a complete restoration of enzyme activity demonstrating that the inactivation is not due to irreversible protein denaturation. A product of the enzymatic reaction, naringenin, provides complete kinetic protection against inactivation by both mercurials. The binding constant (33 microM) for naringenin determined from the concentration dependence of the protection agrees with the inhibition constant (34 microM) for naringenin as a competitive inhibitor of the catalytic reaction. This agreement demonstrates that the observed kinetic protection results from the specific binding of naringenin to the active site. Incubation of native chalcone isomerase with sodium tetrathionate (0.1 M) results in a slow time-dependent loss of enzymatic activity. The inactivation of chalcone isomerase by tetrathionate and N-ethylmaleimide becomes very rapid in the presence of 6 M urea, indicating that the native tertiary structure is responsible for the low reactivity of the enzymatic thiol. The stoichiometric modification of reduced and denatured chalcone isomerase by ({sup 3}H) N-ethylmaleimide indicates that the enzyme contains only a single cysteine residue and does not contain any disulfides. The evidence presented suggests that the only half-cystine residue in chalcone isomerase is located in the active site and thereby provides the first clue to the location of the active site in chalcone isomerase.

  12. An unusual isopentenyl diphosphate isomerase found in the mevalonate pathway gene cluster from Streptomyces sp. strain CL190

    PubMed Central

    Kaneda, Kazuhide; Kuzuyama, Tomohisa; Takagi, Motoki; Hayakawa, Yoichi; Seto, Haruo

    2001-01-01

    A gene cluster encoding five enzymes of the mevalonate pathway had been cloned from Streptomyces sp. strain CL190. This gene cluster contained an additional ORF, orfD, encoding an unknown protein that was detected in some archaebacteria and some Gram-positive bacteria including Staphylococcus aureus. The recombinant product of orfD was purified as a soluble protein and characterized. The molecular mass of the enzyme was estimated to be 37 kDa by SDS-polyacrylamide gel electrophoresis and 155 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a tetramer. The purified enzyme contained flavin mononucleotide (FMN) with the amount per tetramer being 1.4 to 1.6 mol/mol. The enzyme catalyzed the isomerization of isopentenyl diphosphate (IPP) to produce dimethylallyl diphosphate (DMAPP) in the presence of both FMN and NADPH. The Escherichia coli plasmid expressing orfD could complement the disrupted IPP isomerase gene in E. coli. These results indicate that orfD encodes an unusual IPP isomerase showing no sequence similarity to those of IPP isomerases identified to date. Based on the difference in enzymatic properties, we classify the IPP isomerases into two types: Type 2 for FMN- and NAD(P)H-dependent enzymes, and type 1 for the others. In view of the critical role of this isomerase in S. aureus and of the different enzymatic properties of mammalian (type 1) and S. aureus (type 2) isomerases, this unusual enzyme is considered to be a suitable molecular target for the screening of antibacterial drugs specific to S. aureus. PMID:11158573

  13. Steroidomimetic aminomethyl spiroacetals as novel inhibitors of the enzyme Δ8,7-sterol isomerase in cholesterol biosynthesis.

    PubMed

    Krojer, Melanie; Müller, Christoph; Bracher, Franz

    2014-02-01

    Grundmann's ketone is converted to a spiroacetal containing a 5-hydroxymethyl-5-nitro-1,3-dioxane moiety whose hydroxymethyl group can be esterified or directly substituted with primary and secondary amines. Among the resulting aminomethyl spiroacetals, several ones bearing diamino residues were found to be inhibitors of the enzyme Δ8,7-isomerase in cholesterol biosynthesis. The complex bicyclic building block derived from Grundmann's ketone could be replaced by a properly substituted tetraline scaffold, without noteworthy loss in activity. This opens the opportunity to perform further structural modifications for the design of new steroidomimetic inhibitors of human Δ8,7-isomerase.

  14. Protein disulfide isomerases are antibody targets during immune-mediated tumor destruction

    PubMed Central

    Fonseca, Catia; Soiffer, Robert; Ho, Vincent; Vanneman, Matthew; Jinushi, Masahisa; Ritz, Jerome; Neuberg, Donna; Stone, Richard; DeAngelo, Dan

    2009-01-01

    The identification of cancer antigens that contribute to transformation and are linked with immune-mediated tumor destruction is an important goal for immunotherapy. Toward this end, we screened a murine renal cell carcinoma cDNA expression library with sera from mice vaccinated with irradiated tumor cells engineered to secrete granulocyte macrophage colony-stimulating factor (GM-CSF). Multiple nonmutated, overexpressed proteins that function in tumor cell migration, protein/nucleic acid homeostasis, metabolism, and stress responses were detected. Among these, the most frequently recognized clone was protein disulfide isomerase (PDI). High titer antibodies to human PDI were similarly induced in an acute myeloid leukemia patient who achieved a complete response after vac-cination with irradiated, autologous GM-CSF–secreting tumor cells in the setting of nonmyeloablative allogeneic bone marrow transplantation. Moreover, ERp5, a closely related disulfide isomerase involved in major histocompatibility complex (MHC) class I chain-related protein A (MICA) shedding, also evoked potent humoral reactions in diverse solid and hematologic malignancy patients who responded to GM-CSF–secreting tumor cell vaccines or antibody blockade of cytotoxic T lymphocyte–associated antigen 4 (CTLA-4). Together, these findings reveal the unexpected immunogenicity of PDIs and raise the possibility that these gene products might serve as targets for therapeutic monoclonal antibodies. PMID:19008459

  15. Structural insights from a novel invertebrate triosephosphate isomerase from Litopenaeus vannamei.

    PubMed

    Lopez-Zavala, Alonso A; Carrasco-Miranda, Jesus S; Ramirez-Aguirre, Claudia D; López-Hidalgo, Marisol; Benitez-Cardoza, Claudia G; Ochoa-Leyva, Adrian; Cardona-Felix, Cesar S; Diaz-Quezada, Corina; Rudiño-Piñera, Enrique; Sotelo-Mundo, Rogerio R; Brieba, Luis G

    2016-12-01

    Triosephosphate isomerase (TIM; EC 5.3.1.1) is a key enzyme involved in glycolysis and gluconeogenesis. Glycolysis is one of the most regulated metabolic pathways, however little is known about the structural mechanisms for its regulation in non-model organisms, like crustaceans. To understand the structure and function of this enzyme in invertebrates, we obtained the crystal structure of triosephosphate isomerase from the marine Pacific whiteleg shrimp (Litopenaeus vannamei, LvTIM) in complex with its inhibitor 2-phosphogyceric acid (2-PG) at 1.7Å resolution. LvTIM assembles as a homodimer with residues 166-176 covering the active site and residue Glu166 interacting with the inhibitor. We found that LvTIM is the least stable TIM characterized to date, with the lowest range of melting temperatures, and with the lowest activation enthalpy associated with the thermal unfolding process reported. In TIMs dimer stabilization is maintained by an interaction of loop 3 by a set of hydrophobic contacts between subunits. Within these contacts, the side chain of a hydrophobic residue of one subunit fits into a cavity created by a set of hydrophobic residues in the neighboring subunit, via a "ball and socket" interaction. LvTIM presents a Cys47 at the "ball" inter-subunit contact indicating that the character of this residue is responsible for the decrease in dimer stability. Mutational studies show that this residue plays a role in dimer stability but is not a solely determinant for dimer formation.

  16. Sucrose isomerase and its mutants from Erwinia rhapontici can synthesise α-arbutin.

    PubMed

    Zhou, Xing; Zheng, Yuantao; Wei, Xingming; Yang, Kedi; Yang, Xiangkai; Wang, Yuting; Xu, Liming; Du, Liqin; Huang, Ribo

    2011-10-01

    Sucrose isomerase (SI) from Erwinia rhapontici is an intramolecular isomerase that is normally used to synthesise isomaltulose from sucrose by a mechanism of intramolecular transglycosylation. In this study, it was found that SI could synthesise α-arbutin using hydroquinone and sucrose as substrates, via an intermolecular transglycosylation reaction. Five phenylalanine residues (F185, F186, F205, F297, and F321) in the catalytic pocket of SI were chosen for sitedirected mutagenesis. Mutants F185I, F321I, and F321W, whose hydrolytic activities were enhanced after the mutation, could synthesise α-arbutin through intermolecular transglycosylation with a more than two-fold increase in the molar transfer ratio compared with wild type SI. The F297A mutant showed a strong ability to synthesise a novel α-arbutin derivative and a four-fold increase in its specific activity for intermolecular transglycosylation over the wild type. Our findings may lead to a new way to synthesise novel glucoside products such as α-arbutin derivatives by simply manipulating the Phe residues in the catalytic pocket. From the structure superposition, our strategy of manipulating these Phe residues may be applicable to other similar transglycosylating enzymes.

  17. Type II Isopentenyl Diphosphate Isomerase: Probing the Mechanism with Alkyne/Allene Diphosphate Substrate Analogues†

    PubMed Central

    Sharma, Nagendra K.; Pan, Jian-Jung; Poulter, C. Dale

    2010-01-01

    Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the basic five-carbon building blocks of isoprenoid molecules. Two structurally unrelated classes of IDI are known. Type I IPP isomerase (IDI-1) utilizes a divalent metal in a protonation-deprotonation reaction. In contrast, the type II enzyme (IDI-2) requires reduced flavin, raising the possibility that the reaction catalyzed by IDI-2 involves the net addition/abstraction of a hydrogen atom. As part of our studies of the mechanism of isomerization for IDI-2, we synthesized allene and alkyne substrate analogues for the enzyme. These molecules are predicted to be substantially less reactive toward proton addition than IPP and DMAPP, but have similar reactivities toward hydrogen atom addition. This prediction was verified by calculations of gas phase heats of reaction for addition of a proton and of a hydrogen atom to 1-butyne (3) and 1,2-butadiene (4) to form the 1-buten-2-yl carbocation and radical, respectively, and related affinities for 2-methyl-1-butene (5) and 2-methyl-2-butene (6) using G3MP2B3 and CBS-QB3 protocols. Alkyne 1-OPP and allene 2-OPP were not substrates for Thermus thermophilus IDI-2 or Escherichia coli IDI-1, but instead were competitive inhibitors. The experimental and computational results are consistent with a protonation-deprotonation mechanism for the enzyme-catalyzed isomerization of IPP and DMAPP. PMID:20560533

  18. A mutation in the rice chalcone isomerase gene causes the golden hull and internode 1 phenotype.

    PubMed

    Hong, Lilan; Qian, Qian; Tang, Ding; Wang, Kejian; Li, Ming; Cheng, Zhukuan

    2012-07-01

    The biosynthesis of flavonoids, important secondary plant metabolites, has been investigated extensively, but few mutants of genes in this pathway have been identified in rice (Oryza sativa). The rice gold hull and internode (gh) mutants exhibit a reddish-brown pigmentation in the hull and internode and their phenotype has long been used as a morphological marker trait for breeding and genetic study. Here, we characterized that the gh1 mutant was a mutant of the rice chalcone isomerase gene (OsCHI). The result showed that gh1 had a Dasheng retrotransposon inserted in the 5′ UTR of the OsCHI gene, which resulted in the complete loss of OsCHI expression. gh1 exhibited golden pigmentation in hulls and internodes once the panicles were exposed to light. The total flavonoid content in gh1 hulls was increased threefold compared to wild type. Consistent with the gh1 phenotype, OsCHI transcripts were expressed in most tissues of rice and most abundantly in internodes. It was also expressed at high levels in panicles before heading, distributed mainly in lemmas and paleae, but its expression decreased substantially after the panicles emerged from the sheath. OsCHI encodes a protein functionally and structurally conserved to chalcone isomerases in other species. Our findings demonstrated that the OsCHI gene was indispensable for flux of the flavonoid pathway in rice.

  19. In-house SIRAS phasing of the polyunsaturated fatty-acid isomerase from Propionibacterium acnes

    SciTech Connect

    Liavonchanka, Alena; Hornung, Ellen; Feussner, Ivo; Rudolph, Markus

    2006-02-01

    Low iodide concentrations were sufficient to allow SAD and SIRAS phasing of cubic crystals of a novel fatty acid isomerase using Cu Kα radiation. The polyenoic fatty-acid isomerase from Propionibacterium acnes (PAI) catalyzes the double-bond isomerization of linoleic acid to conjugated linoleic acid, which is a dairy- or meat-derived fatty acid in the human diet. PAI was overproduced in Escherichia coli and purified to homogeneity as a yellow-coloured protein. The nature of the bound cofactor was analyzed by absorption and fluorescence spectroscopy. Single crystals of PAI were obtained in two crystal forms. Cubic shaped crystals belong to space group I2{sub 1}3, with a unit-cell parameter of 160.4 Å, and plate-like crystals belong to the monoclinic space group C2, with unit-cell parameters a = 133.7, b = 60.8, c = 72.2 Å, β = 115.8°. Both crystal forms contain one molecule per asymmetric unit and diffract to a resolution of better than 2.0 Å. Initial phases were obtained by SIRAS from in-house data from a cubic crystal that was soaked with an unusually low KI concentration of 0.25 M.

  20. Role of a chalcone isomerase-like protein in flavonoid biosynthesis in Arabidopsis thaliana.

    PubMed

    Jiang, Wenbo; Yin, Qinggang; Wu, Ranran; Zheng, Guangshun; Liu, Jinyue; Dixon, Richard A; Pang, Yongzhen

    2015-12-01

    Flavonoids are important natural products for plant defence and human health. Although almost all the flavonoid pathway genes have been well-documented by biochemical and/or genetic approaches, the role of the Arabidopsis chalcone isomerase-like (CHIL) gene remains unclear. Two chil mutants with a seed colour similar to that of wild-type Arabidopsis have been identified here, but in sharp contrast to the characteristic transparent testa seed phenotype associated with other known flavonoid pathway genes. CHIL loss-of-function mutations led to a strong reduction in the proanthocyanidin and flavonol levels in seeds, but not in the anthocyanin levels in leaves. CHIL over-expression could partially recover the mutant phenotype of the chil mutant and increased both proanthocyanidin and flavonol accumulation in wild-type Arabidopsis. However, the CHIL gene could not rescue the mutant phenotype of TT5 that encodes the intrinsic chalcone isomerase in Arabidopsis. Parallel phenotypical and metabolic analyses of the chil, tt5, chs, and f3h mutants revealed that, genetically, CHIL functions at the same step as TT5. Moreover, it is demonstrated that CHIL co-expresses, co-localizes, and interacts with TT5 in Arabidopsis for flavonoid production. Based on these genetic and metabolic studies, it is concluded that CHIL functions with TT5 to promote flavonoid production, which is a unique enhancer in the flavonoid pathway.

  1. Control of carotenoid biosynthesis through a heme-based cis-trans isomerase

    PubMed Central

    Beltrán, Jesús; Kloss, Brian; Hosler, Jonathan P.; Geng, Jiafeng; Liu, Aimin; Modi, Anuja; Dawson, John H.; Sono, Masanori; Shumskaya, Maria; Ampomah-Dwamena, Charles; Love, James D.; Wurtzel, Eleanore T.

    2015-01-01

    Plants synthesize carotenoids essential for plant development and survival. These metabolites also serve as essential nutrients for human health. The biosynthetic pathway leading to all plant carotenoids occurs in chloroplasts and other plastids and requires 15-cis-ζ-carotene isomerase (Z-ISO). It was not certain whether isomerization was achieved by Z-ISO alone or in combination with other enzymes. Here we show that Z-ISO is a bona fide enzyme and integral membrane protein. Z-ISO independently catalyzes the cis-to-trans isomerization of the 15–15′ C=C bond in 9,15,9′-cis-ζ-carotene to produce the substrate required by the following biosynthetic pathway enzyme. We discovered that isomerization depends upon a ferrous heme b cofactor that undergoes redox-regulated ligand-switching between the heme iron and alternate Z-ISO amino acid residues. Heme b-dependent isomerization of a large, hydrophobic compound in a membrane is unprecedented. As an isomerase, Z-ISO represents a new prototype for heme b proteins and potentially utilizes a novel chemical mechanism. PMID:26075523

  2. Disclosing the essentiality of ribose-5-phosphate isomerase B in Trypanosomatids

    PubMed Central

    Faria, Joana; Loureiro, Inês; Santarém, Nuno; Cecílio, Pedro; Macedo-Ribeiro, Sandra; Tavares, Joana; Cordeiro-da-Silva, Anabela

    2016-01-01

    Ribose-5-phosphate isomerase (RPI) belongs to the non-oxidative branch of the pentose phosphate pathway, catalysing the inter-conversion of D-ribose-5-phosphate and D-ribulose-5-phosphate. Trypanosomatids encode a type B RPI, whereas humans have a structurally unrelated type A, making RPIB worthy of exploration as a potential drug target. Null mutant generation in Leishmania infantum was only possible when an episomal copy of RPIB gene was provided, and the latter was retained both in vitro and in vivo in the absence of drug pressure. This suggests the gene is essential for parasite survival. Importantly, the inability to remove the second allele of RPIB gene in sKO mutants complemented with an episomal copy of RPIB carrying a mutation that abolishes isomerase activity suggests the essentiality is due to its metabolic function. In vitro, sKO promastigotes exhibited no defect in growth, metacyclogenesis or macrophage infection, however, an impairment in intracellular amastigotes’ replication was observed. Additionally, mice infected with sKO mutants rescued by RPIB complementation had a reduced parasite burden in the liver. Likewise, Trypanosoma brucei is resistant to complete RPIB gene removal and mice infected with sKO mutants showed prolonged survival upon infection. Taken together our results genetically validate RPIB as a potential drug target in trypanosomatids. PMID:27230471

  3. Overexpression, purification, crystallization and preliminary diffraction studies of the Protaminobacter rubrum sucrose isomerase SmuA

    SciTech Connect

    Ravaud, Stéphanie; Watzlawick, Hildegard; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

    2006-01-01

    The P. rubrum sucrose isomerase SmuA, a key enzyme in the industrial production of isomaltulose, was crystallized and diffraction data were collected to 1.95 Å resolution. Palatinose (isomaltulose, α-d-glucosylpyranosyl-1,6-d-fructofuranose), a nutritional and acariogenic reducing sugar, is industrially obtained from sucrose by using immobilized cells of Protaminobacter rubrum that produce the sucrose isomerase SmuA. The isomerization of sucrose catalyzed by this enzyme also results in the formation of trehalulose (α-d-glucosylpyranosyl-1,1-d-fructofuranose) in smaller amounts and glucose, fructose and eventually isomaltose as by-products, which lower the yield of the reaction and complicate the recovery of palatinose. The determination of the three-dimensional structure of SmuA will provide a basis for rational protein-engineering studies in order to optimize the industrial production of palatinose. A recombinant form of the 67.3 kDa SmuA enzyme has been crystallized in the native state by the vapour-diffusion method. Crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 61.6, b = 81.4, c = 135.6 Å, and diffract to 1.95 Å resolution on a synchrotron-radiation source.

  4. Human cellular retinaldehyde-binding protein has secondary thermal 9-cis-retinal isomerase activity.

    PubMed

    Bolze, Christin S; Helbling, Rachel E; Owen, Robin L; Pearson, Arwen R; Pompidor, Guillaume; Dworkowski, Florian; Fuchs, Martin R; Furrer, Julien; Golczak, Marcin; Palczewski, Krzysztof; Cascella, Michele; Stocker, Achim

    2014-01-08

    Cellular retinaldehyde-binding protein (CRALBP) chaperones 11-cis-retinal to convert opsin receptor molecules into photosensitive retinoid pigments of the eye. We report a thermal secondary isomerase activity of CRALBP when bound to 9-cis-retinal. UV/vis and (1)H NMR spectroscopy were used to characterize the product as 9,13-dicis-retinal. The X-ray structure of the CRALBP mutant R234W:9-cis-retinal complex at 1.9 Å resolution revealed a niche in the binding pocket for 9-cis-aldehyde different from that reported for 11-cis-retinal. Combined computational, kinetic, and structural data lead us to propose an isomerization mechanism catalyzed by a network of buried waters. Our findings highlight a specific role of water molecules in both CRALBP-assisted specificity toward 9-cis-retinal and its thermal isomerase activity yielding 9,13-dicis-retinal. Kinetic data from two point mutants of CRALBP support an essential role of Glu202 as the initial proton donor in this isomerization reaction.

  5. Human Cellular Retinaldehyde-Binding Protein Has Secondary Thermal 9-cis-Retinal Isomerase Activity

    PubMed Central

    Bolze, Christin S.; Helbling, Rachel E.; Owen, Robin L.; Pearson, Arwen R.; Pompidor, Guillaume; Dworkowski, Florian; Fuchs, Martin R.; Furrer, Julien; Golczak, Marcin; Palczewski, Krzysztof

    2014-01-01

    Cellular retinaldehyde-binding protein (CRALBP) chaperones 11-cis-retinal to convert opsin receptor molecules into photosensitive retinoid pigments of the eye. We report a thermal secondary isomerase activity of CRALBP when bound to 9-cis-retinal. UV/VIS and 1H-NMR spectroscopy were used to characterize the product as 9,13-dicis-retinal. The X-ray structure of the CRALBP mutant R234W:9-cis-retinal complex at 1.9 Å resolution revealed a niche in the binding-pocket for 9-cis-aldehyde different from that reported for 11-cis-retinal. Combined computational, kinetic, and structural data lead us to propose an isomerization mechanism catalyzed by a network of buried waters. Our findings highlight a specific role of water molecules in both CRALBP-assisted specificity towards 9-cis-retinal and its thermal isomerase activity yielding 9,13-dicis-retinal. Kinetic data from two point mutants of CRALBP support an essential role of Glu202 as the initial proton donor in this isomerization reaction. PMID:24328211

  6. TXNDC5, a Newly Discovered Disulfide Isomerase with a Key Role in Cell Physiology and Pathology

    PubMed Central

    Horna-Terrón, Elena; Pradilla-Dieste, Alberto; Sánchez-de-Diego, Cristina; Osada, Jesús

    2014-01-01

    Thioredoxin domain-containing 5 (TXNDC5) is a member of the protein disulfide isomerase family, acting as a chaperone of endoplasmic reticulum under not fully characterized conditions As a result, TXNDC5 interacts with many cell proteins, contributing to their proper folding and correct formation of disulfide bonds through its thioredoxin domains. Moreover, it can also work as an electron transfer reaction, recovering the functional isoform of other protein disulfide isomerases, replacing reduced glutathione in its role. Finally, it also acts as a cellular adapter, interacting with the N-terminal domain of adiponectin receptor. As can be inferred from all these functions, TXNDC5 plays an important role in cell physiology; therefore, dysregulation of its expression is associated with oxidative stress, cell ageing and a large range of pathologies such as arthritis, cancer, diabetes, neurodegenerative diseases, vitiligo and virus infections. Its implication in all these important diseases has made TXNDC5 a susceptible biomarker or even a potential pharmacological target. PMID:25526565

  7. Ethanol production from lignocellulosic hydrolysates using engineered Saccharomyces cerevisiae harboring xylose isomerase-based pathway.

    PubMed

    Ko, Ja Kyong; Um, Youngsoon; Woo, Han Min; Kim, Kyoung Heon; Lee, Sun-Mi

    2016-06-01

    The efficient co-fermentation of glucose and xylose is necessary for the economically feasible bioethanol production from lignocellulosic biomass. Even with xylose utilizing Saccharomyces cerevisiae, the efficiency of the lignocellulosic ethanol production remains suboptimal mainly due to the low conversion yield of xylose to ethanol. In this study, we evaluated the co-fermentation performances of SXA-R2P-E, a recently engineered isomerase-based xylose utilizing strain, in mixed sugars and in lignocellulosic hydrolysates. In a high-sugar fermentation with 70g/L of glucose and 40g/L of xylose, SXA-R2P-E produced 50g/L of ethanol with an yield of 0.43gethanol/gsugars at 72h. From dilute acid-pretreated hydrolysates of rice straw and hardwood (oak), the strain produced 18-21g/L of ethanol with among the highest yield of 0.43-0.46gethanol/gsugars ever reported. This study shows a highly promising potential of a xylose isomerase-expressing strain as an industrially relevant ethanol producer from lignocellulosic hydrolysates.

  8. Structural analysis of arabinose-5-phosphate isomerase from Bacteroides fragilis and functional implications.

    PubMed

    Chiu, Hsiu Ju; Grant, Joanna C; Farr, Carol L; Jaroszewski, Lukasz; Knuth, Mark W; Miller, Mitchell D; Elsliger, Marc André; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

    2014-10-01

    The crystal structure of arabinose-5-phosphate isomerase (API) from Bacteroides fragilis (bfAPI) was determined at 1.7 Å resolution and was found to be a tetramer of a single-domain sugar isomerase (SIS) with an endogenous ligand, CMP-Kdo (cytidine 5'-monophosphate-3-deoxy-D-manno-oct-2-ulosonate), bound at the active site. API catalyzes the reversible isomerization of D-ribulose 5-phosphate to D-arabinose 5-phosphate in the first step of the Kdo biosynthetic pathway. Interestingly, the bound CMP-Kdo is neither the substrate nor the product of the reaction catalyzed by API, but corresponds to the end product in the Kdo biosynthetic pathway and presumably acts as a feedback inhibitor for bfAPI. The active site of each monomer is located in a surface cleft at the tetramer interface between three monomers and consists of His79 and His186 from two different adjacent monomers and a Ser/Thr-rich region, all of which are highly conserved across APIs. Structure and sequence analyses indicate that His79 and His186 may play important catalytic roles in the isomerization reaction. CMP-Kdo mimetics could therefore serve as potent and specific inhibitors of API and provide broad protection against many different bacterial infections.

  9. Structural analysis of arabinose-5-phosphate isomerase from Bacteroides fragilis and functional implications

    PubMed Central

    Chiu, Hsiu-Ju; Grant, Joanna C.; Farr, Carol L.; Jaroszewski, Lukasz; Knuth, Mark W.; Miller, Mitchell D.; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2014-01-01

    The crystal structure of arabinose-5-phosphate isomerase (API) from Bacteroides fragilis (bfAPI) was determined at 1.7 Å resolution and was found to be a tetramer of a single-domain sugar isomerase (SIS) with an endogenous ligand, CMP-Kdo (cytidine 5′-monophosphate-3-deoxy-d-manno-oct-2-ulosonate), bound at the active site. API catalyzes the reversible isomerization of d-ribulose 5-phosphate to d-arabinose 5-phosphate in the first step of the Kdo biosynthetic pathway. Interestingly, the bound CMP-Kdo is neither the substrate nor the product of the reaction catalyzed by API, but corresponds to the end product in the Kdo biosynthetic pathway and presumably acts as a feedback inhibitor for bfAPI. The active site of each monomer is located in a surface cleft at the tetramer interface between three monomers and consists of His79 and His186 from two different adjacent monomers and a Ser/Thr-rich region, all of which are highly conserved across APIs. Structure and sequence analyses indicate that His79 and His186 may play important catalytic roles in the isomerization reaction. CMP-Kdo mimetics could therefore serve as potent and specific inhibitors of API and provide broad protection against many different bacterial infections. PMID:25286848

  10. Protein disulfide isomerase homolog TrPDI2 contributing to cellobiohydrolase production in Trichoderma reesei.

    PubMed

    Wang, Guokun; Lv, Pin; He, Ronglin; Wang, Haijun; Wang, Lixian; Zhang, Dongyuan; Chen, Shulin

    2015-09-01

    The majority of the cysteine residues in the secreted proteins form disulfide bonds via protein disulfide isomerase (PDI)-mediated catalysis, stabilizing the enzyme activity. The role of PDI in cellulase production is speculative, as well as the possibility of PDI as a target for improving enzyme production efficiency of Trichoderma reesei, a widely used producer of enzyme for the production of lignocellulose-based biofuels and biochemicals. Here, we report that a PDI homolog, TrPDI2 in T. reesei exhibited a 36.94% and an 11.81% similarity to Aspergillus niger TIGA and T. reesei PDI1, respectively. The capability of TrPDI2 to recover the activity of reduced and denatured RNase by promoting refolding verified its protein disulfide isomerase activity. The overexpression of Trpdi2 increased the secretion and the activity of CBH1 at the early stage of cellulase induction. In addition, both the expression level and redox state of TrPDI2 responded to cellulase induction in T. reesei, providing sustainable oxidative power to ensure cellobiohydrolase maturation and production. The results suggest that TrPDI2 may contribute to cellobiohydrolase secretion by enhancing the capability of disulfide bond formation, which is essential for protein folding and maturation.

  11. Crystallization and preliminary X-ray diffraction studies of l-rhamnose isomerase from Pseudomonas stutzeri

    SciTech Connect

    Yoshida, Hiromi; Wayoon, Poonperm; Takada, Goro; Izumori, Ken; Kamitori, Shigehiro

    2006-06-01

    Recombinant l-rhamnose isomerase from P. stutzeri has been crystallized. Diffraction data have been collected to 2.0 Å resolution. l-Rhamnose isomerase from Pseudomonas stutzeri (P. stutzeril-RhI) catalyzes not only the reversible isomerization of l-rhamnose to l-rhamnulose, but also isomerization between various rare aldoses and ketoses. Purified His-tagged P. stutzeril-RhI was crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 74.3, b = 104.0, c = 107.0 Å, β = 106.8°. Diffraction data have been collected to 2.0 Å resolution. The molecular weight of the purified P. stutzeril-RhI with a His tag at the C-terminus was confirmed to be 47.7 kDa by MALDI–TOF mass-spectrometric analysis and the asymmetric unit is expected to contain four molecules.

  12. Bioconversion of D-galactose into D-tagatose by expression of L-arabinose isomerase.

    PubMed

    Roh, H J; Kim, P; Park, Y C; Choi, J H

    2000-02-01

    D-Tagatose is a potential bulking agent in food as a non-calorific sweetener. To produce D-tagatose from cheaper resources, plasmids harbouring the L-arabinose isomerase gene (araA) from Escherichia coli, Bacillus subtilis and Salmonella typhimurium were constructed because L-arabinose isomerase was suggested previously as an enzyme that mediates the bioconversion of galactose into tagatose as well as that of arabinose to ribulose. The constructed plasmids were named pTC101, pTC105 and pTC106, containing araA from E. coli, B. subtilis and S. typhimurium respectively. In the cultures of recombinant E. coli with pTC101, pTC105 and pTC106, tagatose was produced from galactose in 9.9, 7.1 and 6.9% yields respectively. The enzyme extract of E. coli with the plasmid pTC101 also converted galactose into tagatose with a 96.4% yield.

  13. Characterization of a monoclonal antibody that specifically inhibits triosephosphate isomerase activity of Taenia solium.

    PubMed

    Víctor, Sanabria-Ayala; Yolanda, Medina-Flores; Araceli, Zavala-Carballo; Lucía, Jiménez; Abraham, Landa

    2013-08-01

    In the present study, we obtained and characterized partially a monoclonal antibody (4H11D10B11 mAb) against triosephosphate isomerase from Taenia solium (TTPI). This antibody recognized the enzyme by both ELISA and western blot and was able to inhibit its enzymatic activity in 74%. Moreover, the antigen-binding fragments (Fabs), products of digestion of the monoclonal antibody with papain, retained almost the same inhibitory effect. We determined the binding site by ELISA; synthetic peptides containing sequences from different non-conserved regions of the TTPI were confronted to the 4H11D10B11 mAb. The epitope recognized by the monoclonal antibody was located on peptide TTPI-56 (ATPAQAQEVHKVVRDWIRKHVDAGIADKARI), and an analysis of mimotopes, obtained with the 4H11D10B11 mAb, suggests that the epitope spans the sequence WIRKHVDAGIAD, residues 193-204 of the enzyme. This epitope is located within helix 6, next to loop 6, an essential active loop during catalysis. The antibody did not recognize triosephosphate isomerase from man and pig, definitive and intermediary hosts of T. solium, respectively. Furthermore, it did not bind to the catalytic site, since kinetic analysis demonstrated that inhibition had a non-competitive profile.

  14. Optimization of lactulose synthesis from whey lactose by immobilized β-galactosidase and glucose isomerase.

    PubMed

    Song, Yoon-Seok; Lee, Hee-Uk; Park, Chulhwan; Kim, Seung-Wook

    2013-03-22

    In the present study, commercially available whey was used as a lactose source, and immobilized β-galactosidase and glucose isomerase were used to synthesize lactulose from whey lactose in the absence of fructose. Optimal reaction conditions, such as lactose concentration, temperature, ionic strength of the buffer, and ratio of immobilized enzymes, were determined to improve lactulose synthesis using immobilized enzymes. Lactulose synthesis using immobilized enzymes improved markedly after optimizing the reaction conditions. When the lactulose synthesis was carried out at 53.5°C using 20% (w/v) whey lactose, 12U/ml of immobilized β-galactosidase and 60U/ml of immobilized glucose isomerase in 100mM sodium phosphate buffer at pH 7.5, the lactulose concentration and specific productivity were 7.68g/l and 0.32mg/Uh, respectively. Additionally, when the immobilized enzymes were reused for lactulose synthesis, their catalytic activity was 57.1% after 7 repeated uses. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Kinetic measurements of phosphoglucose isomerase and phosphomannose isomerase by direct analysis of phosphorylated aldose-ketose isomers using tandem mass spectrometry

    NASA Astrophysics Data System (ADS)

    Gao, Hong; Chen, Ye; Leary, Julie A.

    2005-02-01

    A mass spectrometry based method for the direct determination of kinetic constants for phosphoglucose isomerase (PGI) and phosphomannose isomerase (PMI) is described. PGI catalyzes the interconversion between glucose-6-phosphate (Glc6P) and fructose-6-phosphate (Fru6P) and PMI performs the same function between mannose-6-phosphate (Man6P) and Fru6P. These two enzymes are essential in the pathways of glycolytic or oxidative metabolism of carbohydrates and have been considered as potential therapeutic targets. Traditionally, they are assayed either by spectrophotometric detection of Glc6P with one or more coupling enzymes or by a colorimetric detection of Fru6P. However, no suitable assay for Man6P has been developed yet to study the reaction of PMI in the direction from Fru6P to Man6P. In the work presented herein, a general assay for the isomeric substrate-product pair between Glc6P and Fru6P or between Man6P and Fru6P was developed, with the aim of directly studying the kinetics of PGI and PMI in both directions. The 6-phosphorylated aldose and ketose isomers were distinguished based on their corresponding tandem mass spectra (MS2) obtained on a quadrupole ion trap mass spectrometer, and a multicomponent quantification method was utilized to determine the composition of binary mixtures. Using this method, the conversion between Fru6P and Glc6P and that between Fru6P and Man6P are directly monitored. The equilibrium constants for the reversible reactions catalyzed by PGI and PMI are measured to be 0.3 and 1.1, respectively, and the kinetic parameters for both substrates of PGI and PMI are also determined. The values of KM and Vmax for Fru6P as substrate of PMI are reported to be 0.15 mM and 7.78 [mu]mol/(min mg), respectively. All other kinetic parameters measured correlate well with those obtained using traditional methods, demonstrating the accuracy and reliability of this assay.

  16. Physiological and Pathogenic Roles of Prolyl Isomerase Pin1 in Metabolic Regulations via Multiple Signal Transduction Pathway Modulations

    PubMed Central

    Nakatsu, Yusuke; Matsunaga, Yasuka; Yamamotoya, Takeshi; Ueda, Koji; Inoue, Yuki; Mori, Keiichi; Sakoda, Hideyuki; Fujishiro, Midori; Ono, Hiraku; Kushiyama, Akifumi; Asano, Tomoichiro

    2016-01-01

    Prolyl isomerases are divided into three groups, the FKBP family, Cyclophilin and the Parvulin family (Pin1 and Par14). Among these isomerases, Pin1 is a unique prolyl isomerase binding to the motif including pSer/pThr-Pro that is phosphorylated by kinases. Once bound, Pin1 modulates the enzymatic activity, protein stability or subcellular localization of target proteins by changing the cis- and trans-formations of proline. Several studies have examined the roles of Pin1 in the pathogenesis of cancers and Alzheimer’s disease. On the other hand, recent studies have newly demonstrated Pin1 to be involved in regulating glucose and lipid metabolism. Interestingly, while Pin1 expression is markedly increased by high-fat diet feeding, Pin1 KO mice are resistant to diet-induced obesity, non-alcoholic steatohepatitis and diabetic vascular dysfunction. These phenomena result from the binding of Pin1 to several key factors regulating metabolic functions, which include insulin receptor substrate-1, AMPK, Crtc2 and NF-κB p65. In this review, we focus on recent advances in elucidating the physiological roles of Pin1 as well as the pathogenesis of disorders involving this isomerase, from the viewpoint of the relationships between signal transductions and metabolic functions. PMID:27618008

  17. 40 CFR 174.527 - Phosphomannose isomerase in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... plants are exempt from the requirement of a tolerance when used as plant-incorporated protectant inert... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Phosphomannose isomerase in all plants... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR...

  18. Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors

    PubMed Central

    Yamamoto, Kenta; Wang, Jiguang; Sprinzen, Lisa; Xu, Jun; Haddock, Christopher J; Li, Chen; Lee, Brian J; Loredan, Denis G; Jiang, Wenxia; Vindigni, Alessandro; Wang, Dong; Rabadan, Raul; Zha, Shan

    2016-01-01

    Missense mutations in ATM kinase, a master regulator of DNA damage responses, are found in many cancers, but their impact on ATM function and implications for cancer therapy are largely unknown. Here we report that 72% of cancer-associated ATM mutations are missense mutations that are enriched around the kinase domain. Expression of kinase-dead ATM (AtmKD/-) is more oncogenic than loss of ATM (Atm-/-) in mouse models, leading to earlier and more frequent lymphomas with Pten deletions. Kinase-dead ATM protein (Atm-KD), but not loss of ATM (Atm-null), prevents replication-dependent removal of Topo-isomerase I-DNA adducts at the step of strand cleavage, leading to severe genomic instability and hypersensitivity to Topo-isomerase I inhibitors. Correspondingly, Topo-isomerase I inhibitors effectively and preferentially eliminate AtmKD/-, but not Atm-proficientor Atm-/- leukemia in animal models. These findings identify ATM kinase-domain missense mutations as a potent oncogenic event and a biomarker for Topo-isomerase I inhibitor based therapy. DOI: http://dx.doi.org/10.7554/eLife.14709.001 PMID:27304073

  19. Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors.

    PubMed

    Yamamoto, Kenta; Wang, Jiguang; Sprinzen, Lisa; Xu, Jun; Haddock, Christopher J; Li, Chen; Lee, Brian J; Loredan, Denis G; Jiang, Wenxia; Vindigni, Alessandro; Wang, Dong; Rabadan, Raul; Zha, Shan

    2016-06-15

    Missense mutations in ATM kinase, a master regulator of DNA damage responses, are found in many cancers, but their impact on ATM function and implications for cancer therapy are largely unknown. Here we report that 72% of cancer-associated ATM mutations are missense mutations that are enriched around the kinase domain. Expression of kinase-dead ATM (Atm(KD/-)) is more oncogenic than loss of ATM (Atm(-/-)) in mouse models, leading to earlier and more frequent lymphomas with Pten deletions. Kinase-dead ATM protein (Atm-KD), but not loss of ATM (Atm-null), prevents replication-dependent removal of Topo-isomerase I-DNA adducts at the step of strand cleavage, leading to severe genomic instability and hypersensitivity to Topo-isomerase I inhibitors. Correspondingly, Topo-isomerase I inhibitors effectively and preferentially eliminate Atm(KD/-), but not Atm-proficientor Atm(-/-) leukemia in animal models. These findings identify ATM kinase-domain missense mutations as a potent oncogenic event and a biomarker for Topo-isomerase I inhibitor based therapy.

  20. Open reading frame 176 in the photosynthesis gene cluster of Rhodobacter capsulatus encodes idi, a gene for isopentenyl diphosphate isomerase.

    PubMed Central

    Hahn, F M; Baker, J A; Poulter, C D

    1996-01-01

    Isopentenyl diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway. A database search based on probes from the highly conserved regions in three eukaryotic IPP isomerases revealed substantial similarity with ORF176 in the photosynthesis gene cluster in Rhodobacter capsulatus. The open reading frame was cloned into an Escherichia coli expression vector. The encoded 20-kDa protein, which was purified in two steps by ion exchange and hydrophobic interaction chromatography, catalyzed the interconversion of IPP and dimethylallyl diphosphate. Thus, the photosynthesis gene cluster encodes all of the enzymes required to incorporate IPP into the ultimate carotenoid and bacteriochlorophyll metabolites in R. capsulatus. More recent searches uncovered additional putative open reading frames for IPP isomerase in seed-bearing plants (Oryza sativa, Arabadopsis thaliana, and Clarkia breweri), a worm (Caenorhabiditis elegans), and another eubacterium (Escherichia coli). The R. capsulatus enzyme is the smallest of the IPP isomerases to be identified thus far and may consist mostly of a fundamental catalytic core for the enzyme. PMID:8550491

  1. Single active-site histidine in D-xylose isomerase from Streptomyces violaceoruber. Identification by chemical derivatization and peptide mapping.

    PubMed

    Vangrysperre, W; Ampe, C; Kersters-Hilderson, H; Tempst, P

    1989-10-01

    Group-specific chemical modifications of D-xylose isomerase from Streptomyces violaceruber indicated that complete loss of activity is fully correlated with the acylation of a single histidine. Active-site protection, by the ligand combination of xylitol plus Mg2+, completely blocked diethyl pyrocarbonate derivatization of this particular residue [Vangrysperre, Callens, Kersters-Hilderson & De Bruyne (1988) Biochem. J. 250, 153-160]. Differential peptide mapping between D-xylose isomerase, which has previously been treated with diethyl pyrocarbonate in the presence or absence of xylitol plus Mg2+, allowed specific isolation and sequencing of a peptide containing this active-site histidine. For this purpose we used two essentially new techniques: first, a highly reproducible peptide cleavage protocol for protease-resistant, carbethoxylated proteins with guanidinium hydrochloride as denaturing agent and subtilisin for proteolysis; and second, reverse-phase liquid chromatography with dual-wavelength detection at 214 and 238 nm, and calculation of absorbance ratios. It allowed us to locate the single active-site histidine at position 54 in the primary structure of Streptomyces violaceoruber D-xylose isomerase. The sequence around this residue is conserved in D-xylose isomerases from a diversity of micro-organisms, suggesting that this is a structurally and/or functionally essential part of the molecule.

  2. Purification and characterization of two isoforms of isopentenyl-diphosphate isomerase from elicitor-treated Cinchona robusta cells.

    PubMed

    Ramos-Valdivia, A C; van der Heijden, R; Verpoorte, R; Camara, B

    1997-10-01

    In Cinchona robusta (Rubiaceae) cell suspension cultures, the activity of the enzyme isopentenyl-diphosphate isomerase (isopentenyl-POP isomerase) is transiently induced after addition of a homogenate of the phytopathogenic fungus Phytophthora cinnamomi. The enzyme catalyses the interconversion of isopentenyl-POP and dimethylallyl diphosphate (dimethylallyl-POP) and may be involved in the biosynthesis of anthraquinone phytoalexins that accumulate rapidly after elicitation of Cinchona cells. From elicitor-treated C. robusta cells, two isoforms of isopentenyl-POP isomerase have been purified to apparent homogeneity in four chromatographic steps. The purified forms are monomeric enzymes of 34 kDa (isoform I) and 29 kDa (isoform II), with Km values for isopentenyl-POP of 5.1 microM and 1.0 microM, respectively. Both isoforms require Mn2+ or Mg2+ as cofactor, isoform II showing a preference for Mn2+ with maximum activity at 1.5-2 mM. Isoform I was most active in the presence of 0.5-1.5 mM Mg2+ or in the presence of 0.5 mM Mn2+. A pH optimum of 7-7.8 was found for both forms and both were competitively inhibited by geranyl diphosphate (Ki 96 microM for isoform I) and the transition state analogue 2-(dimethylamino)ethyl diphosphate. Rechromatography of purified isoforms did not indicate any interconversion of both forms. Western blot analysis, using antibodies raised against isopentenyl-POP isomerase purified from Capsicum annuum, showed the presence of both isoforms in the crude protein extracts from C. robusta cells. Isoform II was specifically induced by elicitation, non-treated cells contained low activity of this isoform. The possible role of isopentenyl-POP isomerase in the biosynthesis of anthraquinones is discussed.

  3. Identification of a d-Arabinose-5-Phosphate Isomerase in the Gram-Positive Clostridium tetani.

    PubMed

    Cech, David L; Markin, Katherine; Woodard, Ronald W

    2017-09-01

    d-Arabinose-5-phosphate (A5P) isomerases (APIs) catalyze the interconversion of d-ribulose-5-phosphate and d-arabinose-5-phosphate. Various Gram-negative bacteria, such as the uropathogenic Escherichia coli strain CFT073, contain multiple API paralogs (KdsD, GutQ, KpsF, and c3406) that have been assigned various cellular functions. The d-arabinose-5-phosphate formed by these enzymes seems to play important roles in the biosynthesis of lipopolysaccharide (LPS) and group 2 K-antigen capsules, as well as in the regulation of the cellular d-glucitol uptake and uropathogenic infectivity/virulence. The genome of a Gram-positive pathogenic bacterium, Clostridium tetani, contains a gene encoding a putative API, C. tetani API (CtAPI), even though C. tetani lacks both LPS and capsid biosynthetic genes. To better understand the physiological role of d-arabinose-5-phosphate in this Gram-positive organism, recombinant CtAPI was purified and characterized. CtAPI displays biochemical characteristics similar to those of APIs from Gram-negative organisms and complements the API deficiency of an E. coli API knockout strain. Thus, CtAPI represents the first d-arabinose-5-phosphate isomerase to be identified and characterized from a Gram-positive bacterium.IMPORTANCE The genome of Clostridium tetani, a pathogenic Gram-positive bacterium and the causative agent of tetanus, contains a gene (the CtAPI gene) that shares high sequence similarity with those of genes encoding d-arabinose-5-phosphate isomerases. APIs play an important role within Gram-negative bacteria in d-arabinose-5-phosphate production for lipopolysaccharide biosynthesis, capsule formation, and regulation of cellular d-glucitol uptake. The significance of our research is in identifying and characterizing CtAPI, the first Gram-positive API. Our findings show that CtAPI is specific to the interconversion of arabinose-5-phosphate and ribulose-5-phosphate while having no activity with the other sugars and sugar phosphates

  4. Bioethanol production from steam-pretreated corn stover through an isomerase mediated process.

    PubMed

    De Bari, Isabella; Cuna, Daniela; Di Matteo, Vincenzo; Liuzzi, Federico

    2014-03-25

    Agricultural by-products such as corn stover are considered strategic raw materials for the production of second-generation bioethanol from renewable and non-food sources. This paper describes the conversion of steam-pretreated corn stover to ethanol utilising a multi-step process including enzymatic hydrolysis, isomerisation, and fermentation of mixed hydrolysates with native Saccharomyces cerevisiae. An immobilised isomerase enzyme was used for the xylose isomerisation along with high concentrations of S. cerevisiae. The objective was to assess the extent of simultaneity of the various conversion steps, through a detailed analysis of process time courses, and to test this process scheme for the conversion of lignocellulosic hydrolysates containing several inhibitors of the isomerase enzyme (e.g. metal ions, xylitol and glycerol). The process was tested on two types of hydrolysate after acid-catalysed steam pretreatment: (a) the water soluble fraction (WSF) in which xylose was the largest carbon source and (b) the entire slurry, containing both cellulose and hemicellulose carbohydrates, in which glucose predominated. The results indicated that the ethanol concentration rose when the inoculum concentration was increased in the range 10-75 g/L. However, when xylose was the largest carbon source, the metabolic yields were higher than 0.51g(ethanol)/g(consumed) sugars probably due to the use of yeast internal cellular resources. This phenomenon was not observed in the fermentation of mixed hydrolysates obtained from the entire pretreated product and in which glucose was the largest carbon source. The ethanol yield from biomass suspensions with dry matter (DM) concentrations of 11-12% (w/v) was 70% based on total sugars (glucose, xylose, galactose). The results suggest that xylulose uptake was more effective in mixed hydrolysates containing glucose levels similar to, or higher than, xylose. Analysis of the factors that limit isomerase activity in lignocellulosic

  5. Domain a' of protein disulfide isomerase plays key role in inhibiting alpha-synuclein fibril formation.

    PubMed

    Cheng, Han; Wang, Lei; Wang, Chih-chen

    2010-07-01

    alpha-Synuclein (alpha Syn) is the main component of Lewy bodies formed in midbrain dopaminergic neurons which is a pathological characteristic of Parkinson's disease. It has been recently showed to induce endoplasmic reticulum (ER) stress and impair ER functions. However, the mechanism of how ER responds to alpha Syn toxicity is poorly understood. In the present study, we found that protein disulfide isomerase (PDI), a stress protein abundant in ER, effectively inhibits alpha Syn fibril formation in vitro. In PDI molecule with a structure of abb'xa'c, domain a' was found to be essential and sufficient for PDI to inhibit alpha Syn fibril formation. PDI was further found to be more avid for binding with intermediate species formed during alpha Syn fibril formation, and the binding was more intensive in the later lag phase. Our results provide new insight into the role of PDI in protecting ER from the deleterious effects of misfolded protein accumulation in many neurodegenerative diseases.

  6. Assisted refolding of recombinant prochymosin with the aid of protein disulphide isomerase.

    PubMed Central

    Tang, B; Zhang, S; Yang, K

    1994-01-01

    Protein disulphide isomerase (PDI) was shown to be able to accelerate the refolding of unfolded recombinant prochymosin and to enhance the overall yield of active protein. Unlike previous reports in this study PDI was found to be active at pH values as high as 11. The coincidence of the similar apparent optimum pH values of uncatalysed and PDI-catalysed reactions suggests that conditions favourable to spontaneous refolding of proteins may help PDI to catalyse thiol/disulphide interchange. Under the conditions described here no exogenously added dithiothreitol was required for PDI-catalysed renaturation, implying that the disulphide form of PDI was reduced to its active form by the free thiol groups in prochymosin molecules. PMID:8037666

  7. Novel approach for identifying key residues in enzymatic reactions: proton abstraction in ketosteroid isomerase.

    PubMed

    Ito, Mika; Brinck, Tore

    2014-11-20

    We propose a computationally efficient approach for evaluating the individual contributions of many different residues to the catalytic efficiency of an enzymatic reaction. This approach is based on the fragment molecular orbital (FMO) method, and it defines the energy of a deletion form, i.e., the energy of the system when a particular residue is deleted. Using this approach, we found that, among 10 investigated residues, three, Tyr14, Asp99, and Tyr55, in this order, significantly reduce the activation energy of the proton abstraction from a substrate, cyclopent-2-enone, catalyzed by ketosteroid isomerase (KSI). The relative activation energies estimated in this study are in good agreement with available previous experimental and theoretical data obtained for the similar proton abstraction with a native substrate and substitution mutants of KSI. It was thus indicated that the new approach is efficient for rationally evaluating the catalytic effects of multiple residues on an enzymatic reaction.

  8. Extreme electric fields power catalysis in the active site of ketosteroid isomerase.

    PubMed

    Fried, Stephen D; Bagchi, Sayan; Boxer, Steven G

    2014-12-19

    Enzymes use protein architecture to impose specific electrostatic fields onto their bound substrates, but the magnitude and catalytic effect of these electric fields have proven difficult to quantify with standard experimental approaches. Using vibrational Stark effect spectroscopy, we found that the active site of the enzyme ketosteroid isomerase (KSI) exerts an extremely large electric field onto the C=O chemical bond that undergoes a charge rearrangement in KSI's rate-determining step. Moreover, we found that the magnitude of the electric field exerted by the active site strongly correlates with the enzyme's catalytic rate enhancement, enabling us to quantify the fraction of the catalytic effect that is electrostatic in origin. The measurements described here may help explain the role of electrostatics in many other enzymes and biomolecular systems.

  9. Enzyme-enzyme interaction in the chloroplast: glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase and aldolase.

    PubMed

    Anderson, L E; Goldhaber-Gordon, I M; Li, D; Tang, X Y; Xiang, M; Prakash, N

    1995-01-01

    Apparent physical interaction between pea chloroplast (Pisum sativum L.) glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) and aldolase (EC 4.1.2.13) is seen in phase-partitioning, fluorescent-anisotropy and isoelectric-focusing experiments. Similarly, results obtained in phase-partitioning and isoelectric-focusing experiments indicate physical interaction between aldolase and triose-phosphate isomerase (EC 5.3.1.1). Kinetic experiments suggest that both aldolase-bound glyceraldehyde-3-phosphate can act as substrate for glyceraldehyde-3-phosphate dehydrogenase. These results are consistent with the notion that there is interaction between these three enzymes both during photosynthetic CO2 fixation and during glycolysis in the chloroplast.

  10. Isolation and sequence analysis of the gene encoding triose phosphate isomerase from Zygosaccharomyces bailii.

    PubMed

    Merico, A; Rodrigues, F; Côrte-Real, M; Porro, D; Ranzi, B M; Compagno, C

    2001-06-30

    The ZbTPI1 gene encoding triose phosphate isomerase (TIM) was cloned from a Zygosaccharomyces bailii genomic library by complementation of the Saccharomyces cerevisiae tpi1 mutant strain. The nucleotide sequence of a 1.5 kb fragment showed an open reading frame (ORF) of 746 bp, encoding a protein of 248 amino acid residues. The deduced amino acid sequence shares a high degree of homology with TIMs from other yeast species, including some highly conserved regions. The analysis of the promoter sequence of the ZbTPI1 revealed the presence of putative motifs known to have regulatory functions in S. cerevisiae. The GenBank Accession No. of ZbTPI1 is AF325852.

  11. Triosephosphate Isomerase Gene Characterization and Potential Zoonotic Transmission of Giardia duodenalis

    PubMed Central

    Sulaiman, Irshad M.; Fayer, Ronald; Bern, Caryn; Gilman, Robert H.; Trout, James M.; Schantz, Peter M.; Das, Pradeep; Lal, Altaf A.

    2003-01-01

    To address the source of infection in humans and public health importance of Giardia duodenalis parasites from animals, nucleotide sequences of the triosephosphate isomerase (TPI) gene were generated for 37 human isolates, 15 dog isolates, 8 muskrat isolates, 7 isolates each from cattle and beavers, and 1 isolate each from a rat and a rabbit. Distinct genotypes were found in humans, cattle, beavers, dogs, muskrats, and rats. TPI and small subunit ribosomal RNA (SSU rRNA) gene sequences of G. microti from muskrats were also generated and analyzed. Phylogenetic analysis on the TPI sequences confirmed the formation of distinct groups. Nevertheless, a major group (assemblage B) contained most of the human and muskrat isolates, all beaver isolates, and the rabbit isolate. These data confirm that G. duodenalis from certain animals can potentially infect humans and should be useful in the detection, differentiation, and taxonomy of Giardia spp. PMID:14718089

  12. Production of Ethanol from d-Xylose by Using d-Xylose Isomerase and Yeasts

    PubMed Central

    Gong, Cheng-Shung; Chen, Li-Fu; Flickinger, Michael C.; Chiang, Lin-Chang; Tsao, George T.

    1981-01-01

    d-Xylulose, an intermediate of d-xylose catabolism, was observed to be fermentable to ethanol and carbon dioxide in a yield of greater than 80% by yeasts (including industrial bakers' yeast) under fermentative conditions. This conversion appears to be carried out by many yeasts known for d-glucose fermentation. In some yeasts, xylitol, in addition to ethanol, was produced from d-xylulose. Fermenting yeasts are also able to produce ethanol from d-xylose when d-xylose isomerizing enzyme is present. The results indicate that ethanol could be produced from d-xylose in a yield of greater than 80% by a two-step process. First, d-xylose is converted to d-xylulose by xylose isomerase. d-Xylulose is then fermented to ethanol by yeasts. PMID:16345717

  13. Lack of Variation at Phosphoglucose Isomerase (Pgi) in Bumblebees: Implications for Conservation Genetics Studies

    PubMed Central

    Ellis, Jonathan S.; Turner, Lucy M.; Knight, Mairi E.

    2013-01-01

    Assessing genetic variation underlying ecologically important traits is increasingly of interest and importance in population and conservation genetics. For some groups generally useful markers exist for examining the relative role of selection and drift in shaping genetic diversity e.g. the major histocompatibility complex in vertebrates and self-incompatibility loci in plants. For invertebrates there is no such generally useful locus. However, phosphoglucose isomerase (Pgi) has been proposed as a useful functional marker in the conservation genetics of invertebrates. Where thermal microclimate varies, balanced polymorphisms may be maintained due to trade-offs between thermally stable and kinetically advantageous allelic forms. We here report very low levels of Pgi variation in bumblebees rendering this locus to be of little use as an adaptive marker in a conservation genetics context in this group. Potential explanations for this lack of variation are considered. PMID:23750269

  14. X-ray structure of linalool dehydratase/isomerase from Castellaniella defragrans reveals enzymatic alkene synthesis.

    PubMed

    Weidenweber, Sina; Marmulla, Robert; Ermler, Ulrich; Harder, Jens

    2016-05-01

    Linalool dehydratase/isomerase (Ldi), an enzyme of terpene degradation in Castellaniella defragrans, isomerizes the primary monoterpene alcohol geraniol into the tertiary alcohol (S)-linalool and dehydrates (S)-linalool to the alkene β-myrcene. Here we report on the crystal structures of Ldi with and without terpene substrates, revealing a cofactor-free homopentameric enzyme. The substrates were embedded inside a hydrophobic channel between two monomers of the (α,α)6 barrel fold class and flanked by three clusters of polar residues involved in acid-base catalysis. The detailed view into the active site will guide future biotechnological applications of Ldi, in particular, for industrial butadiene and isoprene production from renewable sources. © 2016 Federation of European Biochemical Societies.

  15. Structural Elucidation of a Small Molecule Inhibitor of Protein Disulfide Isomerase

    PubMed Central

    2015-01-01

    Compound libraries provide a starting point for multiple biological investigations, but the structural integrity of compounds is rarely assessed experimentally until a late stage in the research process. Here, we describe the discovery of a neuroprotective small molecule that was originally incorrectly annotated with a chemical structure. We elucidated the correct structure of the active compound using analytical chemistry, revealing it to be the natural product securinine. We show that securinine is protective in a cell model of Huntington disease and identify the binding site of securinine to its target, protein disulfide isomerase using NMR chemical shift perturbation studies. We show that securinine displays favorable pharmaceutical properties, making it a promising compound for in vivo studies in neurodegenerative disease models. In addition to finding this unexpected activity of securinine, this study provides a systematic roadmap to those who encounter compounds with incorrect structural annotation in the course of screening campaigns. PMID:26500720

  16. Rapid expansion of the protein disulfide isomerase gene family facilitates the folding of venom peptides

    PubMed Central

    Safavi-Hemami, Helena; Li, Qing; Jackson, Ronneshia L.; Song, Albert S.; Boomsma, Wouter; Bandyopadhyay, Pradip K.; Gruber, Christian W.; Purcell, Anthony W.; Yandell, Mark; Olivera, Baldomero M.

    2016-01-01

    Formation of correct disulfide bonds in the endoplasmic reticulum is a crucial step for folding proteins destined for secretion. Protein disulfide isomerases (PDIs) play a central role in this process. We report a previously unidentified, hypervariable family of PDIs that represents the most diverse gene family of oxidoreductases described in a single genus to date. These enzymes are highly expressed specifically in the venom glands of predatory cone snails, animals that synthesize a remarkably diverse set of cysteine-rich peptide toxins (conotoxins). Enzymes in this PDI family, termed conotoxin-specific PDIs, significantly and differentially accelerate the kinetics of disulfide-bond formation of several conotoxins. Our results are consistent with a unique biological scenario associated with protein folding: The diversification of a family of foldases can be correlated with the rapid evolution of an unprecedented diversity of disulfide-rich structural domains expressed by venomous marine snails in the superfamily Conoidea. PMID:26957604

  17. Mannose production from fructose by free and immobilized D-lyxose isomerases from Providencia stuartii.

    PubMed

    Park, Chang-Su; Kwon, Hyun-Jung; Yeom, Soo-Jin; Oh, Deok-Kun

    2010-09-01

    A recombinant D-lyxose isomerase from Providencia stuartii was immobilized on Duolite A568 beads which gave the highest conversion of D-fructose to D-mannose among the various immobilization beads evaluated. Maximum activities of both the free and immobilized enzymes for fructose isomerization were at pH 7.5 and 45 degrees C in the presence of 1 mM Mn(2+). Enzyme half-lives were 14 and 30 h at 35 degrees C and 3.4 and 5.1 h at 45 degrees C, respectively. The immobilized enzyme in 300 g fructose/l (replaced hourly), produced 75 g mannose/l at 35 degrees C = 25% (w/w) yield with a productivity of 75 g mannose l(-1) h(-1) after 23 cycles.

  18. Galactaro δ-Lactone Isomerase: Lactone Isomerization by a Member of the Amidohydrolase Superfamily

    PubMed Central

    2015-01-01

    Agrobacterium tumefaciens strain C58 can utilize d-galacturonate as a sole source of carbon via a pathway in which the first step is oxidation of d-galacturonate to d-galactaro-1,5-lactone. We have identified a novel enzyme, d-galactarolactone isomerase (GLI), that catalyzes the isomerizaton of d-galactaro-1,5-lactone to d-galactaro-1,4-lactone. GLI, a member of the functionally diverse amidohydrolase superfamily, is a homologue of LigI that catalyzes the hydrolysis of 2-pyrone-4,6-dicarboxylate in lignin degradation. The ability of GLI to catalyze lactone isomerization instead of hydrolysis can be explained by the absence of the general basic catalysis used by 2-pyrone-4,6-dicarboxylate lactonase. PMID:24450804

  19. Galactaro δ-lactone isomerase: lactone isomerization by a member of the amidohydrolase superfamily.

    PubMed

    Bouvier, Jason T; Groninger-Poe, Fiona P; Vetting, Matthew; Almo, Steven C; Gerlt, John A

    2014-02-04

    Agrobacterium tumefaciens strain C58 can utilize d-galacturonate as a sole source of carbon via a pathway in which the first step is oxidation of d-galacturonate to D-galactaro-1,5-lactone. We have identified a novel enzyme, D-galactarolactone isomerase (GLI), that catalyzes the isomerizaton of D-galactaro-1,5-lactone to D-galactaro-1,4-lactone. GLI, a member of the functionally diverse amidohydrolase superfamily, is a homologue of LigI that catalyzes the hydrolysis of 2-pyrone-4,6-dicarboxylate in lignin degradation. The ability of GLI to catalyze lactone isomerization instead of hydrolysis can be explained by the absence of the general basic catalysis used by 2-pyrone-4,6-dicarboxylate lactonase.

  20. Dual activity of quinolinate synthase: triose phosphate isomerase and dehydration activities play together to form quinolinate.

    PubMed

    Reichmann, Debora; Couté, Yohann; Ollagnier de Choudens, Sandrine

    2015-10-27

    Quinolinate synthase (NadA) is an Fe4S4 cluster-containing dehydrating enzyme involved in the synthesis of quinolinic acid (QA), the universal precursor of the essential coenzyme nicotinamide adenine dinucleotide. The reaction catalyzed by NadA is not well understood, and two mechanisms have been proposed in the literature that differ in the nature of the molecule (DHAP or G-3P) that condenses with iminoaspartate (IA) to form QA. In this article, using biochemical approaches, we demonstrate that DHAP is the triose that condenses with IA to form QA. The capacity of NadA to use G-3P is due to its previously unknown triose phosphate isomerase activity.

  1. Identification of the critical residues responsible for differential reactivation of the triosephosphate isomerases of two trypanosomes.

    PubMed

    Rodríguez-Bolaños, Monica; Cabrera, Nallely; Perez-Montfort, Ruy

    2016-10-01

    The reactivation of triosephosphate isomerase (TIM) from unfolded monomers induced by guanidine hydrochloride involves different amino acids of its sequence in different stages of protein refolding. We describe a systematic mutagenesis method to find critical residues for certain physico-chemical properties of a protein. The two similar TIMs of Trypanosoma brucei and Trypanosoma cruzi have different reactivation velocities and efficiencies. We used a small number of chimeric enzymes, additive mutants and planned site-directed mutants to produce an enzyme from T. brucei with 13 mutations in its sequence, which reactivates fast and efficiently like wild-type (WT) TIM from T. cruzi, and another enzyme from T. cruzi, with 13 slightly altered mutations, which reactivated slowly and inefficiently like the WT TIM of T. brucei Our method is a shorter alternative to random mutagenesis, saturation mutagenesis or directed evolution to find multiple amino acids critical for certain properties of proteins.

  2. Identification of the critical residues responsible for differential reactivation of the triosephosphate isomerases of two trypanosomes

    PubMed Central

    Rodríguez-Bolaños, Monica; Cabrera, Nallely

    2016-01-01

    The reactivation of triosephosphate isomerase (TIM) from unfolded monomers induced by guanidine hydrochloride involves different amino acids of its sequence in different stages of protein refolding. We describe a systematic mutagenesis method to find critical residues for certain physico-chemical properties of a protein. The two similar TIMs of Trypanosoma brucei and Trypanosoma cruzi have different reactivation velocities and efficiencies. We used a small number of chimeric enzymes, additive mutants and planned site-directed mutants to produce an enzyme from T. brucei with 13 mutations in its sequence, which reactivates fast and efficiently like wild-type (WT) TIM from T. cruzi, and another enzyme from T. cruzi, with 13 slightly altered mutations, which reactivated slowly and inefficiently like the WT TIM of T. brucei. Our method is a shorter alternative to random mutagenesis, saturation mutagenesis or directed evolution to find multiple amino acids critical for certain properties of proteins. PMID:27733588

  3. Inheritance and subcellular localization of triose-phosphate isomerase in dwarf mountain pine (Pinus mugo).

    PubMed

    Odrzykoski, I J

    2001-01-01

    Several trees with expected heterozygous phenotype for triose-phosphate isomerase (TPI) were discovered in a population of dwarf mountain pine (Pinus mugo Turra) from southern Poland. As the inheritance of this enzyme in pines has not been reported, segregation of allelic variants was tested in eight trees with putative heterozygous phenotypes for two loci, TpiA and TPIB: Linkage between these and some other isozyme loci were studied and evidence for linkage has been found between TpiA and PgdA (r = 0.10) and between TpiB and DiaD (r = 0.36), but in single trees only. The subcellular localization of TPI isozymes was determined by comparing isoenzymes from the total extract with those found in fraction enriched in plastids, prepared by differential gradient centrifugation of cellular organelles. The more slowly migrating TPI-B isozyme is located in plastids.

  4. Enzymatic conversion of flavonoids using bacterial chalcone isomerase and enoate reductase.

    PubMed

    Gall, Mechthild; Thomsen, Maren; Peters, Christin; Pavlidis, Ioannis V; Jonczyk, Patrick; Grünert, Philipp P; Beutel, Sascha; Scheper, Thomas; Gross, Egon; Backes, Michael; Geissler, Torsten; Ley, Jakob P; Hilmer, Jens-Michael; Krammer, Gerhard; Palm, Gottfried J; Hinrichs, Winfried; Bornscheuer, Uwe T

    2014-01-27

    Flavonoids are a large group of plant secondary metabolites with a variety of biological properties and are therefore of interest to many scientists, as they can lead to industrially interesting intermediates. The anaerobic gut bacterium Eubacterium ramulus can catabolize flavonoids, but until now, the pathway has not been experimentally confirmed. In the present work, a chalcone isomerase (CHI) and an enoate reductase (ERED) could be identified through whole genome sequencing and gene motif search. These two enzymes were successfully cloned and expressed in Escherichia coli in their active form, even under aerobic conditions. The catabolic pathway of E. ramulus was confirmed by biotransformations of flavanones into dihydrochalcones. The engineered E. coli strain that expresses both enzymes was used for the conversion of several flavanones, underlining the applicability of this biocatalytic cascade reaction. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Thermodynamics of Enzyme-Catalyzed Reactions: Part 5. Isomerases and Ligases

    NASA Astrophysics Data System (ADS)

    Goldberg, Robert N.; Tewari, Yadu B.

    1995-11-01

    Equilibrium constants and enthalpy changes for reactions catalyzed by the isomerase and ligase classes of enzymes have been compiled. For each reaction the following information is given: the reference for the data; the reaction studied; the name of the enzyme used and its Enzyme Commission number; the method of measurement; the conditions of measurement (temperature, pH, ionic strength, and the buffer(s) and cofactor(s) used); the data and an evaluation of it; and, sometimes, commentary on the data and on any corrections which have been applied to it or any calculations for which the data have been used. The data from 176 references have been examined and evaluated. Chemical Abstract Service registry numbers are given for the substances involved in these various reactions. There is a cross reference between the substances and the Enzyme Commission numbers of the enzymes used to catalyze the reactions in which the substances participate.

  6. Redox-coupled structural changes of the catalytic a' domain of protein disulfide isomerase.

    PubMed

    Inagaki, Koya; Satoh, Tadashi; Yagi-Utsumi, Maho; Le Gulluche, Anne-Charlotte; Anzai, Takahiro; Uekusa, Yoshinori; Kamiya, Yukiko; Kato, Koichi

    2015-09-14

    Protein disulfide isomerase functions as a folding catalyst in the endoplasmic reticulum. Its b' and a' domains provide substrate-binding sites and undergo a redox-dependent domain rearrangement coupled to an open-closed structural change. Here we determined the first solution structure of the a' domain in its oxidized form and thereby demonstrate that oxidation of the a' domain induces significant conformational changes not only in the vicinity of the active site but also in the distal b'-interfacial segment. Based on these findings, we propose that this conformational transition triggers the domain segregation coupled with the exposure of the hydrophobic surface. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  7. Production of ethanol from D-xylose by using D-xylose isomerase and yeasts

    SciTech Connect

    Gong, C.S.; Chem, L.F.; Flickinger, M.C.; Chiang, L.C.; Tsao, G.T.

    1981-02-01

    D-xylulose, an intermediate of D-xylose catabolism, was observed to be fermentable to ethanol and carbon dioxide in a yield of greater than 80% by yeasts (including industrial bakers' yeast) under fermentative conditions. This conversion appears to be carried out by many yeasts known for D-glucose fermentation. In some yeasts, xylitol, in addition to ethanol, was produced from D-xylulose. Fermenting yeasts are also able to produce ethanol from D-xylose when D-xylose isomerizing enzyme is present. The results indicate that ethanol could be produced from D-xylose in a yield of greater than 80% by a two-step process. First. D-xylose is converted to D-xylulose by xylose isomerase. D-xylulose is then fermented to ethanol by yeasts.

  8. Lack of variation at phosphoglucose isomerase (pgi) in bumblebees: implications for conservation genetics studies.

    PubMed

    Ellis, Jonathan S; Turner, Lucy M; Knight, Mairi E

    2013-01-01

    Assessing genetic variation underlying ecologically important traits is increasingly of interest and importance in population and conservation genetics. For some groups generally useful markers exist for examining the relative role of selection and drift in shaping genetic diversity e.g. the major histocompatibility complex in vertebrates and self-incompatibility loci in plants. For invertebrates there is no such generally useful locus. However, phosphoglucose isomerase (Pgi) has been proposed as a useful functional marker in the conservation genetics of invertebrates. Where thermal microclimate varies, balanced polymorphisms may be maintained due to trade-offs between thermally stable and kinetically advantageous allelic forms. We here report very low levels of Pgi variation in bumblebees rendering this locus to be of little use as an adaptive marker in a conservation genetics context in this group. Potential explanations for this lack of variation are considered.

  9. Mannose supplements induce embryonic lethality and blindness in phosphomannose isomerase hypomorphic mice

    PubMed Central

    Sharma, Vandana; Nayak, Jonamani; DeRossi, Charles; Charbono, Adriana; Ichikawa, Mie; Ng, Bobby G.; Grajales-Esquivel, Erika; Srivastava, Anand; Wang, Ling; He, Ping; Scott, David A.; Russell, Joseph; Contreras, Emily; Guess, Cherise M.; Krajewski, Stan; Del Rio-Tsonis, Katia; Freeze, Hudson H.

    2014-01-01

    Patients with congenital disorder of glycosylation (CDG), type Ib (MPI-CDG or CDG-Ib) have mutations in phosphomannose isomerase (MPI) that impair glycosylation and lead to stunted growth, liver dysfunction, coagulopathy, hypoglycemia, and intestinal abnormalities. Mannose supplements correct hypoglycosylation and most symptoms by providing mannose-6-P (Man-6-P) via hexokinase. We generated viable Mpi hypomorphic mice with residual enzymatic activity comparable to that of patients, but surprisingly, these mice appeared completely normal except for modest (∼15%) embryonic lethality. To overcome this lethality, pregnant dams were provided 1–2% mannose in their drinking water. However, mannose further reduced litter size and survival to weaning by 40 and 66%, respectively. Moreover, ∼50% of survivors developed eye defects beginning around midgestation. Mannose started at birth also led to eye defects but had no effect when started after eye development was complete. Man-6-P and related metabolites accumulated in the affected adult eye and in developing embryos and placentas. Our results demonstrate that disturbing mannose metabolic flux in mice, especially during embryonic development, induces a highly specific, unanticipated pathological state. It is unknown whether mannose is harmful to human fetuses during gestation; however, mothers who are at risk for having MPI-CDG children and who consume mannose during pregnancy hoping to benefit an affected fetus in utero should be cautious.—Sharma, V., Nayak, J., DeRossi, C., Charbono, A., Ichikawa, M., Ng, B. G., Grajales-Esquivel, E., Srivastava, A., Wang, L., He, P., Scott, D. A., Russell, J., Contreras, E., Guess, C. M., Krajewski, S., Del Rio-Tsonis, K., Freeze, H. H. Mannose supplements induce embryonic lethality and blindness in phosphomannose isomerase hypomorphic mice. PMID:24421398

  10. Concerted Proton Transfer Mechanism of Clostridium thermocellum Ribose-5-phosphate Isomerase

    PubMed Central

    Wang, Jun; Yang, Weitao

    2013-01-01

    Ribose-5-phosphate isomerase (Rpi) catalyzes the interconversion of D-ribose-5-phosphate and D-ribulose-5-phosphate and plays an essential role in the pentose phosphate pathway and the Calvin cycle of photosynthesis. RpiB, one of the two isoforms of Rpi, is also a potential drug target for some pathogenic bacteria. Clostridium thermocellum ribose-5-phosphate isomerase (CtRpi), belonging to RpiB family, has recently been employed to the industrial production of rare sugars because of it fast reactions kinetics and narrow substrate specificity. It is known this enzyme adopts proton transfer mechanism. It was suggested that the deprotonated Cys65 attracts the proton at C2 of substrate to initiate the isomerization reaction and this step is the rate-limiting step. However the elaborate catalytic mechanism is still unclear. We have performed quantum mechanical/molecular mechanical simulations of this rate-limiting step of the reaction catalyzed by CtRpi with the substrate D-ribose. Our results demonstrate that the deprotonated Cys65 is not a stable reactant. Instead, our calculations revealed a concerted proton-transfer mechanism: Asp8, a highly conserved residue in the RpiB family performs as the base to abstract the proton at Cys65 and Cys65 in turn abstracts the proton of the D-ribose simultaneously. Moreover, we found Thr67 cannot catalyze the proton transfer from O2 to O1 of the D-ribose alone. Water molecule(s) may assist this proton transfer with Thr67. Our findings lead to a clear understanding of the catalysis mechanism of RpiB family and should guide the experiments to increase the catalysis efficiency. This study also highlights the importance of initial protonation states of cysteines. PMID:23875675

  11. Substrate specificity of platypus venom L-to-D-peptide isomerase.

    PubMed

    Bansal, Paramjit S; Torres, Allan M; Crossett, Ben; Wong, Karen K Y; Koh, Jennifer M S; Geraghty, Dominic P; Vandenberg, Jamie I; Kuchel, Philip W

    2008-04-04

    The L-to-D-peptide isomerase from the venom of the platypus (Ornithorhyncus anatinus) is the first such enzyme to be reported for a mammal. In delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used N-terminal segments of defensin-like peptides DLP-2 and DLP-4 and natriuretic peptide OvCNP from the venom as substrates. The DLP analogues IMFsrs and ImFsrs (srs is a solubilizing chain; lowercase letters denote D-amino acid) were effective substrates for the isomerase; it appears to recognize the N-terminal tripeptide sequence Ile-Xaa-Phe-. A suite of 26 mutants of these hexapeptides was synthesized by replacing the second residue (Met) with another amino acid, viz. Ala, alpha-aminobutyric acid, Ile, Leu, Lys, norleucine, Phe, Tyr, and Val. It was shown that mutant peptides incorporating norleucine and Phe are substrates and exhibit L- or D-amino acid isomerization, but mutant peptides that contain residues with shorter, beta-branched or long side chains with polar terminal groups, viz. Ala, alpha-aminobutyric acid, Ile, Val, Leu, Lys, and Tyr, respectively, are not substrates. It was demonstrated that at least three N-terminal amino acid residues are absolutely essential for L-to-D-isomerization; furthermore, the third amino acid must be a Phe residue. None of the hexapeptides based on LLH, the first three residues of OvCNP, were substrates. A consistent 2-base mechanism is proposed for the isomerization; abstraction of a proton by 1 base is concomitant with delivery of a proton by the conjugate acid of a second base.

  12. Characterization of an isopentenyl diphosphate isomerase involved in the juvenile hormone pathway in Aedes aegypti.

    PubMed

    Diaz, Miguel E; Mayoral, Jaime G; Priestap, Horacio; Nouzova, Marcela; Rivera-Perez, Crisalejandra; Noriega, Fernando G

    2012-10-01

    Isopentenyl diphosphate isomerase (IPPI) is an enzyme involved in the synthesis of juvenile hormone (JH) in the corpora allata (CA) of insects. IPPI catalyzes the conversion of isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP); afterward IPP and DMAPP condense in a head-to-tail manner to produce geranyl diphosphate (GPP), this head-to-tail condensation can be repeated, by the further reaction of GPP with IPP, yielding the JH precursor farnesyl diphosphate. An IPPI expressed sequence tag (EST) was obtained from an Aedes aegypti corpora-allata + corpora cardiaca library. Its full-length cDNA encodes a 244-aa protein that shows a high degree of similarity with type I IPPIs from other organisms, particularly for those residues that have important roles in catalysis, metal coordination and interaction with the diphosphate moiety of the IPP. Heterologous expression produced a recombinant protein that metabolized IPP into DMAPP; treatment of DMAPP with phosphoric acid produced isoprene, a volatile compound that was measured with an assay based on a solid-phase micro extraction protocol and direct analysis by gas chromatography. A. aegypti IPPI (AaIPPI) required Mg(2+) or Mn(2+) but not Zn(2+) for full activity and it was entirely inhibited by iodoacetamide. Real time PCR experiments showed that AaIPPI is highly expressed in the CA. Changes in AaIPPI mRNA levels in the CA in the pupal and adult female mosquito corresponded well with changes in JH synthesis (Li et al., 2003). This is the first molecular and functional characterization of an isopentenyl diphosphate isomerase involved in the production of juvenile hormone in the CA of an insect.

  13. Improved xylose fermentation of Kluyveromyces marxianus at elevated temperature through construction of a xylose isomerase pathway.

    PubMed

    Wang, Rongliang; Li, Lulu; Zhang, Biao; Gao, Xiaolian; Wang, Dongmei; Hong, Jiong

    2013-08-01

    To improve the xylose fermentation ability of Kluyveromyces marxianus, a xylose assimilation pathway through xylose isomerase was constructed. The genes encoding xylose reductase (KmXyl1) and xylitol dehydrogenase (KmXyl2) were disrupted in K. marxianus YHJ010 and the resultant strain was named YRL002. A codon-optimized xylose isomerase gene from Orpinomyces was transformed into K. marxianus YRL002 and expressed under GAPDH promoter. The transformant was adapted in the SD medium containing 1 % casamino acid with 2 % xylose as sole carbon source. After 32 times of trans-inoculation, a strain named YRL005, which can grow at a specific growth rate of 0.137/h with xylose as carbon source, was obtained. K. marxianus YRL005 could ferment 30.15 g/l of xylose and produce 11.52 g/l ethanol with a yield of 0.38 g/g, production rate of 0.069 g/l/h at 42 °C, and also could ferment 16.60 g/l xylose to produce 5.21 g/l ethanol with a yield of 0.31 g/g, and production rate of 0.054 g/l h at 45 °C. Co-fermentation with 2 % glucose could not improve the amount and yield of ethanol fermented from xylose obviously, but it could improve the production rate. Furthermore, K. marxianus YRL005 can ferment with the corn cob hydrolysate, which contained 20.04 g/l xylose to produce 8.25 g/l ethanol. It is a good platform to construct thermo-tolerant xylose fermentation yeast.

  14. Polyols accumulated in ribose-5-phosphate isomerase deficiency increase mitochondrial superoxide production and improve antioxidant defenses in rats' prefrontal cortex.

    PubMed

    Stone, V; Kudo, K Y; August, P M; Marcelino, T B; Matté, C

    2014-10-01

    The ribose-5-phosphate isomerase deficiency is an inherited condition, which results in cerebral d-arabitol and ribitol accumulation. Patients present leukoencephalopathy, mental retardation, and psychomotor impairment. Considering that the pathophysiology of this disorder is still unclear, and literature are sparse and contradictory, reporting pro and antioxidant activities of polyols, the main objective of this study was to investigate some parameters of oxidative homeostasis of prefrontal cortex of rats incubated with d-arabitol and ribitol. We found evidences that ribitol promoted an increase in antioxidant enzymes activity (superoxide dismutase, catalase, and glutathione peroxidase), probably secondary to enhanced production of superoxide radical, measured by flow cytometry. Oxidation of proteins and lipids was not induced by polyols. Our data allow us to conclude that, at least in our methodological conditions, arabitol and ribitol probably have a secondary effect on the pathophysiology of ribose-5-phosphate isomerase deficiency. Copyright © 2014 ISDN. Published by Elsevier Ltd. All rights reserved.

  15. Rational design of Bacillus stearothermophilus US100 L-arabinose isomerase: potential applications for D-tagatose production.

    PubMed

    Rhimi, Moez; Aghajari, Nushin; Juy, Michel; Chouayekh, Hichem; Maguin, Emmanuelle; Haser, Richard; Bejar, Samir

    2009-05-01

    L-arabinose isomerases catalyze the bioconversion of D-galactose into D-tagatose. With the aim of producing an enzyme optimized for D-tagatose production, three Bacillus stearothermophilus US100 L-arabinose isomerase mutants were constructed, purified and characterized. Our results indicate that mutant Q268K was significantly more acidotolerant and more stable at acidic pH than the wild-type enzyme. The N175H mutant has a broad optimal temperature range from 50 to 65 degrees C. With the aim of constructing an acidotolerant mutant working at relatively low temperatures we generated the Q268K/N175H construct. This double mutant displays an optimal pH in the range 6.0-7.0 and an optimal activity around 50-65 degrees C, temperatures at which the enzyme was stable without addition of metal ions.

  16. Sequential oxygenation of linoleic acid in the fungus Gaeumannomyces graminis: stereochemistry of dioxygenase and hydroperoxide isomerase reactions.

    PubMed

    Hamberg, M; Zhang, L Y; Brodowsky, I D; Oliw, E H

    1994-02-15

    Linoleic acid is sequentially oxygenated to (7S,8S)-dihydroxylinoleic acid by dioxygenase and hydroperoxide isomerase activities present in the fungus Gaeumannomyces graminis (Brodowsky, I. D., Hamberg, M., and Oliw, E. H., J. Biol. Chem. 267, 14738-14745 (1992)). Linoleic acids stereospecifically deuterated at C-7 and C-8 were prepared by biological desaturation of the corresponding stearates and used to determine the stereochemistry of the hydrogen abstractions occurring in the dioxygenase- and hydroperoxide isomerase-catalyzed reactions. The dioxygenase reaction was found to involve stereospecific abstraction of the pro-S hydrogen from C-8 followed by antarafacial insertion of dioxygen to produce (8R)-hydroperoxylinoleic acid. The hydroperoxide isomerase reaction consisted of conversion of (8R)-hydroperoxylinoleic acid into (7S,8S)-dihydroxylinoleic acid by stereospecific elimination of the pro-S hydrogen from C-7 and intramolecular suprafacial insertion of oxygen at C-7. Accordingly, during the conversion of linoleic acid into (8R)-hydroperoxylinoleic acid, the absolute configuration of C-8 was inverted, while the conversion of (8R)-hydroperoxylinoleic acid into (7S,8S)-dihydroxylinoleic acid occurred with retention of absolute configuration at C-7.

  17. Substrate specificity of a galactose 6-phosphate isomerase from Lactococcus lactis that produces d-allose from d-psicose.

    PubMed

    Park, Ha-Young; Park, Chang-Su; Kim, Hye-Jung; Oh, Deok-Kun

    2007-10-15

    We purified recombinant galactose 6-phosphate isomerase (LacAB) from Lactococcus lactis using HiTrap Q HP and Phenyl-Sepharose columns. The purified LacAB had a final specific activity of 1.79units/mg to produce d-allose. The molecular mass of native galactose 6-phosphate isomerase was estimated at 135.5kDa using Sephacryl S-300 gel filtration, and the enzyme exists as a hetero-octamer of LacA and LacB subunits. The activity of galactose 6-phosphate isomerase was maximal at pH 7.0 and 30 degrees C, and enzyme activity was independent of metal ions. When 100g/L of d-psicose was used as the substrate, 25g/L of d-allose and 13g/L of d-altrose were simultaneously produced at pH 7.0 and 30 degrees C after 12h of incubation. The enzyme had broad specificity for various aldoses and ketoses. The interconversion of sugars with the same configuration except at the C2 position was driven by using a large amount of enzyme in extended reactions. The interconversion occurred via two isomerization reactions, i.e., the interconversion of d-allose<-->d-psicose<-->d-altrose, and d-allose to d-psicose reaction was faster than d-altrose to d-psicose reaction.

  18. Revisiting the mechanistic basis of the French Paradox: Red wine inhibits the activity of protein disulfide isomerase in vitro.

    PubMed

    Galinski, Christine N; Zwicker, Jeffrey I; Kennedy, Daniel R

    2016-01-01

    Although epidemiologic evidence points to cardioprotective activity of red wine, the mechanistic basis for antithrombotic activity has not been established. Quercetin and related flavonoids are present in high concentrations in red but not white wine. Quercetin-glycosides were recently shown to prevent thrombosis in animal models through the inhibition of extracellular protein disulfide isomerase (PDI). We evaluated whether red or white wine inhibited PDI activity in vitro. Quercetin levels in red and white wines were measured by HPLC analysis. Inhibition of PDI activity by red and white wines was assessed by an insulin reduction turbidity assay at various concentrations of wine. PDI inhibition was confirmed using a reduced peptide that contained a disulfide containing peptide as a substrate. The inhibition of PDI related thiol isomerases ERp5 and ERp57 was also assessed. We observed a dose-dependent decrease of PDI activity for a variety of red but not white wines. Red wine diluted to 3% final concentration resulted in over 80% inhibition of PDI activity by insulin reductase assay for all varieties tested. This inhibition was also observed in the peptide based assay. Red grape juice yielded similar results but ethanol alone did not affect PDI activity. Interestingly, red wine also inhibited the PDI related thiol isomerases ERp5 and ERp57, albeit to a lesser degree than PDI. PDI activity is inhibited by red wine and grape juice, identifying a potentially novel mechanism underlying the cardiovascular benefits attributed to wine consumption. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. FK506 binding protein mutational analysis. Defining the surface residue contributions to stability of the calcineurin co-complex.

    PubMed

    Futer, O; DeCenzo, M T; Aldape, R A; Livingston, D J

    1995-08-11

    The 12- and 13-kDa FK506 binding proteins (FKBP12 and FKBP13) are cis-trans peptidyl-prolyl isomerases that bind the macrolides FK506 (Tacrolimus) and rapamycin (Sirolimus). The FKBP12.FK506 complex is immunosuppressive, acting as an inhibitor of the protein phosphatase calcineurin. We have examined the role of the key surface residues of FKBP12 and FKBP13 in calcineurin interactions by generating substitutions at these residues by site-directed mutagenesis. All mutants are active catalysts of the prolyl isomerase reaction, and bind FK506 or rapamycin with high affinity. Mutations at FKBP12 residues Asp-37, Arg-42, His-87, and Ile-90 decrease calcineurin affinity of the mutant FKBP12.FK506 complex by as much as 2600-fold in the case of I90K. Replacement of three FKBP13 surface residues (Gln-50, Ala-95, and Lys-98) with the corresponding homologous FKBP12 residues (Arg-42, His-87, and Ile-90) generates an FKBP13 variant that is equivalent to FKBP12 in its affinity for FK506, rapamycin, and calcineurin. These results confirm the role of two loop regions of FKBP12 (residues 40-44 and 84-91) as part of the effector face that interacts with calcineurin.

  20. Responses of Mytilus galloprovincialis to bacterial challenges by metabolomics and proteomics.

    PubMed

    Ji, Chenglong; Wu, Huifeng; Wei, Lei; Zhao, Jianmin; Wang, Qing; Lu, Hongjian

    2013-08-01

    Pathogens can cause diseases and lead to massive mortalities of aquaculture animals and substantial economic loss. In this work, we studied the responses induced by Micrococcus luteus and Vibrio anguillarum in gill of mussel Mytilus galloprovincialis at protein and metabolite levels. Metabolic biomarkers (e.g., amino acids, betaine, ATP) suggested that both M. luteus and V. anguillarum induced disturbances in energy metabolism and osmotic regulation. The unique and some more remarkably altered metabolic biomarkers (threonine, alanine, aspartate, taurine, succinate) demonstrated that V. anguillarum could cause more severe disturbances in osmotic regulation and energy metabolism. Proteomic biomarkers (e.g., goose-type lysozyme 2, matrilin, ependymin-related protein, peptidyl-prolyl cis-trans isomerases) indicated that M. luteus caused immune stress, and disturbances in signaling pathways and protein synthesis. However, V. anguillarum mainly induced oxidative stress and disturbance in energy metabolism in mussel gills indicated by altered procollagen-proline dioxygenase, protein disulfide isomerase, nucleoside diphosphate kinases, electron transfer flavoprotein and glutathione S-transferase. This work confirmed that an integration of proteomics and metabolomics could provide an insightful view into the effects of pathogens to the marine mussel M. galloprovincialis.

  1. Ligand-independent assembly of recombinant human CD1 by using oxidative refolding chromatography

    PubMed Central

    Altamirano, Myriam M.; Woolfson, Adrian; Donda, Alena; Shamshiev, Abdijapar; Briseño-Roa, Luis; Foster, Nicholas W.; Veprintsev, Dmitry B.; De Libero, Gennaro; Fersht, Alan R.; Milstein, César

    2001-01-01

    CD1 is an MHC class I-like antigen-presenting molecule consisting of a heavy chain and β2-microglobulin light chain. The in vitro refolding of synthetic MHC class I molecules has always required the presence of ligand. We report here the use of a folding method using an immobilized chaperone fragment, a protein disulphide isomerase, and a peptidyl-prolyl cis-trans isomerase (oxidative refolding chromatography) for the fast and efficient assembly of ligand-free and ligand-associated CD1a and CD1b, starting with material synthesized in Escherichia coli. The results suggest that “empty” MHC class I-like molecules can assemble and remain stable at physiological temperatures in the absence of ligand. The use of oxidative refolding chromatography thus is extended to encompass complex multisubunit proteins and specifically to members of the extensive, functionally diverse and important immunoglobulin supergene family of proteins, including those for which a ligand has yet to be identified. PMID:11248071

  2. Structure and dynamics of Pin1 during catalysis by NMR

    PubMed Central

    Labeikovsky, Wladimir; Eisenmesser, Elan Z.; Bosco, Daryl A.; Kern, Dorothee

    2009-01-01

    The link between internal enzyme motions and catalysis is poorly understood. Correlated motions in the us-ms timescale may be critical for enzyme function. We have characterized the backbone dynamics of the peptidyl-prolyl isomerase Pin1 catalytic domain in the free state and during catalysis. Pin1 is a prolyl isomerase of the parvulin family and specifically catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds. Pin1 has been shown to be essential for cell-cycle progression and to interact with the neuronal tau protein inhibiting its aggregation into fibrillar tangles as found in Alzheimer’s disease. 15N relaxation dispersion measurements performed on Pin1 during catalysis reveal conformational exchange processes in the microsecond timescale. A subset of active site residues undergo kinetically similar exchange processes even in the absence of substrate, suggesting that this area is already “primed” for catalysis. Furthermore, structural data of the turning-over enzyme were obtained through inter- and intra-molecular NOEs. This analysis together with a characterization of the substrate concentration dependence of the conformational exchange allowed distinguishing of regions of the enzyme active site that are affected primarily by substrate binding versus substrate isomerization. Together these data suggest a model for the reaction trajectory of Pin1 catalysis. PMID:17316687

  3. Structural Basis for High-Affinity Peptide Inhibition of Human Pin1

    PubMed Central

    Zhang, Yan; Daum, Sebastian; Wildemann, Dirk; Zhou, Xiao Zhen; Verdecia, Mark A.; Bowman, Marianne E.; Lücke, Christian; Hunter, Tony; Lu, Kun-Ping; Fischer, Gunter; Noel, Joseph P.

    2009-01-01

    Human Pin1 is a key regulator of cell-cycle progression and plays growth-promoting roles in human cancers. High-affinity inhibitors of Pin1 may provide a unique opportunity for disrupting oncogenic pathways. Here we report two high-resolution X-ray crystal structures of human Pin1 bound to non-natural peptide inhibitors. The structures of the bound high-affinity peptides identify a type-I β-turn conformation for Pin1 prolyl peptide isomerase domain–peptide binding and an extensive molecular interface for high-affinity recognition. Moreover, these structures suggest chemical elements that may further improve the affinity and pharmacological properties of future peptide-based Pin inhibitors. Finally, an intramolecular hydrogen bond observed in both peptide complexes mimics the cyclic conformation of FK506 and rapamycin. Both FK506 and rapamycin are clinically important inhibitors of other peptidyl-prolyl cis-trans isomerases. This comparative discovery suggests that a cyclic peptide polyketide bridge, like that found in FK506 and rapamycin or a similar linkage, may significantly improve the binding affinity of structure-based Pin1 inhibitors. PMID:17518432

  4. Pin1 down-regulates transforming growth factor-beta (TGF-beta) signaling by inducing degradation of Smad proteins.

    PubMed

    Nakano, Ayako; Koinuma, Daizo; Miyazawa, Keiji; Uchida, Takafumi; Saitoh, Masao; Kawabata, Masahiro; Hanai, Jun-ichi; Akiyama, Hirotada; Abe, Masahiro; Miyazono, Kohei; Matsumoto, Toshio; Imamura, Takeshi

    2009-03-06

    Transforming growth factor-beta (TGF-beta) is crucial in numerous cellular processes, such as proliferation, differentiation, migration, and apoptosis. TGF-beta signaling is transduced by intracellular Smad proteins that are regulated by the ubiquitin-proteasome system. Smad ubiquitin regulatory factor 2 (Smurf2) prevents TGF-beta and bone morphogenetic protein signaling by interacting with Smads and inducing their ubiquitin-mediated degradation. Here we identified Pin1, a peptidylprolyl cis-trans isomerase, as a novel protein binding Smads. Pin1 interacted with Smad2 and Smad3 but not Smad4; this interaction was enhanced by the phosphorylation of (S/T)P motifs in the Smad linker region. (S/T)P motif phosphorylation also enhanced the interaction of Smad2/3 with Smurf2. Pin1 reduced Smad2/3 protein levels in a manner dependent on its peptidyl-prolyl cis-trans isomerase activity. Knockdown of Pin1 increased the protein levels of endogenous Smad2/3. In addition, Pin1 both enhanced the interaction of Smurf2 with Smads and enhanced Smad ubiquitination. Pin1 inhibited TGF-beta-induced transcription and gene expression, suggesting that Pin1 negatively regulates TGF-beta signaling by down-regulating Smad2/3 protein levels via induction of Smurf2-mediated ubiquitin-proteasomal degradation.

  5. Investigation of the effect of nitrogen on severity of Fusarium head blight in barley.

    PubMed

    Yang, Fen; Jensen, Jens D; Spliid, Niels Henrik; Svensson, Birte; Jacobsen, Susanne; Jørgensen, Lise Nistrup; Jørgensen, Hans J L; Collinge, David B; Finnie, Christine

    2010-02-10

    The effect of nitrogen on Fusarium Head Blight (FHB) in a susceptible barley cultivar was investigated using gel-based proteomics. Barley grown with either 15 or 100kgha(-1)N fertilizer was inoculated with Fusarium graminearum (Fg). The storage protein fraction did not change significantly in response either to N level or Fg, whereas eighty protein spots in the water-soluble albumin fraction increased and 108 spots decreased more than two-fold in intensity in response to Fg. Spots with greater intensity in infected plants contained fungal proteins (9 spots) and proteolytic fragments of plant proteins (65 spots). Identified fungal proteins included two superoxide dismutases, L-xylulose reductase in two spots, peptidyl prolyl cis-trans isomerase and triosephosphate isomerase, and proteins of unknown function. Spots decreasing in intensity in response to Fg contained plant proteins possibly degraded by fungal proteases. Greater spot volume changes occurred in response to Fg in plants grown with low nitrogen, although proteomes of uninfected plants were similar for both treatments. Correlation of proteome changes with measurement of Fusarium-damaged kernels, fungal biomass and mycotoxin levels indicated that increased Fusarium infection occurred in barley with low N and suggests control of N fertilization as a possible way to minimise FHB in barley.

  6. Endoplasmic reticulum stress regulates rat mandibular cartilage thinning under compressive mechanical stress.

    PubMed

    Li, Huang; Zhang, Xiang-Yu; Wu, Tuo-Jiang; Cheng, Wei; Liu, Xin; Jiang, Ting-Ting; Wen, Juan; Li, Jie; Ma, Qiao-Ling; Hua, Zi-Chun

    2013-06-21

    Compressive mechanical stress-induced cartilage thinning has been characterized as a key step in the progression of temporomandibular joint diseases, such as osteoarthritis. However, the regulatory mechanisms underlying this loss have not been thoroughly studied. Here, we used an established animal model for loading compressive mechanical stress to induce cartilage thinning in vivo. The mechanically stressed mandibular chondrocytes were then isolated to screen potential candidates using a proteomics approach. A total of 28 proteins were identified that were directly or indirectly associated with endoplasmic reticulum stress, including protein disulfide-isomerase, calreticulin, translationally controlled tumor protein, and peptidyl-prolyl cis/trans-isomerase protein. The altered expression of these candidates was validated at both the mRNA and protein levels. The induction of endoplasmic reticulum stress by mechanical stress loading was confirmed by the activation of endoplasmic reticulum stress markers, the elevation of the cytoplasmic Ca(2+) level, and the expansion of endoplasmic reticulum membranes. More importantly, the use of a selective inhibitor to block endoplasmic reticulum stress in vivo reduced the apoptosis observed at the early stages of mechanical stress loading and inhibited the proliferation observed at the later stages of mechanical stress loading. Accordingly, the use of the inhibitor significantly restored cartilage thinning. Taken together, these results demonstrated that endoplasmic reticulum stress is significantly activated in mechanical stress-induced mandibular cartilage thinning and, more importantly, that endoplasmic reticulum stress inhibition alleviates this loss, suggesting a novel pharmaceutical strategy for the treatment of mechanical stress-induced temporomandibular joint diseases.

  7. Proteomic analysis of stress-related proteins in transgenic broccoli harboring a gene for cytokinin production during postharvest senescence.

    PubMed

    Liu, Mao-Sen; Li, Hui-Chun; Chang, You-Min; Wu, Min-Tze; Chen, Long-Fang Oliver

    2011-09-01

    Our previous study revealed a cytokinin-related retardation of post-harvest floret yellowing in transgenic broccoli (Brassica oleracea var. italica) that harbored the bacterial isopentenyltransferase (ipt) gene. We aimed to investigate the underlining mechanism of this delayed post-harvest senescence. We used 2D electrophoresis and liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry for a proteomics analysis of heads of ipt-transgenic and non-transgenic inbred lines of broccoli at harvest and after four days post-harvest storage. At harvest, we found an accumulation of stress-responsive proteins involved in maintenance of protein folding (putative protein disulfide isomerase, peptidyl-prolyl cis-trans isomerase and chaperonins), scavenging of reactive oxygen species (Mn superoxide dismutase), and stress protection [myrosinase-binding protein, jasmonate inducible protein, dynamin-like protein, NADH dehydrogenase (ubiquinone) Fe-S protein 1 and stress-inducible tetratricopeptide repeat-containing protein]. After four days' post-harvest storage of non-transgenic broccoli florets, the levels of proteins involved in protein folding and carbon fixation were decreased, which indicates cellular degradation and a change in metabolism toward senescence. In addition, staining for antioxidant enzyme activity of non-transgenic plants after post-harvest storage revealed a marked decrease in activity of Fe-superoxide dismutase and ascorbate peroxidase. Thus, the accumulation of stress-responsive proteins and antioxidant enzyme activity in ipt-transgenic broccoli are most likely associated with retardation of post-harvest senescence.

  8. Prolyl cis-trans isomerization as a molecular timer.

    PubMed

    Lu, Kun Ping; Finn, Greg; Lee, Tae Ho; Nicholson, Linda K

    2007-10-01

    Proline is unique in the realm of amino acids in its ability to adopt completely distinct cis and trans conformations, which allows it to act as a backbone switch that is controlled by prolyl cis-trans isomerization. This intrinsically slow interconversion can be catalyzed by the evolutionarily conserved group of peptidyl prolyl cis-trans isomerase enzymes. These enzymes include cyclophilins and FK506-binding proteins, which are well known for their isomerization-independent role as cellular targets for immunosuppressive drugs. The significance of enzyme-catalyzed prolyl cis-trans isomerization as an important regulatory mechanism in human physiology and pathology was not recognized until the discovery of the phosphorylation-specific prolyl isomerase Pin1. Recent studies indicate that both phosphorylation-dependent and phosphorylation-independent prolyl cis-trans isomerization can act as a novel molecular timer to help control the amplitude and duration of a cellular process, and prolyl cis-trans isomerization might be a new target for therapeutic interventions.

  9. The Mechanism of the Reaction Catalyzed by Uronate Isomerase Illustrates How an Isomerase May Have Evolved from a Hydrolase within the Amidohydrolase Superfamily

    SciTech Connect

    Nguyen, T.; Fedorov, A; Williams, L; Fedorov, E; Li, Y; Xu, C; Almo, S; Raushel, F

    2009-01-01

    Uronate isomerase (URI) catalyzes the reversible isomerization of d-glucuronate to d-fructuronate and of d-galacturonate to d-tagaturonate. URI is a member of the amidohydrolase superfamily (AHS), a highly divergent group of enzymes that catalyze primarily hydrolytic reactions. The chemical mechanism and active site structure of URI were investigated in an attempt to improve our understanding of how an active site template that apparently evolved to catalyze hydrolytic reactions has been reforged to catalyze an isomerization reaction. The pH-rate profiles for kcat and kcat/Km for URI from Escherichia coli are bell-shaped and indicate that one group must be unprotonated and another residue must be protonated for catalytic activity. Primary isotope effects on the kinetic constants with [2-2H]-d-glucuronate and the effects of changes in solvent viscosity are consistent with product release being the rate-limiting step. The X-ray structure of Bh0493, a URI from Bacillus halodurans, was determined in the presence of the substrate d-glucuronate. The bound complex showed that the mononuclear metal center in the active site is ligated to the C-6 carboxylate and the C-5 hydroxyl group of the substrate. This hydroxyl group is also hydrogen bonded to Asp-355 in the same orientation as the hydroxide or water is bound in those members of the AHS that catalyze hydrolytic reactions. In addition, the C-2 and C-3 hydroxyl groups of the substrate are hydrogen bonded to Arg-357 and the carbonyl group at C-1 is hydrogen bonded to Tyr-50. A chemical mechanism is proposed that utilizes a proton transfer from C-2 of d-glucuronate to C-1 that is initiated by the combined actions of Asp-355 from the end of ?-strand 8 and the C-5 hydroxyl of the substrate that is bound to the metal ion. The formation of the proposed cis-enediol intermediate is further facilitated by the shuttling of the proton between the C-2 and C-1 oxygens by the conserved Tyr-50 and/or Arg-355.

  10. Erythritol feeds the pentose phosphate pathway via three new isomerases leading to D-erythrose-4-phosphate in Brucella

    PubMed Central

    Barbier, Thibault; Collard, François; Zúñiga-Ripa, Amaia; Moriyón, Ignacio; Godard, Thibault; Becker, Judith; Wittmann, Christoph; Van Schaftingen, Emile; Letesson, Jean-Jacques

    2014-01-01

    Erythritol is an important nutrient for several α-2 Proteobacteria, including N2-fixing plant endosymbionts and Brucella, a worldwide pathogen that finds this four-carbon polyol in genital tissues. Erythritol metabolism involves phosphorylation to l-erythritol-4-phosphate by the kinase EryA and oxidation of the latter to l-3-tetrulose 4-phosphate by the dehydrogenase EryB. It is accepted that further steps involve oxidation by the putative dehydrogenase EryC and subsequent decarboxylation to yield triose-phosphates. Accordingly, growth on erythritol as the sole C source should require aldolase and fructose-1,6-bisphosphatase to produce essential hexose-6-monophosphate. However, we observed that a mutant devoid of fructose-1,6-bisphosphatases grew normally on erythritol and that EryC, which was assumed to be a dehydrogenase, actually belongs to the xylose isomerase superfamily. Moreover, we found that TpiA2 and RpiB, distant homologs of triose phosphate isomerase and ribose 5-phosphate isomerase B, were necessary, as previously shown for Rhizobium. By using purified recombinant enzymes, we demonstrated that l-3-tetrulose-4-phosphate was converted to d-erythrose 4-phosphate through three previously unknown isomerization reactions catalyzed by EryC (tetrulose-4-phosphate racemase), TpiA2 (d-3-tetrulose-4-phosphate isomerase; renamed EryH), and RpiB (d-erythrose-4-phosphate isomerase; renamed EryI), a pathway fully consistent with the isotopomer distribution of the erythrose-4-phosphate-derived amino acids phenylalanine and tyrosine obtained from bacteria grown on 13C-labeled erythritol. d-Erythrose-4-phosphate is then converted by enzymes of the pentose phosphate pathway to glyceraldehyde 3-phosphate and fructose 6-phosphate, thus bypassing fructose-1,6-bisphosphatase. This is the first description to our knowledge of a route feeding carbohydrate metabolism exclusively via d-erythrose 4-phosphate, a pathway that may provide clues to the preferential metabolism of

  11. Metabolic fate of unsaturated glucuronic/iduronic acids from glycosaminoglycans: molecular identification and structure determination of streptococcal isomerase and dehydrogenase.

    PubMed

    Maruyama, Yukie; Oiki, Sayoko; Takase, Ryuichi; Mikami, Bunzo; Murata, Kousaku; Hashimoto, Wataru

    2015-03-06

    Glycosaminoglycans in mammalian extracellular matrices are degraded to their constituents, unsaturated uronic (glucuronic/iduronic) acids and amino sugars, through successive reactions of bacterial polysaccharide lyase and unsaturated glucuronyl hydrolase. Genes coding for glycosaminoglycan-acting lyase, unsaturated glucuronyl hydrolase, and the phosphotransferase system are assembled into a cluster in the genome of pathogenic bacteria, such as streptococci and clostridia. Here, we studied the streptococcal metabolic pathway of unsaturated uronic acids and the structure/function relationship of its relevant isomerase and dehydrogenase. Two proteins (gbs1892 and gbs1891) of Streptococcus agalactiae strain NEM316 were overexpressed in Escherichia coli, purified, and characterized. 4-Deoxy-l-threo-5-hexosulose-uronate (Dhu) nonenzymatically generated from unsaturated uronic acids was converted to 2-keto-3-deoxy-d-gluconate via 3-deoxy-d-glycero-2,5-hexodiulosonate through successive reactions of gbs1892 isomerase (DhuI) and gbs1891 NADH-dependent reductase/dehydrogenase (DhuD). DhuI and DhuD enzymatically corresponded to 4-deoxy-l-threo-5-hexosulose-uronate ketol-isomerase (KduI) and 2-keto-3-deoxy-d-gluconate dehydrogenase (KduD), respectively, involved in pectin metabolism, although no or low sequence identity was observed between DhuI and KduI or between DhuD and KduD, respectively. Genes for DhuI and DhuD were found to be included in the streptococcal genetic cluster, whereas KduI and KduD are encoded in clostridia. Tertiary and quaternary structures of DhuI and DhuD were determined by x-ray crystallography. Distinct from KduI β-barrels, DhuI adopts an α/β/α-barrel structure as a basic scaffold similar to that of ribose 5-phosphate isomerase. The structure of DhuD is unable to accommodate the substrate/cofactor, suggesting that conformational changes are essential to trigger enzyme catalysis. This is the first report on the bacterial metabolism of

  12. Erythritol feeds the pentose phosphate pathway via three new isomerases leading to D-erythrose-4-phosphate in Brucella.

    PubMed

    Barbier, Thibault; Collard, François; Zúñiga-Ripa, Amaia; Moriyón, Ignacio; Godard, Thibault; Becker, Judith; Wittmann, Christoph; Van Schaftingen, Emile; Letesson, Jean-Jacques

    2014-12-16

    Erythritol is an important nutrient for several α-2 Proteobacteria, including N2-fixing plant endosymbionts and Brucella, a worldwide pathogen that finds this four-carbon polyol in genital tissues. Erythritol metabolism involves phosphorylation to L-erythritol-4-phosphate by the kinase EryA and oxidation of the latter to L-3-tetrulose 4-phosphate by the dehydrogenase EryB. It is accepted that further steps involve oxidation by the putative dehydrogenase EryC and subsequent decarboxylation to yield triose-phosphates. Accordingly, growth on erythritol as the sole C source should require aldolase and fructose-1,6-bisphosphatase to produce essential hexose-6-monophosphate. However, we observed that a mutant devoid of fructose-1,6-bisphosphatases grew normally on erythritol and that EryC, which was assumed to be a dehydrogenase, actually belongs to the xylose isomerase superfamily. Moreover, we found that TpiA2 and RpiB, distant homologs of triose phosphate isomerase and ribose 5-phosphate isomerase B, were necessary, as previously shown for Rhizobium. By using purified recombinant enzymes, we demonstrated that L-3-tetrulose-4-phosphate was converted to D-erythrose 4-phosphate through three previously unknown isomerization reactions catalyzed by EryC (tetrulose-4-phosphate racemase), TpiA2 (D-3-tetrulose-4-phosphate isomerase; renamed EryH), and RpiB (D-erythrose-4-phosphate isomerase; renamed EryI), a pathway fully consistent with the isotopomer distribution of the erythrose-4-phosphate-derived amino acids phenylalanine and tyrosine obtained from bacteria grown on (13)C-labeled erythritol. D-erythrose-4-phosphate is then converted by enzymes of the pentose phosphate pathway to glyceraldehyde 3-phosphate and fructose 6-phosphate, thus bypassing fructose-1,6-bisphosphatase. This is the first description to our knowledge of a route feeding carbohydrate metabolism exclusively via D-erythrose 4-phosphate, a pathway that may provide clues to the preferential metabolism of

  13. Pin1 Modulates the Synaptic Content of NMDA Receptors via Prolyl-Isomerization of PSD-95.

    PubMed

    Antonelli, Roberta; De Filippo, Roberto; Middei, Silvia; Stancheva, Stefka; Pastore, Beatrice; Ammassari-Teule, Martine; Barberis, Andrea; Cherubini, Enrico; Zacchi, Paola

    2016-05-18

    Phosphorylation of serine/threonine residues preceding a proline regulates the fate of its targets through postphosphorylation conformational changes catalyzed by the peptidyl-prolyl cis-/trans isomerase Pin1. By flipping the substrate between two different functional conformations, this enzyme exerts a fine-tuning of phosphorylation signals. Pin1 has been detected in dendritic spines and shafts where it regulates protein synthesis required to sustain the late phase of long-term potentiation (LTP). Here, we demonstrate that Pin1 residing in postsynaptic structures can interact with postsynaptic density protein-95 (PSD-95), a key scaffold protein that anchors NMDA receptors (NMDARs) in PSD via GluN2-type receptor subunits. Pin1 recruitment by PSD-95 occurs at specific serine-threonine/proline consensus motifs localized in the linker region connecting PDZ2 to PDZ3 domains. Upon binding, Pin1 triggers structural changes in PSD-95, thus negatively affecting its ability to interact with NMDARs. In electrophysiological experiments, larger NMDA-mediated synaptic currents, evoked in CA1 principal cells by Schaffer collateral stimulation, were detected in hippocampal slices obtained from Pin1(-/-) mice compared with controls. Similar results were obtained in cultured hippocampal cells expressing a PSD-95 mutant unable to undergo prolyl-isomerization, thus indicating that the action of Pin1 on PSD-95 is critical for this effect. In addition, an enhancement in spine density and size was detected in CA1 principal cells of Pin1(-/-) or in Thy-1GFP mice treated with the pharmacological inhibitor of Pin1 catalytic activity PiB.Our data indicate that Pin1 controls synaptic content of NMDARs via PSD-95 prolyl-isomerization and the expression of dendritic spines, both required for LTP maintenance. PSD-95, a membrane-associated guanylate kinase, is the major scaffolding protein at excitatory postsynaptic densities and a potent regulator of synaptic strength and plasticity. The

  14. Functions of FKBP12 and Mitochondrial Cyclophilin Active Site Residues In Vitro and In Vivo in Saccharomyces cerevisiae

    PubMed Central

    Dolinski, Kara; Scholz, Christian; Muir, R. Scott; Rospert, Sabine; Schmid, Franz X.; Cardenas, Maria E.; Heitman, Joseph

    1997-01-01

    Cyclophilin and FK506 binding protein (FKBP) accelerate cis–trans peptidyl-prolyl isomerization and bind to and mediate the effects of the immunosuppressants cyclosporin A and FK506. The normal cellular functions of these proteins, however, are unknown. We altered the active sites of FKBP12 and mitochondrial cyclophilin from the yeast Saccharomyces cerevisiae by introducing mutations previously reported to inactivate these enzymes. Surprisingly, most of these mutant enzymes were biologically active in vivo. In accord with previous reports, all of the mutant enzymes had little or no detectable prolyl isomerase activity in the standard peptide substrate-chymotrypsin coupled in vitro assay. However, in a variation of this assay in which the protease is omitted, the mutant enzymes exhibited substantial levels of prolyl isomerase activity (5–20% of wild-type), revealing that these mutations confer sensitivity to protease digestion and that the classic in vitro assay for prolyl isomerase activity may be misleading. In addition, the mutant enzymes exhibited near wild-type activity with two protein substrates, dihydrofolate reductase and ribonuclease T1, whose folding is accelerated by prolyl isomerases. Thus, a number of cyclophilin and FKBP12 “active-site” mutants previously identified are largely active but protease sensitive, in accord with our findings that these mutants display wild-type functions in vivo. One mitochondrial cyclophilin mutant (R73A), and also the wild-type human FKBP12 enzyme, catalyze protein folding in vitro but lack biological activity in vivo in yeast. Our findings provide evidence that both prolyl isomerase activity and other structural features are linked to FKBP and cyclophilin in vivo functions and suggest caution in the use of these active-site mutations to study FKBP and cyclophilin functions. PMID:9362068

  15. Inhibitors of protein disulfide isomerase suppress apoptosis induced by misfolded proteins

    PubMed Central

    Hoffstrom, Benjamin G.; Kaplan, Anna; Letso, Reka; Schmid, Ralf; Turmel, Gregory J.; Lo, Donald C.; Stockwell, Brent R.

    2010-01-01

    A hallmark of many neurodegenerative diseases is accumulation of misfolded proteins within neurons, leading to cellular dysfunction and cell death. Although several mechanisms have been proposed to link protein misfolding to cellular toxicity, the connection remains enigmatic. Here, we report a cell death pathway involving protein disulfide isomerase (PDI), a protein chaperone that catalyzes isomerization, reduction, and oxidation of disulfides. Through a small-molecule-screening approach, we discovered five structurally distinct compounds that prevent apoptosis induced by mutant huntingtin protein. Using modified Huisgen cycloaddition chemistry, we then identified PDI as the molecular target of these small molecules. Expression of polyglutamine-expanded huntingtin exon 1 in PC12 cells caused PDI to accumulate at mitochondrial-associated-ER-membranes and trigger apoptotic cell death, via mitochondrial outer membrane permeabilization. Inhibiting PDI in rat brain cells suppressed the toxicity of mutant huntingtin exon1 and Aβ peptides processed from the amyloid precursor protein. This pro-apoptotic function of PDI provides a new mechanism linking protein misfolding and apoptotic cell death. PMID:21079601

  16. Identification of triosephosphate isomerase as a novel allergen in Octopus fangsiao.

    PubMed

    Yang, Yang; Chen, Zhong-Wei; Hurlburt, Barry K; Li, Gui-Ling; Zhang, Yong-Xia; Fei, Dan-Xia; Shen, Hai-Wang; Cao, Min-Jie; Liu, Guang-Ming

    2017-02-13

    Octopus is an important mollusk in human dietary for its nutritional value, however it also causes allergic reactions in humans. Major allergens from octopus have been identified, while the knowledge of novel allergens remains poor. In the present study, a novel allergen with molecular weight of 28kDa protein was purified from octopus (Octopus fangsiao) and identified as triosephosphate isomerase (TIM) by mass spectrometry. TIM aggregated beyond 45°C, and its IgE-binding activity was affected under extreme pH conditions due to the altered secondary structure. In simulated gastric fluid digestion, TIM can be degraded into small fragments, while retaining over 80% of the IgE-binding activity. The full-length cDNA of O. fangsiao TIM (1140bp) was cloned, which encodes 247 amino acid residues, and the entire recombinant TIM was successfully expressed in Escherichia coli BL21, which showed similar immunoreactivity to the native TIM. Different intensity of cross-reactivity among TIM from related species revealed the complexity of its epitopes. Eight linear epitopes of TIM were predicted following bioinformatic analysis. Furthermore, a conformational epitope (A71G74S69D75T73F72V67) was confirmed by the phage display technology. The results revealed the physicochemical and immunological characteristics of TIM, which is significant in the development of hyposensitivity food and allergy diagnosis.

  17. Crystal structure of native RPE65, the retinoid isomerase of the visual cycle

    SciTech Connect

    Kiser, Philip D.; Golczak, Marcin; Lodowski, David T.; Chance, Mark R.; Palczewski, Krzysztof

    2009-12-01

    Vertebrate vision is maintained by the retinoid (visual) cycle, a complex enzymatic pathway that operates in the retina to regenerate the visual chromophore, 11-cis-retinal. A key enzyme in this pathway is the microsomal membrane protein RPE65. This enzyme catalyzes the conversion of all-trans-retinyl esters to 11-cis-retinol in the retinal pigment epithelium (RPE). Mutations in RPE65 are known to be responsible for a subset of cases of the most common form of childhood blindness, Leber congenital amaurosis (LCA). Although retinoid isomerase activity has been attributed to RPE65, its catalytic mechanism remains a matter of debate. Also, the manner in which RPE65 binds to membranes and extracts retinoid substrates is unclear. To gain insight into these questions, we determined the crystal structure of native bovine RPE65 at 2.14-{angstrom} resolution. The structural, biophysical, and biochemical data presented here provide the framework needed for an in-depth understanding of the mechanism of catalytic isomerization and membrane association, in addition to the role mutations that cause LCA have in disrupting protein function.

  18. Protein disulfide isomerase externalization in endothelial cells follows classical and unconventional routes.

    PubMed

    Araujo, Thaís L S; Zeidler, Julianna D; Oliveira, Percíllia V S; Dias, Matheus H; Armelin, Hugo A; Laurindo, Francisco R M

    2017-02-01

    Extracellular protein disulfide isomerase (PDIA1) pool mediates thrombosis and vascular remodeling, however its externalization mechanisms remain unclear. We performed systematic pharmacological screening of secretory pathways affecting extracellular PDIA1 in endothelial cells (EC). We identified cell-surface (csPDIA1) and secreted non-particulated PDIA1 pools in EC. Such Golgi bypass also occurred for secreted PDIA1 in EC at baseline or after PMA, thrombin or ATP stimulation. Inhibitors of Type I, II and III unconventional routes, secretory lysosomes and recycling endosomes, including syntaxin-12 deletion, did not impair EC PDIA1 externalization. This suggests predominantly Golgi-independent unconventional secretory route(s), which were GRASP55-independent. Also, these data reinforce a vesicular-type traffic for PDIA1. We further showed that PDIA1 traffic is ATP-independent, while actin or tubulin cytoskeletal disruption markedly increased EC PDIA1 secretion. Clathrin inhibition enhanced extracellular soluble PDIA1, suggesting dynamic cycling. Externalized PDIA1 represents <2% of intracellular PDIA1. PDIA1 was robustly secreted by physiological levels of arterial laminar shear in EC and supported alpha 5 integrin thiol oxidation. Such results help clarify signaling and homeostatic mechanisms involved in multiple (patho)physiological extracellular PDIA1 functions. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Analysis of the interaction of calcitriol with the disulfide isomerase ERp57

    NASA Astrophysics Data System (ADS)

    Gaucci, Elisa; Raimondo, Domenico; Grillo, Caterina; Cervoni, Laura; Altieri, Fabio; Nittari, Giulio; Eufemi, Margherita; Chichiarelli, Silvia

    2016-11-01

    Calcitriol, the active form of vitamin D3, can regulate the gene expression through the binding to the nuclear receptor VDR, but it can also display nongenomic actions, acting through a membrane-associated receptor, which has been discovered as the disulfide isomerase ERp57. The aim of our research is to identify the binding sites for calcitriol in ERp57 and to analyze their interaction. We first studied the interaction through bioinformatics and fluorimetric analyses. Subsequently, we focused on two protein mutants containing the predicted interaction domains with calcitriol: abb’-ERp57, containing the first three domains, and a’-ERp57, the fourth domain only. To consolidate the achievements we used the calorimetric approach to the whole protein and its mutants. Our results allow us to hypothesize that the interaction with the a’ domain contributes to a greater extent than the other potential binding sites to the dissociation constant, calculated as a Kd of about 10‑9 M.

  20. A novel function of tissue-type transglutaminase: protein disulphide isomerase.

    PubMed Central

    Hasegawa, Go; Suwa, Motoi; Ichikawa, Yasuo; Ohtsuka, Tetsuro; Kumagai, Satoru; Kikuchi, Masashi; Sato, Yoshitaka; Saito, Yuji

    2003-01-01

    We have found that tissue-type transglutaminase (tTG), also called TGc, TGase2 and Galpha(h), has the activity of protein disulphide isomerase (PDI). We have shown that tTG converts completely reduced/denatured inactive RNase A molecule to the native active enzyme. The PDI activity of tTG was strongly inhibited by bacitracin, which is a frequently used inhibitor of conventional PDI activity. It was substantially inhibited by the simultaneous presence of other potential substrate proteins such as completely reduced BSA, but not by native BSA. This activity was especially high in the presence of GSSG, but not GSH. The addition of GSH to the reaction mixture in the presence of GSSG at a fixed concentration up to at least 200-fold excess did not very substantially inhibit the PDI activity. It is possible that tTG can exert PDI activity in a fairly reducing environment like cytosol, where most of tTG is found. It is quite obvious from the following observations that PDI activity of tTG is catalysed by a domain different from that used for the transglutaminase reaction. Although the alkylation of Cys residues in tTG completely abolished the transglutaminase activity, as was expected, it did not affect the PDI activity at all. This PDI activity did not require the presence of Ca(2+). It was not inhibited by nucleotides including GTP at all, unlike the other activity of tTG. PMID:12737632

  1. Elicitor-mediated induction of chalcone isomerase in Phaseolus vulgaris cell suspension cultures.

    PubMed

    Dixon, R A; Gerrish, C; Lamb, C J; Robbins, M P

    1983-12-01

    Approximately fourfold increases in the extractable activity of the enzyme chalcone isomerase (CHI, EC 5.5.1.6) were observed within 24 h of treatment of cell suspension cultures of Phaseolus vulgaris with a crude elicitor preparation heatreleased from the cell walls of the bean pathogen Colletotrichum lindemuthianum. The induction of CHI activity was highly dependent upon elicitor concentration, with maximum induction occurring in two discrete concentration ranges. A basal half-life for CHI>32 h in control cultures was determined by labelling with (2)H from (2)H2O followed by analysis of the equilibrium distribution of enzyme activity in CsCl density gradients. Comparative density labelling indicated that at both the lower and higher effective elicitor concentrations, the induced appearance of CHI activity was the result of an apparent initial activation of pre-existing enzyme followed by an increase in the rate of de-novo synthesis of the enzyme as compared with non-elicited controls. The increased appearance of the enzyme over the first 8 h in elicitor-treated cultures was inhibited by cycloheximide, cordycepin and actinomycin D. The results are discussed in relation to the mechanisms of co-ordinate enzyme induction operating in French-bean cell cultures exposed to fungal elicitors.

  2. Characterization of Triosephosphate Isomerase Mutants with Reduced Enzyme Activity in Mus Musculus

    PubMed Central

    Merkle, S.; Pretsch, W.

    1989-01-01

    Four heterozygous triosephosphate isomerase (TPI) mutants with approximately 50% reduced activity in blood compared to wild type were detected in offspring of 1-ethyl-1-nitrosourea treated male mice. Breeding experiments displayed an autosomal, dominant mode of inheritance for the mutations. All mutations were found to be homozygous lethal at an early postimplantation stage of embryonic development, probably due to a total lack of TPI activity and consequently to the inability to utilize glucose as a source of metabolic energy. Although activity alteration was also found in liver, lung, kidney, spleen, heart, brain and muscle the TPI deficiency in heterozygotes has no influence on the following physiological traits: hematological parameters, plasma glucose, glucose consumption of blood cells, body weight and organo-somatic indices of liver, spleen, heart, kidney and lung. Biochemical investigations of TPI in the four mutant lines indicated no difference of physicochemical properties compared to the wild type. Results from immunoinactivation assays indicate that the decrease of enzyme activity corresponds to a decrease in the level of an immunologically active moiety. It is suggested that the mutations have affected the Tpi-1 structural locus and resulted in alleles which produce no detectable enzyme activity and no immunologically cross-reacting material. The study furthermore suggests one functional TPI gene per haploid genome in the erythrocyte and seven other tested organs of the mouse. PMID:2693209

  3. A direct, continuous, sensitive assay for protein disulphide-isomerase based on fluorescence self-quenching.

    PubMed

    Raturi, Arun; Vacratsis, Panayiotis O; Seslija, Dana; Lee, Lana; Mutus, Bulent

    2005-10-15

    PDI (protein disulphide-isomerase) activity is generally monitored by insulin turbidity assay or scrambled RNase assay, both of which are performed by UV-visible spectroscopy. In this paper, we present a sensitive fluorimetric assay for continuous determination of disulphide reduction activity of PDI. This assay utilizes the pseudo-substrate diabz-GSSG [where diabz stands for di-(o-aminobenzoyl)], which is formed by the reaction of isatoic anhydride with the two free N-terminal amino groups of GSSG. The proximity of two benzoyl groups leads to quenching of the diabz-GSSG fluorescence by approx. 50% in comparison with its non-disulphide-linked form, abz-GSH (where abz stands for o-aminobenzoyl). Therefore the PDI-dependent disulphide reduction can be monitored by the increase in fluorescence accompanying the loss of proximity-quenching upon conversion of diabz-GSSG into abz-GSH. The apparent K(m) of PDI for diabz-GSSG was estimated to be approx. 15 muM. Unlike the insulin turbidity assay and scrambled RNase assay, the diabz-GSSG-based assay was shown to be effective in determining a single turnover of enzyme in the absence of reducing agents with no appreciable blank rates. The assay is simple to perform and very sensitive, with an estimated detection limit of approx. 2.5 nM PDI, enabling its use for the determination of platelet surface PDI activity in crude sample preparations.

  4. Triosephosphate Isomerase and Filamin C Share Common Epitopes as Novel Allergens of Procambarus clarkii.

    PubMed

    Yang, Yang; Zhang, Yong-Xia; Liu, Meng; Maleki, Soheila J; Zhang, Ming-Li; Liu, Qing-Mei; Cao, Min-Jie; Su, Wen-Jin; Liu, Guang-Ming

    2017-02-01

    Triosephosphate isomerase (TIM) is a key enzyme in glycolysis and has been identified as an allergen in saltwater products. In this study, TIM with a molecular mass of 28 kDa was purified from the freshwater crayfish (Procambarus clarkii) muscle. A 90-kDa protein that showed IgG/IgE cross-reactivity with TIM was purified and identified as filamin C (FLN c), which is an actin-binding protein. TIM showed similar thermal and pH stability with better digestion resistance compared with FLN c. The result of the surface plasmon resonance (SPR) experiment demonstrated the infinity of anti-TIM polyclonal antibody (pAb) to both TIM and FLN c. Five linear and 3 conformational epitopes of TIM, as well as 9 linear and 10 conformational epitopes of FLN c, were mapped by phage display. Epitopes of TIM and FLN c demonstrated the sharing of certain residues; the occurrence of common epitopes in the two allergens accounts for their cross-reactivity.

  5. Prolyl-isomerase Pin1 controls Notch3 protein expression and regulates T-ALL progression

    PubMed Central

    Franciosa, G; Diluvio, G; Gaudio, F Del; Giuli, M V; Palermo, R; Grazioli, P; Campese, A F; Talora, C; Bellavia, D; D'Amati, G; Besharat, Z M; Nicoletti, C; Siebel, C W; Choy, L; Rustighi, A; Sal, G Del; Screpanti, I; Checquolo, S

    2016-01-01

    Deregulated Notch signaling is associated with T-cell Acute Lymphoblastic Leukemia (T-ALL) development and progression. Increasing evidence reveals that Notch pathway has an important role in the invasion ability of tumor cells, including leukemia, although the underlying molecular mechanisms remain mostly unclear. Here, we show that Notch3 is a novel target protein of the prolyl-isomerase Pin1, which is able to regulate Notch3 protein processing and to stabilize the cleaved product, leading to the increased expression of the intracellular domain (N3IC), finally enhancing Notch3-dependent invasiveness properties. We demonstrate that the combined inhibition of Notch3 and Pin1 in the Notch3-overexpressing human leukemic TALL-1 cells reduces their high invasive potential, by decreasing the expression of the matrix metalloprotease MMP9. Consistently, Pin1 depletion in a mouse model of Notch3-induced T-ALL, by reducing N3IC expression and signaling, impairs the expansion/invasiveness of CD4+CD8+ DP cells in peripheral lymphoid and non-lymphoid organs. Notably, in in silico gene expression analysis of human T-ALL samples we observed a significant correlation between Pin1 and Notch3 expression levels, which may further suggest a key role of the newly identified Notch3-Pin1 axis in T-ALL aggressiveness and progression. Thus, combined suppression of Pin1 and Notch3 proteins may be exploited as an additional target therapy for T-ALL. PMID:26876201

  6. Human Eosinophil Leukocytes Express Protein Disulfide Isomerase in Secretory Granules and Vesicles: Ultrastructural Studies.

    PubMed

    Dias, Felipe F; Amaral, Kátia B; Carmo, Lívia A S; Shamri, Revital; Dvorak, Ann M; Weller, Peter F; Melo, Rossana C N

    2014-06-01

    Protein disulfide isomerase (PDI) has fundamental roles in the oxidative folding of proteins in the endoplasmic reticulum (ER) of eukaryotic cells. The study of this molecule has been attracting considerable attention due to its association with other cell functions and human diseases. In leukocytes, such as neutrophils, PDI is involved with cell adhesion, signaling and inflammation. However, the expression of PDI in other leukocytes, such as eosinophils, important cells in inflammatory, allergic and immunomodulatory responses, remains to be defined. Here we used different approaches to investigate PDI expression within human eosinophils. Western blotting and flow cytometry demonstrated high PDI expression in both unstimulated and CCL11/eotaxin-1-stimulated eosinophils, with similar levels in both conditions. By using an immunogold electron microscopy technique that combines better epitope preservation and secondary Fab-fragments of antibodies linked to 1.4-nm gold particles for optimal access to microdomains, we identified different intracellular sites for PDI. In addition to predictable strong PDI labeling at the nuclear envelope, other unanticipated sites, such as secretory granules, lipid bodies and vesicles, including large transport vesicles (eosinophil sombrero vesicles), were also labeled. Thus, we provide the first identification of PDI in human eosinophils, suggesting that this molecule may have additional/specific functions in these leukocytes.

  7. Mapping of domains on HIV envelope protein mediating association with calnexin and protein-disulfide isomerase.

    PubMed

    Papandréou, Marie-Jeanne; Barbouche, Rym; Guieu, Régis; Rivera, Santiago; Fantini, Jacques; Khrestchatisky, Michel; Jones, Ian M; Fenouillet, Emmanuel

    2010-04-30

    The cell catalysts calnexin (CNX) and protein-disulfide isomerase (PDI) cooperate in establishing the disulfide bonding of the HIV envelope (Env) glycoprotein. Following HIV binding to lymphocytes, cell-surface PDI also reduces Env to induce the fusogenic conformation. We sought to define the contact points between Env and these catalysts to illustrate their potential as therapeutic targets. In lysates of Env-expressing cells, 15% of the gp160 precursor, but not gp120, coprecipitated with CNX, whereas only 0.25% of gp160 and gp120 coprecipitated with PDI. Under in vitro conditions, which mimic the Env/PDI interaction during virus/cell contact, PDI readily associated with Env. The domains of Env interacting in cellulo with CNX or in vitro with PDI were then determined using anti-Env antibodies whose binding site was occluded by CNX or PDI. Antibodies against domains V1/V2, C2, and the C terminus of V3 did not bind CNX-associated Env, whereas those against C1, V1/V2, and the CD4-binding domain did not react with PDI-associated Env. In addition, a mixture of the latter antibodies interfered with PDI-mediated Env reduction. Thus, Env interacts with intracellular CNX and extracellular PDI via discrete, largely nonoverlapping, regions. The sites of interaction explain the mode of action of compounds that target these two catalysts and may enable the design of further new competitive agents.

  8. Effects of a Buried Cysteine-To-Serine Mutation on Yeast Triosephosphate Isomerase Structure and Stability

    PubMed Central

    Hernández-Santoyo, Alejandra; Domínguez-Ramírez, Lenin; Reyes-López, César A.; González-Mondragón, Edith; Hernández-Arana, Andrés; Rodríguez-Romero, Adela

    2012-01-01

    All the members of the triosephosphate isomerase (TIM) family possess a cystein residue (Cys126) located near the catalytically essential Glu165. The evolutionarily conserved Cys126, however, does not seem to play a significant role in the catalytic activity. On the other hand, substitution of this residue by other amino acid residues destabilizes the dimeric enzyme, especially when Cys is replaced by Ser. In trying to assess the origin of this destabilization we have determined the crystal structure of Saccharomyces cerevisiae TIM (ScTIM) at 1.86 Å resolution in the presence of PGA, which is only bound to one subunit. Comparisons of the wild type and mutant structures reveal that a change in the orientation of the Ser hydroxyl group, with respect to the Cys sulfhydryl group, leads to penetration of water molecules and apparent destabilization of residues 132–138. The latter results were confirmed by means of Molecular Dynamics, which showed that this region, in the mutated enzyme, collapses at about 70 ns. PMID:22949845

  9. [Bioconversion of D-fructose to D-allose by novel isomerases].

    PubMed

    Bai, Wei; Zhu, Yueming; Men, Yan; Li, Xiaobo; Izumori, Ken; Sun, Yuanxia

    2012-04-01

    Rare sugar is a kind of important low-energy monosaccharide that is rarely found in nature and difficult to synthesize chemically. D-allose, a six-carbon aldose, is an important rare sugar with unique physiological functions. It is radical scavenging active and can inhibit cancer cell proliferation. To obtain D-allose, the microorganisms deriving D-psicose 3-epimerase (DPE) and L-rhamnose isomerase (L-RhI) have drawn intense attention. In this paper, DPE from Clostridium cellulolyticum H10 was cloned and expressed in Bacillus subtilis, and L-RhI from Bacillus subtilis 168 was cloned and expressed in Escherichia coli BL21 (DE3). The obtained crude DPE and L-RhI were then purified through a HisTrap HP affinity chromatography column and an anion-exchange chromatography column. The purified DPE and L-RhI were employed for the production of rare sugars at last, in which DPE catalyzed D-fructose into D-psicose while L-RhI converted D-psicose into D-allose. The conversion of D-fructose into D-psicose by DPE was 27.34%, and the conversion of D-psicose into D-allose was 34.64%.

  10. Inhibition of triosephosphate isomerase by phosphoenolpyruvate in the feedback-regulation of glycolysis.

    PubMed

    Grüning, Nana-Maria; Du, Dijun; Keller, Markus A; Luisi, Ben F; Ralser, Markus

    2014-03-05

    The inhibition of triosephosphate isomerase (TPI) in glycolysis by the pyruvate kinase (PK) substrate phosphoenolpyruvate (PEP) results in a newly discovered feedback loop that counters oxidative stress in cancer and actively respiring cells. The mechanism underlying this inhibition is illuminated by the co-crystal structure of TPI with bound PEP at 1.6 Å resolution, and by mutational studies guided by the crystallographic results. PEP is bound to the catalytic pocket of TPI and occludes substrate, which accounts for the observation that PEP competitively inhibits the interconversion of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Replacing an isoleucine residue located in the catalytic pocket of TPI with valine or threonine altered binding of substrates and PEP, reducing TPI activity in vitro and in vivo. Confirming a TPI-mediated activation of the pentose phosphate pathway (PPP), transgenic yeast cells expressing these TPI mutations accumulate greater levels of PPP intermediates and have altered stress resistance, mimicking the activation of the PK-TPI feedback loop. These results support a model in which glycolytic regulation requires direct catalytic inhibition of TPI by the pyruvate kinase substrate PEP, mediating a protective metabolic self-reconfiguration of central metabolism under conditions of oxidative stress.

  11. Protein disulphide isomerase as a target for nanoparticle-mediated sensitisation of cancer cells to radiation

    NASA Astrophysics Data System (ADS)

    Taggart, L. E.; McMahon, S. J.; Butterworth, K. T.; Currell, F. J.; Schettino, G.; Prise, K. M.

    2016-05-01

    Radiation resistance and toxicity in normal tissues are limiting factors in the efficacy of radiotherapy. Gold nanoparticles (GNPs) have been shown to be effective at enhancing radiation-induced cell death, and were initially proposed to physically enhance the radiation dose deposited. However, biological responses of GNP radiosensitization based on physical assumptions alone are not predictive of radiosensitisation and therefore there is a fundamental research need to determine biological mechanisms of response to GNPs alone and in combination with ionising radiation. This study aimed to identify novel mechanisms of cancer cell radiosensitisation through the use of GNPs, focusing on their ability to induce cellular oxidative stress and disrupt mitochondrial function. Using N-acetyl-cysteine, we found mitochondrial oxidation to be a key event prior to radiation for the radiosensitisation of cancer cells and suggests the overall cellular effects of GNP radiosensitisation are a result of their interaction with protein disulphide isomerase (PDI). This investigation identifies PDI and mitochondrial oxidation as novel targets for radiosensitisation.

  12. Functional Role of the Disulfide Isomerase ERp57 in Axonal Regeneration

    PubMed Central

    Woehlbier, Ute; Rozas, Pablo; Andreu, Catherine; Medinas, Danilo; Valdés, Pamela; Osorio, Fabiola; Mercado, Gabriela; Vidal, René L.; Kerr, Bredford; Court, Felipe A.; Hetz, Claudio

    2015-01-01

    ERp57 (also known as grp58 and PDIA3) is a protein disulfide isomerase that catalyzes disulfide bonds formation of glycoproteins as part of the calnexin and calreticulin cycle. ERp57 is markedly upregulated in most common neurodegenerative diseases downstream of the endoplasmic reticulum (ER) stress response. Despite accumulating correlative evidence supporting a neuroprotective role of ERp57, the contribution of this foldase to the physiology of the nervous system remains unknown. Here we developed a transgenic mouse model that overexpresses ERp57 in the nervous system under the control of the prion promoter. We analyzed the susceptibility of ERp57 transgenic mice to undergo neurodegeneration. Unexpectedly, ERp57 overexpression did not affect dopaminergic neuron loss and striatal denervation after injection of a Parkinson’s disease-inducing neurotoxin. In sharp contrast, ERp57 transgenic animals presented enhanced locomotor recovery after mechanical injury to the sciatic nerve. These protective effects were associated with enhanced myelin removal, macrophage infiltration and axonal regeneration. Our results suggest that ERp57 specifically contributes to peripheral nerve regeneration, whereas its activity is dispensable for the survival of a specific neuronal population of the central nervous system. These results demonstrate for the first time a functional role of a component of the ER proteostasis network in peripheral nerve regeneration. PMID:26361352

  13. Local variability of the phosphoglycerate kinase-triosephosphate isomerase fusion protein from Thermotoga maritima MSB 8.

    PubMed

    Wassenberg, D; Wuhrer, M; Beaucamp, N; Schurig, H; Wozny, M; Reusch, D; Fabry, S; Jaenicke, R

    2001-04-01

    The pgk-tpi gene locus of Thermotoga maritima encodes both phosphoglycerate kinase (PGK) and a bienzyme complex consisting of a fusion protein of PGK with triosephosphate isomerase (TIM). No separate tpi gene for TIM is present in T. maritima. A frame-shift at the end of the pgk gene has been previously proposed as a mechanism to regulate the expression of the two protein variants [Schurig et al., EMBO J. 14 (1995), 442-451]. Surprisingly, the complete T. maritima genome was found to contain a pgk-tpi sequence not requiring the proposed frameshift mechanism. To clarify the apparent discrepancy, a variety of DNA sequencing techniques were applied, disclosing an anomalous local variability in the pgk-tpi fusion region. The comparison of different DNA samples and the mass spectrometric analysis of the amino acid sequence of the natural fusion protein from T. maritima MSB8 confirmed the local variability of the DNA variants. Since not all peptide masses could be assigned, further variations are conceivable, suggesting an even higher heterogeneity of the T. maritima MSB8 strain.

  14. Competitive inhibition of phosphoglucose isomerase of apple leaves by sorbitol 6-phosphate.

    PubMed

    Zhou, Rui; Cheng, Lailiang

    2008-06-16

    Apple leaf cytosolic phosphoglucose isomerase (PGI, EC 5.3.1.9) was purified to an apparent homogeneity with a specific activity of 2456 units/mg protein, and chloroplastic PGI was partially purified to a specific activity of 72 units/mg protein to characterize their biochemical properties. These two isoforms showed differential responses to heat treatment; incubation at 50 degrees C for 10 min resulted in a complete loss of the chloroplastic PGI activity, whereas the cytosolic PGI only lost 50% of its activity. Apple cytosolic PGI is a dimeric enzyme with a molecular mass of 66 kDa for each monomer. The activity of both isoforms was strongly inhibited by erythrose 4-phosphate (E4P) with a K(i) of 1.2 and 3.0 microM for the cytosolic PGI and chloroplastic PGI, respectively. Sorbitol 6-phosphate (Sor6P), an intermediate in sorbitol biosynthesis, was found to be a competitive inhibitor for both cytosolic and chloroplastic PGIs with a K(i) of 61 and 40 microM, respectively. PGIs from both spinach and tomato leaves were also inhibited by Sor6P in a similar manner. The possible physiological significance of this finding is discussed.

  15. Protein disulfide isomerase secretion following vascular injury initiates a regulatory pathway for thrombus formation

    PubMed Central

    Bowley, Sheryl R.; Fang, Chao; Merrill-Skoloff, Glenn; Furie, Barbara C.; Furie, Bruce

    2017-01-01

    Protein disulfide isomerase (PDI), secreted by platelets and endothelial cells on vascular injury, is required for thrombus formation. Using PDI variants that form mixed disulfide complexes with their substrates, we identify by kinetic trapping multiple substrate proteins, including vitronectin. Plasma vitronectin does not bind to αvβ3 or αIIbβ3 integrins on endothelial cells and platelets. The released PDI reduces disulfide bonds on plasma vitronectin, enabling vitronectin to bind to αVβ3 and αIIbβ3. In vivo studies of thrombus generation in mice demonstrate that vitronectin rapidly accumulates on the endothelium and the platelet thrombus following injury. This process requires PDI activity and promotes platelet accumulation and fibrin generation. We hypothesize that under physiologic conditions in the absence of secreted PDI, thrombus formation is suppressed and maintains a quiescent, patent vasculature. The release of PDI during vascular injury may serve as a regulatory switch that allows activation of proteins, among them vitronectin, critical for thrombus formation. PMID:28218242

  16. Cloning and characterization of a sucrose isomerase from Erwinia rhapontici NX-5 for isomaltulose hyperproduction.

    PubMed

    Li, Sha; Cai, Heng; Qing, Yujia; Ren, Ben; Xu, Hong; Zhu, Hongyang; Yao, Jun

    2011-01-01

    The sucrose isomerase (SIase) gene from an efficient strain of Erwinia rhapontici NX-5 for isomaltulose hyperproduction was cloned and overexpressed in Escherichia coli. Protein sequence alignment revealed that SIase was a member of the glycoside hydrolase 13 family. The molecular mass of the purified recombinant protein was estimated at 66 kDa by SDS-PAGE. The SIase had an optimal pH and temperature of 5.0 and 30 °C, respectively, with a K (m) of 257 mmol/l and V (max) of 48.09 μmol/l/s for sucrose. To the best of our knowledge, the recombinant SIase has the most acidic optimum pH for isomaltulose synthesis. When the recombinant E. coli (pET22b- palI) cells were used for isomaltulose synthesis, almost complete conversion of sucrose (550 g/l solution) to isomaltulose was achieved in 1.5 h with high isomaltulose yields (87%). The immobilized E. coli cells remained stable for more than 30 days in a "batch"-type enzyme reactor. This indicated that the recombinant SIase could continuously and efficiently produce isomaltulose.

  17. Functional characterization of the sucrose isomerase responsible for trehalulose production in plant-associated Pectobacterium species.

    PubMed

    Nam, Cheon-Hyeon; Seo, Dong-Ho; Jung, Jong-Hyun; Koh, Young-Jin; Jung, Jae-Sung; Heu, Sunggi; Oh, Chang-Sik; Park, Cheon-Seok

    2014-02-05

    Fifty-three plant-associated microorganisms were investigated for their ability to convert sucrose to its isomers. These microorganisms included one Dickeya zeae isolate and 7 Enterobacter, 3 Pantoea, and 43 Pectobacterium species. Eleven out of the 53 strains (21%) showed the ability to transform sucrose to isomaltulose and trehalulose. Among those, Pectobacterium carotovorum KKH 3-1 showed the highest bioconversion yield (97.4%) from sucrose to its isomers. In this strain, the addition of up to 14% sucrose in the medium enhanced sucrose isomerase (SIase) production. The SIase activity at 14% sucrose (47.6 U/mg dcw) was about 3.6-fold higher than that of the negative control (13.3 U/mg dcw at 0% sucrose). The gene encoding SIase, which is comprised a 1776 bp open reading frame (ORF) encoding 591 amino acids, was cloned from P. carotovorum KKH 3-1 and expressed in Escherichia coli. The recombinant SIase (PCSI) was shown to have optimum activity at pH 6.0 and 40 °C. The reaction temperature significantly affected the ratio of sucrose isomers produced by PCSI. The amount of trehalulose increased from 47.5% to 79.1% as temperature was lowered from 50 °C to 30 °C, implying that SIase activity can be controlled by reaction temperature.

  18. The Ess1 prolyl isomerase: Traffic cop of the RNA polymerase II transcription

    PubMed Central

    Hanes, Steven D.

    2014-01-01

    Ess1 is a prolyl isomerase that regulates the structure and function of eukaryotic RNA polymerase II. Ess1 works by catalyzing the cis/trans conversion of pSer5–Pro6 bonds, and to a lesser extent pSer2–Pro3 bonds, within the carboxy-terminal domain (CTD) of Rpb1, the largest subunit of RNA pol II. Ess1 is conserved in organisms ranging from yeast to humans. In budding yeast, Ess1 is essential for growth and is required for efficient transcription initiation and termination, RNA processing, and suppression of cryptic transcription. In mammals, Ess1 (called Pin1) functions in a variety of pathways, including transcription, but it is not essential. Recent work has shown that Ess1 coordinates the binding and release of CTD-binding proteins that function as co-factors in the RNA pol II complex. In this way, Ess1 plays an integral role in writing (and reading) the so-called CTD code to promote production of mature RNA pol II transcripts including non-coding RNAs and mRNAs. PMID:24530645

  19. Novel giardicidal compounds bearing proton pump inhibitor scaffold proceeding through triosephosphate isomerase inactivation.

    PubMed

    Hernández-Ochoa, B; Navarrete-Vázquez, G; Nava-Zuazo, C; Castillo-Villanueva, A; Méndez, S T; Torres-Arroyo, A; Gómez-Manzo, S; Marcial-Quino, J; Ponce-Macotela, M; Rufino-González, Y; Martínez-Gordillo, M; Palencia-Hernández, G; Esturau-Escofet, N; Calderon-Jaimes, E; Oria-Hernández, J; Reyes-Vivas, H

    2017-08-10

    Giardiasis is a worldwide parasitic disease that affects mainly children and immunosuppressed people. Side effects and the emergence of resistance over current used drugs make imperative looking for new antiparasitics through discovering of new biological targets and designing of novel drugs. Recently, it has determined that gastric proton-pump inhibitors (PPI) have anti-giardiasic activity. The glycolytic enzyme, triosephosphate isomerase (GlTIM), is one of its potential targets. Therefore, we employed the scaffold of PPI to design new compounds aimed to increase their antigiardial capacity by inactivating GlTIM. Here we demonstrated that two novel PPI-derivatives (BHO2 and BHO3), have better anti-giardiasic activity than omeprazole in concentrations around 120-130 µM, without cytotoxic effect on mammal cell cultures. The derivatives inactivated GlTIM through the chemical modification of Cys222 promoting local structural changes in the enzyme. Furthermore, derivatives forms adducts linked to Cys residues through a C-S bond. We demonstrated that PPI can be used as scaffolds to design better antiparasitic molecules; we also are proposing a molecular mechanism of reaction for these novel derivatives.

  20. Potential Role of Protein Disulfide Isomerase in Metabolic Syndrome-Derived Platelet Hyperactivity

    PubMed Central

    Gaspar, Renato Simões

    2016-01-01

    Metabolic Syndrome (MetS) has become a worldwide epidemic, alongside with a high socioeconomic cost, and its diagnostic criteria must include at least three out of the five features: visceral obesity, hypertension, dyslipidemia, insulin resistance, and high fasting glucose levels. MetS shows an increased oxidative stress associated with platelet hyperactivation, an essential component for thrombus formation and ischemic events in MetS patients. Platelet aggregation is governed by the peroxide tone and the activity of Protein Disulfide Isomerase (PDI) at the cell membrane. PDI redox active sites present active cysteine residues that can be susceptible to changes in plasma oxidative state, as observed in MetS. However, there is a lack of knowledge about the relationship between PDI and platelet hyperactivation under MetS and its metabolic features, in spite of PDI being a mediator of important pathways implicated in MetS-induced platelet hyperactivation, such as insulin resistance and nitric oxide dysfunction. Thus, the aim of this review is to analyze data available in the literature as an attempt to support a possible role for PDI in MetS-induced platelet hyperactivation. PMID:28053690

  1. Overexpression, purification, crystallization and preliminary diffraction studies of the Protaminobacter rubrum sucrose isomerase SmuA.

    PubMed

    Ravaud, Stéphanie; Watzlawick, Hildegard; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

    2006-01-01

    Palatinose (isomaltulose, alpha-D-glucosylpyranosyl-1,6-D-fructofuranose), a nutritional and acariogenic reducing sugar, is industrially obtained from sucrose by using immobilized cells of Protaminobacter rubrum that produce the sucrose isomerase SmuA. The isomerization of sucrose catalyzed by this enzyme also results in the formation of trehalulose (alpha-D-glucosylpyranosyl-1,1-D-fructofuranose) in smaller amounts and glucose, fructose and eventually isomaltose as by-products, which lower the yield of the reaction and complicate the recovery of palatinose. The determination of the three-dimensional structure of SmuA will provide a basis for rational protein-engineering studies in order to optimize the industrial production of palatinose. A recombinant form of the 67.3 kDa SmuA enzyme has been crystallized in the native state by the vapour-diffusion method. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 61.6, b = 81.4, c = 135.6 A, and diffract to 1.95 A resolution on a synchrotron-radiation source.

  2. Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site

    PubMed Central

    Sigala, Paul A.; Fafarman, Aaron T.; Schwans, Jason P.; Fried, Stephen D.; Fenn, Timothy D.; Caaveiro, Jose M. M.; Pybus, Brandon; Ringe, Dagmar; Petsko, Gregory A.; Boxer, Steven G.; Herschlag, Daniel

    2013-01-01

    Hydrogen bond networks are key elements of protein structure and function but have been challenging to study within the complex protein environment. We have carried out in-depth interrogations of the proton transfer equilibrium within a hydrogen bond network formed to bound phenols in the active site of ketosteroid isomerase. We systematically varied the proton affinity of the phenol using differing electron-withdrawing substituents and incorporated site-specific NMR and IR probes to quantitatively map the proton and charge rearrangements within the network that accompany incremental increases in phenol proton affinity. The observed ionization changes were accurately described by a simple equilibrium proton transfer model that strongly suggests the intrinsic proton affinity of one of the Tyr residues in the network, Tyr16, does not remain constant but rather systematically increases due to weakening of the phenol–Tyr16 anion hydrogen bond with increasing phenol proton affinity. Using vibrational Stark spectroscopy, we quantified the electrostatic field changes within the surrounding active site that accompany these rearrangements within the network. We were able to model these changes accurately using continuum electrostatic calculations, suggesting a high degree of conformational restriction within the protein matrix. Our study affords direct insight into the physical and energetic properties of a hydrogen bond network within a protein interior and provides an example of a highly controlled system with minimal conformational rearrangements in which the observed physical changes can be accurately modeled by theoretical calculations. PMID:23798390

  3. Molecular dynamics simulations of "loop closing" in the enzyme triose phosphate isomerase.

    PubMed

    Brown, F K; Kollman, P A

    1987-12-05

    We present molecular dynamics simulations on the active site region of dimeric triose phosphate isomerase (TIM) using the co-ordinates of native chicken muscle TIM as a starting point and performing simulations with no substrate, with dihydroxyacetone phosphate (DHAP), the natural substrate, and with dihydroxyacetone sulfate (DHAS), a substrate analog. Whereas most of the protein moves less than 1 A during the simulation, some residues in the active site loop move more than 8 A during the 10.5 picoseconds of dynamics for each of the simulations. Most interestingly, the nature of the loop motion depends on the substrate, with the largest motion found in the presence of DHAP, and only in the presence of DHAP does the loop move to "close off" the active site pocket. The final structure found for the DHAP-chicken TIM complex is qualitatively similar to that described by Alber et al. for DHAP-yeast TIM. Simulations on the monomeric protein gives insight into why the molecule is active only as a dimer.

  4. Prolyl Isomerase Pin1 Regulates Neuronal Differentiation via β-Catenin

    PubMed Central

    Nakamura, Kazuhiro; Kosugi, Isao; Lee, Daniel Y.; Hafner, Angela; Sinclair, David A.

    2012-01-01

    The Wnt/β-catenin pathway promotes proliferation of neural progenitor cells (NPCs) at early stages and induces neuronal differentiation from NPCs at late stages, but the molecular mechanisms that control this stage-specific response are unclear. Pin1 is a prolyl isomerase that regulates cell signaling uniquely by controlling protein conformation after phosphorylation, but its role in neuronal differentiation is not known. Here we found that whereas Pin1 depletion suppresses neuronal differentiation, Pin1 overexpression enhances it, without any effects on gliogenesis from NPCs in vitro. Consequently, Pin1-null mice have significantly fewer upper layer neurons in the motor cortex and severely impaired motor activity during the neonatal stage. A proteomic approach identified β-catenin as a major substrate for Pin1 in NPCs, in which Pin1 stabilizes β-catenin. As a result, Pin1 knockout leads to reduced β-catenin during differentiation but not proliferation of NPCs in developing brains. Importantly, defective neuronal differentiation in Pin1 knockout NPCs is fully rescued in vitro by overexpression of β-catenin but not a β-catenin mutant that fails to act as a Pin1 substrate. These results show that Pin1 is a novel regulator of NPC differentiation by acting on β-catenin and provides a new postphosphorylation signaling mechanism to regulate developmental stage-specific functioning of β-catenin signaling in neuronal differentiation. PMID:22645310

  5. Arabidopsis Responds to Alternaria alternata Volatiles by Triggering Plastid Phosphoglucose Isomerase-Independent Mechanisms1[OPEN

    PubMed Central

    Sánchez-López, Ángela María; Bahaji, Abdellatif; De Diego, Nuria; Baslam, Marouane; Li, Jun; Almagro, Goizeder; García-Gómez, Pablo; Ricarte-Bermejo, Adriana; Novák, Ondřej; Spíchal, Lukáš; Ciordia, Sergio; Mena, María Carmen

    2016-01-01

    Volatile compounds (VCs) emitted by phylogenetically diverse microorganisms (including plant pathogens and microbes that do not normally interact mutualistically with plants) promote photosynthesis, growth, and the accumulation of high levels of starch in leaves through cytokinin (CK)-regulated processes. In Arabidopsis (Arabidopsis thaliana) plants not exposed to VCs, plastidic phosphoglucose isomerase (pPGI) acts as an important determinant of photosynthesis and growth, likely as a consequence of its involvement in the synthesis of plastidic CKs in roots. Moreover, this enzyme plays an important role in connecting the Calvin-Benson cycle with the starch biosynthetic pathway in leaves. To elucidate the mechanisms involved in the responses of plants to microbial VCs and to investigate the extent of pPGI involvement, we characterized pPGI-null pgi1-2 Arabidopsis plants cultured in the presence or absence of VCs emitted by Alternaria alternata. We found that volatile emissions from this fungal phytopathogen promote growth, photosynthesis, and the accumulation of plastidic CKs in pgi1-2 leaves. Notably, the mesophyll cells of pgi1-2 leaves accumulated exceptionally high levels of starch following VC exposure. Proteomic analyses revealed that VCs promote global changes in the expression of proteins involved in photosynthesis, starch metabolism, and growth that can account for the observed responses in pgi1-2 plants. The overall data show that Arabidopsis plants can respond to VCs emitted by phytopathogenic microorganisms by triggering pPGI-independent mechanisms. PMID:27663407

  6. Human Polymorphisms in the Glutathione Transferase Zeta 1/Maleylacetoacetate Isomerase Gene Influence the Toxicokinetics of Dichloroacetate

    PubMed Central

    Shroads, Albert L.; Langaee, Taimour; Coats, Bonnie S.; Kurtz, Tracie L.; Bullock, John R.; Weithorn, David; Gong, Yan; Wagner, David A.; Ostrov, David A.; Johnson, Julie A.; Stacpoole, Peter W.

    2013-01-01

    Dichloroacetate (DCA), a chemical relevant to environmental science and allopathic medicine, is dehalogenated by the bifunctional enzyme glutathione transferase zeta (GSTz1) maleylacetoacetate isomerase (MAAI), the penultimate enzyme in the phenylalanine/tyrosine catabolic pathway. The authors postulated that polymorphisms in GSTz1/MAAI modify the toxicokinetics of DCA. GSTz1/MAAI haplotype significantly affected the kinetics and biotransformation of 1,2-13C-DCA when it was administered at either environmentally (μg/kg/d) or clinically (mg/kg/d) relevant doses. GSTz1/MAAI haplotype also influenced the urinary accumulation of potentially toxic tyrosine metabolites. Atomic modeling revealed that GSTz1/MAAI variants associated with the slowest rates of DCA metabolism induced structural changes in the enzyme homodimer, predicting protein instability or abnormal protein-protein interactions. Knowledge of the GSTz1/MAAI haplotype can be used prospectively to identify individuals at potential risk of DCA’s adverse side effects from environmental or clinical exposure or who may exhibit aberrant amino acid metabolism in response to dietary protein. PMID:21642471

  7. Overexpression, purification, crystallization and preliminary diffraction studies of the Protaminobacter rubrum sucrose isomerase SmuA

    PubMed Central

    Ravaud, Stéphanie; Watzlawick, Hildegard; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

    2006-01-01

    Palatinose (isomaltulose, α-d-glucosylpyranosyl-1,6-d-fructofuranose), a nutritional and acariogenic reducing sugar, is industrially obtained from sucrose by using immobilized cells of Protaminobacter rubrum that produce the sucrose isomerase SmuA. The isomerization of sucrose catalyzed by this enzyme also results in the formation of trehalulose (α-d-glucosylpyranosyl-1,1-d-fructofuranose) in smaller amounts and glucose, fructose and eventually isomaltose as by-products, which lower the yield of the reaction and complicate the recovery of palatinose. The determination of the three-dimensional structure of SmuA will provide a basis for rational protein-engineering studies in order to optimize the industrial production of palatinose. A recombinant form of the 67.3 kDa SmuA enzyme has been crystallized in the native state by the vapour-diffusion method. Crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 61.6, b = 81.4, c = 135.6 Å, and diffract to 1.95 Å resolution on a synchrotron-radiation source. PMID:16511267

  8. Mannose supplements induce embryonic lethality and blindness in phosphomannose isomerase hypomorphic mice.

    PubMed

    Sharma, Vandana; Nayak, Jonamani; DeRossi, Charles; Charbono, Adriana; Ichikawa, Mie; Ng, Bobby G; Grajales-Esquivel, Erika; Srivastava, Anand; Wang, Ling; He, Ping; Scott, David A; Russell, Joseph; Contreras, Emily; Guess, Cherise M; Krajewski, Stan; Del Rio-Tsonis, Katia; Freeze, Hudson H

    2014-04-01

    Patients with congenital disorder of glycosylation (CDG), type Ib (MPI-CDG or CDG-Ib) have mutations in phosphomannose isomerase (MPI) that impair glycosylation and lead to stunted growth, liver dysfunction, coagulopathy, hypoglycemia, and intestinal abnormalities. Mannose supplements correct hypoglycosylation and most symptoms by providing mannose-6-P (Man-6-P) via hexokinase. We generated viable Mpi hypomorphic mice with residual enzymatic activity comparable to that of patients, but surprisingly, these mice appeared completely normal except for modest (~15%) embryonic lethality. To overcome this lethality, pregnant dams were provided 1-2% mannose in their drinking water. However, mannose further reduced litter size and survival to weaning by 40 and 66%, respectively. Moreover, ~50% of survivors developed eye defects beginning around midgestation. Mannose started at birth also led to eye defects but had no effect when started after eye development was complete. Man-6-P and related metabolites accumulated in the affected adult eye and in developing embryos and placentas. Our results demonstrate that disturbing mannose metabolic flux in mice, especially during embryonic development, induces a highly specific, unanticipated pathological state. It is unknown whether mannose is harmful to human fetuses during gestation; however, mothers who are at risk for having MPI-CDG children and who consume mannose during pregnancy hoping to benefit an affected fetus in utero should be cautious.

  9. Characterization of stress and methylglyoxal inducible triose phosphate isomerase (OscTPI) from rice

    PubMed Central

    Sharma, Shweta; Mustafiz, Ananda; Singla-Pareek, Sneh L.; Shankar Srivastava, Prem; Sopory, Sudhir Kumar

    2012-01-01

    As compared with plant system, triose phosphate isomerase (TPI), a crucial enzyme of glycolysis, has been well studied in animals. In order to characterize TPI in plants, a full-length cDNA encoding OscTPI was cloned from rice and expressed in E. coli. The recombinant OscTPI was purified to homogeneity and it showed Km value of 0.1281 ± 0.025 µM, and the Vmax value of 138.7 ± 16 µmol min−1mg−1 which is comparable to the kinetic values studied in other plants. The OscTPI was found to be exclusively present in the cytoplasm when checked with the various methods. Functional assay showed that OscTPI could complement a TPI mutation in yeast. Real time PCR analysis revealed that OscTPI transcript level was regulated in response to various abiotic stresses. Interestingly, it was highly induced under different concentration of methylglyoxal (MG) stress in a concentration dependent manner. There was also a corresponding increase in the protein and the enzyme activity of OscTPI both in shoot and root tissues under MG stress. Our result shows that increases in MG leads to the increase in TPI which results in decrease of DHAP and consequently decrease in the level of toxic MG. PMID:22902706

  10. Prolyl-isomerase Pin1 controls normal and cancer stem cells of the breast

    PubMed Central

    Rustighi, Alessandra; Zannini, Alessandro; Tiberi, Luca; Sommaggio, Roberta; Piazza, Silvano; Sorrentino, Giovanni; Nuzzo, Simona; Tuscano, Antonella; Eterno, Vincenzo; Benvenuti, Federica; Santarpia, Libero; Aifantis, Iannis; Rosato, Antonio; Bicciato, Silvio; Zambelli, Alberto; Del Sal, Giannino

    2014-01-01

    Mammary epithelial stem cells are fundamental to maintain tissue integrity. Cancer stem cells (CSCs) are implicated in both treatment resistance and disease relapse, and the molecular bases of their malignant properties are still poorly understood. Here we show that both normal stem cells and CSCs of the breast are controlled by the prolyl-isomerase Pin1. Mechanistically, following interaction with Pin1, Notch1 and Notch4, key regulators of cell fate, escape from proteasomal degradation by their major ubiquitin-ligase Fbxw7α. Functionally, we show that Fbxw7α acts as an essential negative regulator of breast CSCs' expansion by restraining Notch activity, but the establishment of a Notch/Pin1 active circuitry opposes this effect, thus promoting breast CSCs self-renewal, tumor growth and metastasis in vivo. In human breast cancers, despite Fbxw7α expression, high levels of Pin1 sustain Notch signaling, which correlates with poor prognosis. Suppression of Pin1 holds promise in reverting aggressive phenotypes, through CSC exhaustion as well as recovered drug sensitivity carrying relevant implications for therapy of breast cancers. PMID:24357640

  11. The critical role of the loops of triosephosphate isomerase for its oligomerization, dynamics, and functionality

    PubMed Central

    Katebi, Ataur R; Jernigan, Robert L

    2014-01-01

    Triosephosphate isomerase (TIM) catalyzes the reaction to convert dihydroxyacetone phosphate into glyceraldehyde 3-phosphate, and vice versa. In most organisms, its functional oligomeric state is a homodimer; however, tetramer formation in hyperthermophiles is required for functional activity. The tetrameric TIM structure also provides added stability to the structure, enabling it to function at more extreme temperatures. We apply Principal Component Analysis to find that the TIM structure space is clearly divided into two groups—the open and the closed TIM structures. The distribution of the structures in the open set is much sparser than that in the closed set, showing a greater conformational diversity of the open structures. We also apply the Elastic Network Model to four different TIM structures—an engineered monomeric structure, a dimeric structure from a mesophile—Trypanosoma brucei, and two tetrameric structures from hyperthermophiles Thermotoga maritima and Pyrococcus woesei. We find that dimerization not only stabilizes the structures, it also enhances their functional dynamics. Moreover, tetramerization of the hyperthermophilic structures increases their functional loop dynamics, enabling them to function in the destabilizing environment of extreme temperatures. Computations also show that the functional loop motions, especially loops 6 and 7, are highly coordinated. In summary, our computations reveal the underlying mechanism of the allosteric regulation of the functional loops of the TIM structures, and show that tetramerization of the structure as found in the hyperthermophilic organisms is required to maintain the coordination of the functional loops at a level similar to that in the dimeric mesophilic structure. PMID:24318986

  12. Evaluating the catalytic contribution from the oxyanion hole in ketosteroid isomerase.

    PubMed

    Schwans, Jason P; Sunden, Fanny; Gonzalez, Ana; Tsai, Yingssu; Herschlag, Daniel

    2011-12-21

    Prior site-directed mutagenesis studies in bacterial ketosteroid isomerase (KSI) reported that substitution of both oxyanion hole hydrogen bond donors gives a 10(5)- to 10(8)-fold rate reduction, suggesting that the oxyanion hole may provide the major contribution to KSI catalysis. But these seemingly conservative mutations replaced the oxyanion hole hydrogen bond donors with hydrophobic side chains that could lead to suboptimal solvation of the incipient oxyanion in the mutants, thereby potentially exaggerating the apparent energetic benefit of the hydrogen bonds relative to water-mediated hydrogen bonds in solution. We determined the functional and structural consequences of substituting the oxyanion hole hydrogen bond donors and several residues surrounding the oxyanion hole with smaller residues in an attempt to create a local site that would provide interactions more analogous to those in aqueous solution. These more drastic mutations created an active-site cavity estimated to be ~650 Å(3) and sufficient for occupancy by 15-17 water molecules and led to a rate decrease of only ~10(3)-fold for KSI from two different species, a much smaller effect than that observed from more traditional conservative mutations. The results underscore the strong context dependence of hydrogen bond energetics and suggest that the oxyanion hole provides an important, but moderate, catalytic contribution relative to the interactions in the corresponding solution reaction.

  13. Structural and biochemical characterization of a recombinant triosephosphate isomerase from Rhipicephalus (Boophilus) microplus.

    PubMed

    Moraes, Jorge; Arreola, Rodrigo; Cabrera, Nallely; Saramago, Luiz; Freitas, Daniela; Masuda, Aoi; da Silva Vaz, Itabajara; Tuena de Gomez-Puyou, Marietta; Perez-Montfort, Ruy; Gomez-Puyou, Armando; Logullo, Carlos

    2011-06-01

    Triosephosphate isomerase (TIM) is an enzyme with a role in glycolysis and gluconeogenesis by catalyzing the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This enzyme has been used as a target in endoparasite drug development. In this work we cloned, expressed, purified and studied kinetic and structural characteristics of TIM from tick embryos, Rhipicephalus (Boophilus) microplus (BmTIM). The Km and Vmax of the recombinant BmTIM with glyceraldehyde 3-phosphate as substrate, were 0.47 mM and 6031 μmol min⁻¹ mg protein⁻¹, respectively. The resolution of the diffracted crystal was estimated to be 2.4 Å and the overall data showed that BmTIM is similar to other reported dimeric TIMs. However, we found that, in comparison to other TIMs, BmTIM has the highest content of cysteine residues (nine cysteine residues per monomer). Only two cysteines could make disulfide bonds in monomers of BmTIM. Furthermore, BmTIM was highly sensitive to the action of the thiol reagents dithionitrobenzoic acid and methyl methane thiosulfonate, suggesting that there are five cysteines exposed in each dimer and that these residues could be employed in the development of species-specific inhibitors.

  14. Enhancement of flavone levels through overexpression of chalcone isomerase in hairy root cultures of Scutellaria baicalensis.

    PubMed

    Park, Nam Il; Xu, Hui; Li, Xiaohua; Kim, Sun-Ju; Park, Sang Un

    2011-09-01

    A complementary DNA (cDNA) encoding Scutellaria baicalensis chalcone isomerase (SbCHI) was isolated using rapid amplification of cDNA ends polymerase chain reaction. After the treatment of wounding or methyl jasmonate, SbCHI transcripts were increased in S. baicalensis cell suspensions. SbCHI-overexpressed and SbCH-silenced transgenic hairy root lines were established by using an Agrobacterium rhizogenes-mediated system. SbCHI-overexpressed hairy root lines not only enhanced SbCHI gene expression but also produced more flavones (i.e., baicalin, baicalein, and wogonin) than the control hairy root line. In contrast, SbCHI-silenced hairy root lines reduced SbCHI transcripts and flavone production compared to those of the control hairy roots. In addition, the amount of wogonin in all hairy root cultures was increased compared to that of wild-type roots of S. baicalensis. Finally, this study showed the importance of CHI in flavone biosynthesis and the efficiency of metabolic engineering in S. baicalensis hairy roots.

  15. Analysis of the interaction of calcitriol with the disulfide isomerase ERp57

    PubMed Central

    Gaucci, Elisa; Raimondo, Domenico; Grillo, Caterina; Cervoni, Laura; Altieri, Fabio; Nittari, Giulio; Eufemi, Margherita; Chichiarelli, Silvia

    2016-01-01

    Calcitriol, the active form of vitamin D3, can regulate the gene expression through the binding to the nuclear receptor VDR, but it can also display nongenomic actions, acting through a membrane-associated receptor, which has been discovered as the disulfide isomerase ERp57. The aim of our research is to identify the binding sites for calcitriol in ERp57 and to analyze their interaction. We first studied the interaction through bioinformatics and fluorimetric analyses. Subsequently, we focused on two protein mutants containing the predicted interaction domains with calcitriol: abb’-ERp57, containing the first three domains, and a’-ERp57, the fourth domain only. To consolidate the achievements we used the calorimetric approach to the whole protein and its mutants. Our results allow us to hypothesize that the interaction with the a’ domain contributes to a greater extent than the other potential binding sites to the dissociation constant, calculated as a Kd of about 10−9 M. PMID:27897272

  16. Mapping Soluble Guanylyl Cyclase and Protein Disulfide Isomerase Regions of Interaction.

    PubMed

    Heckler, Erin J; Kholodovych, Vladyslav; Jain, Mohit; Liu, Tong; Li, Hong; Beuve, Annie

    2015-01-01

    Soluble guanylyl cyclase (sGC) is a heterodimeric nitric oxide (NO) receptor that produces cyclic GMP. This signaling mechanism is a key component in the cardiovascular system. NO binds to heme in the β subunit and stimulates the catalytic conversion of GTP to cGMP several hundred fold. Several endogenous factors have been identified that modulate sGC function in vitro and in vivo. In previous work, we determined that protein disulfide isomerase (PDI) interacts with sGC in a redox-dependent manner in vitro and that PDI inhibited NO-stimulated activity in cells. To our knowledge, this was the first report of a physical interaction between sGC and a thiol-redox protein. To characterize this interaction between sGC and PDI, we first identified peptide linkages between sGC and PDI, using a lysine cross-linking reagent and recently developed mass spectrometry analysis. Together with Flag-immunoprecipitation using sGC domain deletions, wild-type (WT) and mutated PDI, regions of sGC involved in this interaction were identified. The observed data were further explored with computational modeling to gain insight into the interaction mechanism between sGC and oxidized PDI. Our results indicate that PDI interacts preferentially with the catalytic domain of sGC, thus providing a mechanism for PDI inhibition of sGC. A model in which PDI interacts with either the α or the β catalytic domain is proposed.

  17. Mannose Phosphate Isomerase Regulates Fibroblast Growth Factor Receptor Family Signaling and Glioma Radiosensitivity

    PubMed Central

    Cazet, Aurélie; Charest, Jonathan; Bennett, Daniel C.; Sambrooks, Cecilia Lopez; Contessa, Joseph N.

    2014-01-01

    Asparagine-linked glycosylation is an endoplasmic reticulum co- and post- translational modification that enables the transit and function of receptor tyrosine kinase (RTK) glycoproteins. To gain insight into the regulatory role of glycosylation enzymes on RTK function, we investigated shRNA and siRNA knockdown of mannose phosphate isomerase (MPI), an enzyme required for mature glycan precursor biosynthesis. Loss of MPI activity reduced phosphorylation of FGFR family receptors in U-251 and SKMG-3 malignant glioma cell lines and also resulted in significant decreases in FRS2, Akt, and MAPK signaling. However, MPI knockdown did not affect ligand-induced activation or signaling of EGFR or MET RTKs, suggesting that FGFRs are more susceptible to MPI inhibition. The reductions in FGFR signaling were not caused by loss of FGF ligands or receptors, but instead were caused by interference with receptor dimerization. Investigations into the cellular consequences of MPI knockdown showed that cellular programs driven by FGFR signaling, and integral to the clinical progression of malignant glioma, were impaired. In addition to a blockade of cellular migration, MPI knockdown also significantly reduced glioma cell clonogenic survival following ionizing radiation. Therefore our results suggest that targeted inhibition of enzymes required for cell surface receptor glycosylation can be manipulated to produce discrete and limited consequences for critical client glycoproteins expressed by tumor cells. Furthermore, this work identifies MPI as a potential enzymatic target for disrupting cell surface receptor-dependent survival signaling and as a novel approach for therapeutic radiosensitization. PMID:25314669

  18. Prolyl isomerase Pin1 regulates axon guidance by stabilizing CRMP2A selectively in distal axons

    PubMed Central

    Balastik, Martin; Zhou, Xiao Zhen; Alberich-Jorda, Meritxell; Weissova, Romana; Žiak, Jakub; Pazyra-Murphy, Maria F.; Cosker, Katharina E; Machonova, Olga; Kozmikova, Iryna; Chen, Chun-Hau; Pastorino, Lucia; Asara, John M.; Cole, Adam; Sutherland, Calum; Segal, Rosalind A.; Lu, Kun Ping

    2015-01-01

    SUMMARY Axon guidance relies on precise translation of the gradients of the extracellular signals into local changes of cytoskeletal dynamics, but the molecular mechanisms regulating dose-dependent responses of growth cones are still poorly understood. Here we show that during embryonic development in growing axons low level of Semaphorin3A stimulation is buffered by the prolyl isomerase Pin1. We demonstrate, that Pin1 stabilizes CDK5-phosphorylated CRMP2A, the major isoform of CRMP2 in distal axons. Consequently, Pin1 knockdown or knockout reduces CRMP2A level specifically in distal axons and inhibits axon growth, which can be fully rescued by Pin1 or CRMP2A expression. Moreover, Pin1 knockdown or knockout increases sensitivity to Sema3A-induced growth cone collapse in vitro and in vivo leading to developmental abnormalities in axon guidance. These results identify an important isoform-specific function and regulation of CRMP2A in controlling axon growth, and uncover Pin1-catalyzed prolyl isomerization as a regulatory mechanism in axon guidance. PMID:26489457

  19. Diversifying selection underlies the origin of allozyme polymorphism at the phosphoglucose isomerase locus in Tigriopus californicus.

    PubMed

    Schoville, Sean D; Flowers, Jonathan M; Burton, Ronald S

    2012-01-01

    The marine copepod Tigriopus californicus lives in intertidal rock pools along the Pacific coast, where it exhibits strong, temporally stable population genetic structure. Previous allozyme surveys have found high frequency private alleles among neighboring subpopulations, indicating that there is limited genetic exchange between populations. Here we evaluate the factors responsible for the diversification and maintenance of alleles at the phosphoglucose isomerase (Pgi) locus by evaluating patterns of nucleotide variation underlying previously identified allozyme polymorphism. Copepods were sampled from eleven sites throughout California and Baja California, revealing deep genetic structure among populations as well as genetic variability within populations. Evidence of recombination is limited to the sample from Pescadero and there is no support for linkage disequilibrium across the Pgi locus. Neutrality tests and codon-based models of substitution suggest the action of natural selection due to elevated non-synonymous substitutions at a small number of sites in Pgi. Two sites are identified as the charge-changing residues underlying allozyme polymorphisms in T. californicus. A reanalysis of allozyme variation at several focal populations, spanning a period of 26 years and over 200 generations, shows that Pgi alleles are maintained without notable frequency changes. Our data suggest that diversifying selection accounted for the origin of Pgi allozymes, while McDonald-Kreitman tests and the temporal stability of private allozyme alleles suggests that balancing selection may be involved in the maintenance of amino acid polymorphisms within populations.

  20. Diversifying Selection Underlies the Origin of Allozyme Polymorphism at the Phosphoglucose Isomerase Locus in Tigriopus californicus

    PubMed Central

    Schoville, Sean D.; Flowers, Jonathan M.; Burton, Ronald S.

    2012-01-01

    The marine copepod Tigriopus californicus lives in intertidal rock pools along the Pacific coast, where it exhibits strong, temporally stable population genetic structure. Previous allozyme surveys have found high frequency private alleles among neighboring subpopulations, indicating that there is limited genetic exchange between populations. Here we evaluate the factors responsible for the diversification and maintenance of alleles at the phosphoglucose isomerase (Pgi) locus by evaluating patterns of nucleotide variation underlying previously identified allozyme polymorphism. Copepods were sampled from eleven sites throughout California and Baja California, revealing deep genetic structure among populations as well as genetic variability within populations. Evidence of recombination is limited to the sample from Pescadero and there is no support for linkage disequilibrium across the Pgi locus. Neutrality tests and codon-based models of substitution suggest the action of natural selection due to elevated non-synonymous substitutions at a small number of sites in Pgi. Two sites are identified as the charge-changing residues underlying allozyme polymorphisms in T. californicus. A reanalysis of allozyme variation at several focal populations, spanning a period of 26 years and over 200 generations, shows that Pgi alleles are maintained without notable frequency changes. Our data suggest that diversifying selection accounted for the origin of Pgi allozymes, while McDonald-Kreitman tests and the temporal stability of private allozyme alleles suggests that balancing selection may be involved in the maintenance of amino acid polymorphisms within populations. PMID:22768211

  1. Role of loop-loop interactions in coordinating motions and enzymatic function in triosephosphate isomerase.

    PubMed

    Wang, Yan; Berlow, Rebecca B; Loria, J Patrick

    2009-06-02

    The enzyme triosephosphate isomerase (TIM) has been used as a model system for understanding the relationship between protein sequence, structure, and biological function. The sequence of the active site loop (loop 6) in TIM is directly correlated with a conserved motif in loop 7. Replacement of loop 7 of chicken TIM with the corresponding loop 7 sequence from an archaeal homologue caused a 10(2)-fold loss in enzymatic activity, a decrease in substrate binding affinity, and a decrease in thermal stability. Isotope exchange studies performed by one-dimensional (1)H NMR showed that the substrate-derived proton in the enzyme is more susceptible to solvent exchange for DHAP formation in the loop 7 mutant than for WT TIM. TROSY-Hahn Echo and TROSY-selected R(1rho) experiments indicate that upon mutation of loop 7, the chemical exchange rate for active site loop motion is nearly doubled and that the coordinated motion of loop 6 is reduced relative to that of the WT. Temperature dependent NMR experiments show differing activation energies for the N- and C-terminal hinges in this mutant enzyme. Together, these data suggest that interactions between loop 6 and loop 7 are necessary to provide the proper chemical context for the enzymatic reaction to occur and that the interactions play a significant role in modulating the chemical dynamics near the active site.

  2. Hyphal tip extension in Aspergillus nidulans requires the manA gene, which encodes phosphomannose isomerase.

    PubMed Central

    Smith, D J; Payton, M A

    1994-01-01

    A strain of Aspergillus nidulans carrying a temperature-sensitive mutation in the manA gene produces cell walls depleted of D-mannose and forms hyphal tip balloons at the restrictive temperature (B.P. Valentine and B.W. Bainbridge, J. Gen. Microbiol. 109:155-168, 1978). We have isolated and characterized the manA gene and physically located it between 3.5 and 5.5 kb centromere distal of the riboB locus on chromosome VIII. The manA gene contains four introns and encodes a 50.6-kDa protein which has significant sequence identity to type I phosphomannose isomerase proteins from other eukaryotes. We have constructed by integrative transformation a null mutation in the manA gene which can only be maintained in a heterokaryotic strain with wild-type manA+ nuclei. Thus, a manA null mutation is lethal in A. nidulans. The phenotype of the mutation was analyzed in germinating conidia. Such conidia are able to commence germination but swell abnormally, sometimes producing a misshapen germ tube, before growth ceases. The reason for the lethality is probably the lack of synthesis of mannose-containing cell wall polymers that must be required for normal cell wall development in growing hyphae. Images PMID:8065336

  3. Understanding protein lids: kinetic analysis of active hinge mutants in triosephosphate isomerase.

    PubMed

    Sun, J; Sampson, N S

    1999-08-31

    In previous work we tested what three amino acid sequences could serve as a protein hinge in triosephosphate isomerase [Sun, J., and Sampson, N. S. (1998) Protein Sci. 7, 1495-1505]. We generated a genetic library encoding all 8000 possible 3 amino acid combinations at the C-terminal hinge and selected for those combinations of amino acids that formed active mutants. These mutants were classified into six phylogenetic families. Two families resembled wild-type hinges, and four families represented new types of hinges. In this work, the kinetic characteristics and thermal stabilities of mutants representing each of these families were determined in order to understand what properties make an efficient protein hinge, and why all of the families are not observed in nature. From a steady-state kinetic analysis of our mutants, it is clear that the partitioning between protonation of intermediate to form product and intermediate release from the enzyme surface to form methylglyoxal (a decomposition product) is not affected. The two most impaired mutants undergo a change in rate-limiting step from enediol formation to dihydroxyacetone phosphate binding. Thus, it appears that k(cat)/K(m)'s are reduced relative to wild type as a result of slower Michaelis complex formation and dissociation, rather than increased loop opening speed.

  4. Cellular and biochemical characterization of two closely related triosephosphate isomerases from Trichomonas vaginalis.

    PubMed

    Figueroa-Angulo, Elisa E; Estrella-Hernández, Priscila; Salgado-Lugo, Holjes; Ochoa-Leyva, Adrián; Gómez Puyou, Armando; Campos, Silvia S; Montero-Moran, Gabriela; Ortega-López, Jaime; Saab-Rincón, Gloria; Arroyo, Rossana; Benítez-Cardoza, Claudia G; Brieba, Luis G

    2012-11-01

    The glycolytic enzyme triosephosphate isomerase catalyses the isomerization between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Here we report that Trichomonas vaginalis contains 2 fully functional tpi genes. Both genes are located in separated chromosomal context with different promoter regulatory elements and encode ORFs of 254 amino acids; the only differences between them are the character of 4 amino acids located in α-helices 1, 2 and 8. Semi-quantitative RT-PCR assays showed that tpi2 transcript is approximately 3·3-fold more abundant than tpi1. Using an anti-TvTIM2 polyclonal antibody it was demonstrated that TIM proteins have a cytoplasmic localization and both enzymes are able to complement an Escherichia coli strain carrying a deletion of its endogenous tpi gene. Both TIM proteins assemble as dimers and their secondary structure assessment is essentially identical to TIM from Saccharomyces cerevisiae. The kinetic catalytic constants of the recombinant enzymes using glyceraldehyde-3-phosphate as substrate are similar to the catalytic constants of TIMs from other organisms including parasitic protozoa. As T. vaginalis depends on glycolysis for ATP production, we speculate 2 possible reasons to maintain a duplicated tpi copy on its genome: an increase in gene dosage or an early event of neofunctionalization of TIM as a moonlighting protein.

  5. Reduction of PDC1 expression in S. cerevisiae with xylose isomerase on xylose medium.

    PubMed

    Kim, Dong Min; Choi, Seung-Hyun; Ko, Byung Sam; Jeong, Gwon-Young; Jang, Han-Bit; Han, Jae-Gun; Jeong, Kyung-Hwan; Lee, Hyeon-Yong; Won, Yonggwan; Kim, Il-Chul

    2012-01-01

    Ethanol production using hemicelluloses has recently become a focus of many researchers. In order to promote D: -xylose fermentation, we cloned the bacterial xylA gene encoding for xylose isomerase with 434 amino acid residues from Agrobacterium tumefaciens, and successfully expressed it in Saccharomyces cerevisiae, a non-xylose assimilating yeast. The recombinant strain S. cerevisiae W303-1A/pAGROXI successfully colonized a minimal medium containing D: -xylose as a sole carbon source and was capable of growth in minimal medium containing 2% xylose via aerobic shake cultivation. Although the recombinant strain assimilates D: -xylose, its ethanol productivity is quite low during fermentation with D: -xylose alone. In order to ascertain the key enzyme in ethanol production from D: -xylose, we checked the expression levels of the gene clusters involved in the xylose assimilating pathway. Among the genes classified into four groups by their expression patterns, the mRNA level of pyruvate decarboxylase (PDC1) was reduced dramatically in xylose media. This reduced expression of PDC1, an enzyme which converts pyruvate to acetaldehyde, may cause low ethanol productivity in xylose medium. Thus, the enhancement of PDC1 gene expression may provide us with a useful tool for the fermentation of ethanol from hemicellulose.

  6. Enhanced activity and stability of L-arabinose isomerase by immobilization on aminopropyl glass.

    PubMed

    Zhang, Ye-Wang; Jeya, Marimuthu; Lee, Jung-Kul

    2011-03-01

    Immobilization of Bacillus licheniformis L: -arabinose isomerase (BLAI) on aminopropyl glass modified with glutaraldehyde (4 mg protein g support⁻¹) was found to enhance the enzyme activity. The immobilization yield of BLAI was proportional to the quantity of amino groups on the surface of support. Reducing particle size increased the adsorption capacity (q(m)) and affinity (k(a)). The pH and temperature for immobilization were optimized to be pH 7.1 and 33 °C using response surface methodology (RSM). The immobilized enzyme was characterized and compared to the free enzyme. There is no change in optimal pH and temperature before and after immobilization. However, the immobilized BLAI enzyme achieved 145% of the activity of the free enzyme. Correspondingly, the catalytic efficiency (k(cat)/K(m)) was improved 1.47-fold after immobilization compared to the free enzyme. The thermal stability was improved 138-fold (t₁/₂) increased from 2 to 275 h) at 50 °C following immobilization.

  7. Structures and functions of protein disulfide isomerase family members involved in proteostasis in the endoplasmic reticulum.

    PubMed

    Okumura, Masaki; Kadokura, Hiroshi; Inaba, Kenji

    2015-06-01

    The endoplasmic reticulum (ER) is an essential cellular compartment in which an enormous number of secretory and cell surface membrane proteins are synthesized and subjected to cotranslational or posttranslational modifications, such as glycosylation and disulfide bond formation. Proper maintenance of ER protein homeostasis (sometimes termed proteostasis) is essential to avoid cellular stresses and diseases caused by abnormal proteins. Accumulating knowledge of cysteine-based redox reactions catalyzed by members of the protein disulfide isomerase (PDI) family has revealed that these enzymes play pivotal roles in productive protein folding accompanied by disulfide formation, as well as efficient ER-associated degradation accompanied by disulfide reduction. Each of PDI family members forms a protein-protein interaction with a preferential partner to fulfill a distinct function. Multiple redox pathways that utilize PDIs appear to function synergistically to attain the highest quality and productivity of the ER, even under various stress conditions. This review describes the structures, physiological functions, and cooperative actions of several essential PDIs, and provides important insights into the elaborate proteostatic mechanisms that have evolved in the extremely active and stress-sensitive ER. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Structural basis of redox-dependent substrate binding of protein disulfide isomerase

    PubMed Central

    Yagi-Utsumi, Maho; Satoh, Tadashi; Kato, Koichi

    2015-01-01

    Protein disulfide isomerase (PDI) is a multidomain enzyme, operating as an essential folding catalyst, in which the b′ and a′ domains provide substrate binding sites and undergo an open–closed domain rearrangement depending on the redox states of the a′ domain. Despite the long research history of this enzyme, three-dimensional structural data remain unavailable for its ligand-binding mode. Here we characterize PDI substrate recognition using α-synuclein (αSN) as the model ligand. Our nuclear magnetic resonance (NMR) data revealed that the substrate-binding domains of PDI captured the αSN segment Val37–Val40 only in the oxidized form. Furthermore, we determined the crystal structure of an oxidized form of the b′–a′ domains in complex with an undecapeptide corresponding to this segment. The peptide-binding mode observed in the crystal structure with NMR validation, was characterized by hydrophobic interactions on the b′ domain in an open conformation. Comparison with the previously reported crystal structure indicates that the a′ domain partially masks the binding surface of the b′ domain, causing steric hindrance against the peptide in the reduced form of the b′–a′ domains that exhibits a closed conformation. These findings provide a structural basis for the mechanism underlying the redox-dependent substrate binding of PDI. PMID:26350503

  9. Quercetin-3-rutinoside Inhibits Protein Disulfide Isomerase by Binding to Its b′x Domain*

    PubMed Central

    Lin, Lin; Gopal, Srila; Sharda, Anish; Passam, Freda; Bowley, Sheryl R.; Stopa, Jack; Xue, Guangpu; Yuan, Cai; Furie, Barbara C.; Flaumenhaft, Robert; Huang, Mingdong; Furie, Bruce

    2015-01-01

    Quercetin-3-rutinoside inhibits thrombus formation in a mouse model by inhibiting extracellular protein disulfide isomerase (PDI), an enzyme required for platelet thrombus formation and fibrin generation. Prior studies have identified PDI as a potential target for novel antithrombotic agents. Using a fluorescence enhancement-based assay and isothermal calorimetry, we show that quercetin-3-rutinoside directly binds to the b′ domain of PDI with a 1:1 stoichiometry. The binding of quercetin-3-rutinoside to PDI induces a more compact conformation and restricts the conformational flexibility of PDI, as revealed by small angle x-ray scattering. The binding sites of quercetin-3-rutinoside to PDI were determined by studying its interaction with isolated fragments of PDI. Quercetin-3-rutinoside binds to the b′x domain of PDI. The infusion of the b′x fragment of PDI rescued thrombus formation that was inhibited by quercetin-3-rutinoside in a mouse thrombosis model. This b′x fragment does not possess reductase activity and, in the absence of quercetin-3-rutinoside, does not affect thrombus formation in vivo. The isolated b′ domain of PDI has potential as an antidote to reverse the antithrombotic effect of quercetin-3-rutinoside by binding and neutralizing quercetin-3-rutinoside. PMID:26240139

  10. Quercetin-3-rutinoside Inhibits Protein Disulfide Isomerase by Binding to Its b'x Domain.

    PubMed

    Lin, Lin; Gopal, Srila; Sharda, Anish; Passam, Freda; Bowley, Sheryl R; Stopa, Jack; Xue, Guangpu; Yuan, Cai; Furie, Barbara C; Flaumenhaft, Robert; Huang, Mingdong; Furie, Bruce

    2015-09-25

    Quercetin-3-rutinoside inhibits thrombus formation in a mouse model by inhibiting extracellular protein disulfide isomerase (PDI), an enzyme required for platelet thrombus formation and fibrin generation. Prior studies have identified PDI as a potential target for novel antithrombotic agents. Using a fluorescence enhancement-based assay and isothermal calorimetry, we show that quercetin-3-rutinoside directly binds to the b' domain of PDI with a 1:1 stoichiometry. The binding of quercetin-3-rutinoside to PDI induces a more compact conformation and restricts the conformational flexibility of PDI, as revealed by small angle x-ray scattering. The binding sites of quercetin-3-rutinoside to PDI were determined by studying its interaction with isolated fragments of PDI. Quercetin-3-rutinoside binds to the b'x domain of PDI. The infusion of the b'x fragment of PDI rescued thrombus formation that was inhibited by quercetin-3-rutinoside in a mouse thrombosis model. This b'x fragment does not possess reductase activity and, in the absence of quercetin-3-rutinoside, does not affect thrombus formation in vivo. The isolated b' domain of PDI has potential as an antidote to reverse the antithrombotic effect of quercetin-3-rutinoside by binding and neutralizing quercetin-3-rutinoside. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Structural and biochemical characterization of a recombinant triosephosphate isomerase from Rhipicephalus (Boophilus) microplus

    SciTech Connect

    Moraes, Jorge; Arreola, Rodrigo; Cabrera, Nallely; Saramago, Luiz; Freitas, Daniela; Masuda, Aoi; da Silva Vaz Jr., Itabajara; Tuena de Gomez-Puyou, Marietta; Perez-Montfort, Ruy; Gomez-Puyou, Armando; Logullo, Carlos

    2012-02-06

    Triosephosphate isomerase (TIM) is an enzyme with a role in glycolysis and gluconeogenesis by catalyzing the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This enzyme has been used as a target in endoparasite drug development. In this work we cloned, expressed, purified and studied kinetic and structural characteristics of TIM from tick embryos, Rhipicephalus (Boophilus) microplus (BmTIM). The Km and Vmax of the recombinant BmTIM with glyceraldehyde 3-phosphate as substrate, were 0.47 mM and 6031 {micro}mol min{sup -1} mg protein{sup -1}, respectively. The resolution of the diffracted crystal was estimated to be 2.4 {angstrom} and the overall data showed that BmTIM is similar to other reported dimeric TIMs. However, we found that, in comparison to other TIMs, BmTIM has the highest content of cysteine residues (nine cysteine residues per monomer). Only two cysteines could make disulfide bonds in monomers of BmTIM. Furthermore, BmTIM was highly sensitive to the action of the thiol reagents dithionitrobenzoic acid and methyl methane thiosulfonate, suggesting that there are five cysteines exposed in each dimer and that these residues could be employed in the development of species-specific inhibitors.

  12. Molecular dynamics study of triosephosphate isomerase from Trypanosoma cruzi in water/decane mixtures.

    PubMed

    Díaz-Vergara, Norma; Piñeiro, Angel

    2008-03-20

    A comprehensive study of the triosephosphate isomerase from the parasite Trypanosoma cruzi (TcTIM) in water, in decane, and in three water/decane mixtures was performed using molecular dynamics (MD) simulations in a time scale of 40 ns. The structure and dynamics of the enzyme, as well as the solvent molecules' distribution and mobility, were analyzed in detail. In the presence of decane, the amplitudes of the most important internal motions of the enzyme backbone were observed to depend on the solvent concentration: the higher the water concentration, the greater the amplitudes. Contrary to this trend, the amplitudes of the TcTIM motions in pure water were similar to those of the simulation with the lowest water concentration. The enzyme was observed to be almost motionless in pure decane due to a sharp increase of the number of intramolecular hydrogen bonds. This caused a contraction of the enzyme structure accompanied by a loss of secondary structure and of a decrease of the hydrophilic solvent accessible surface. A similar behavior, although to a lesser extent, was observed in the simulation at the lowest water concentration. Our results suggest that the presence of decane molecules located at specific sites of the enzyme might accelerate its internal movements, although a minimum number of water molecules is needed for the protein to keep its structure and dynamics. Altogether, this work provides new insight into protein and water behavior in organic solvents as well as into the dynamics of TcTIM itself.

  13. Glucose-6-Phosphate Isomerase (G6PI) Mediates Hypoxia-Induced Angiogenesis in Rheumatoid Arthritis

    PubMed Central

    Lu, Ying; Yu, Shan-Shan; Zong, Ming; Fan, Sha-Sha; Lu, Tian-Bao; Gong, Ru-Han; Sun, Li-Shan; Fan, Lie-Ying

    2017-01-01

    The higher level of Glucose-6-phosphate isomerase (G6PI) has been found in both synovial tissue and synovial fluid of rheumatoid arthritis (RA) patients, while the function of G6PI in RA remains unclear. Herein we found the enrichment of G6PI in microvascular endothelial cells of synovial tissue in RA patients, where a 3% O2 hypoxia environment has been identified. In order to determine the correlation between the high G6PI level and the low oxygen concentration in RA, a hypoxia condition (~3% O2) in vitro was applied to mimic the RA environment in vivo. Hypoxia promoted cellular proliferation of rheumatoid arthritis synovial fibroblasts (RASFs), and induced cell migration and angiogenic tube formation of human dermal microvascular endothelial cells (HDMECs), which were accompanied with the increased expression of G6PI and HIF-1α. Through application of G6PI loss-of-function assays, we confirmed the requirement of G6PI expression for those hypoxia-induced phenotype in RA. In addition, we demonstrated for the first time that G6PI plays key roles in regulating VEGF secretion from RASFs to regulate the hypoxia-induced angiogenesis in RA. Taken together, we demonstrated a novel pathway regulating hypoxia-induced angiogenesis in RA mediated by G6PI. PMID:28067317

  14. The flavonoid biosynthetic enzyme chalcone isomerase modulates terpenoid production in glandular trichomes of tomato.

    PubMed

    Kang, Jin-Ho; McRoberts, John; Shi, Feng; Moreno, Javier E; Jones, A Daniel; Howe, Gregg A

    2014-03-01

    Flavonoids and terpenoids are derived from distinct metabolic pathways but nevertheless serve complementary roles in mediating plant interactions with the environment. Here, we show that glandular trichomes of the anthocyanin free (af) mutant of cultivated tomato (Solanum lycopersicum) fail to accumulate both flavonoids and terpenoids. This pleiotropic metabolic deficiency was associated with loss of resistance to native populations of coleopteran herbivores under field conditions. We demonstrate that Af encodes an isoform (SlCHI1) of the flavonoid biosynthetic enzyme chalcone isomerase (CHI), which catalyzes the conversion of naringenin chalcone to naringenin and is strictly required for flavonoid production in multiple tissues of tomato. Expression of the wild-type SlCHI1 gene from its native promoter complemented the anthocyanin deficiency in af. Unexpectedly, the SlCHI1 transgene also complemented the defect in terpenoid production in glandular trichomes. Our results establish a key role for SlCHI1 in flavonoid production in tomato and reveal a link between CHI1 and terpenoid production. Metabolic coordination of the flavonoid and terpenoid pathways may serve to optimize the function of trichome glands in dynamic environments.

  15. Identification and analysis of residues contained on β → α loops of the dual-substrate (βα)8 phosphoribosyl isomerase A specific for its phosphoribosyl anthranilate isomerase activity

    PubMed Central

    Noda-García, Lianet; Camacho-Zarco, Aldo R; Verdel-Aranda, Karina; Wright, Helena; Soberón, Xavier; Fülöp, Vilmos; Barona-Gómez, Francisco

    2010-01-01

    A good model to experimentally explore evolutionary hypothesis related to enzyme function is the ancient-like dual-substrate (βα)8 phosphoribosyl isomerase A (PriA), which takes part in both histidine and tryptophan biosynthesis in Streptomyces coelicolor and related organisms. In this study, we determined the Michaelis–Menten enzyme kinetics for both isomerase activities in wild-type PriA from S. coelicolor and in selected single-residue monofunctional mutants, identified after Escherichia coli in vivo complementation experiments. Structural and functional analyses of a hitherto unnoticed residue contained on the functionally important β → α loop 5, namely, Arg139, which was postulated on structural grounds to be important for the dual-substrate specificity of PriA, is presented for the first time. Indeed, enzyme kinetics analyses done on the mutant variants PriA_Ser81Thr and PriA_Arg139Asn showed that these residues, which are contained on β → α loops and in close proximity to the N-terminal phosphate-binding site, are essential solely for the phosphoribosyl anthranilate isomerase activity of PriA. Moreover, analysis of the X-ray crystallographic structure of PriA_Arg139Asn elucidated at 1.95 Å herein strongly implicates the occurrence of conformational changes in this β → α loop as a major structural feature related to the evolution of the dual-substrate specificity of PriA. It is suggested that PriA has evolved by tuning a fine energetic balance that allows the sufficient degree of structural flexibility needed for accommodating two topologically dissimilar substrates—within a bifunctional and thus highly constrained active site—without compromising its structural stability. PMID:20066665

  16. The Protein Disulfide Isomerase gene family in bread wheat (T. aestivum L.)

    PubMed Central

    2010-01-01

    Background The Protein Disulfide Isomerase (PDI) gene family encodes several PDI and PDI-like proteins containing thioredoxin domains and controlling diversified metabolic functions, including disulfide bond formation and isomerisation during protein folding. Genomic, cDNA and promoter sequences of the three homoeologous wheat genes encoding the "typical" PDI had been cloned and characterized in a previous work. The purpose of present research was the cloning and characterization of the complete set of genes encoding PDI and PDI like proteins in bread wheat (Triticum aestivum cv Chinese Spring) and the comparison of their sequence, structure and expression with homologous genes from other plant species. Results Eight new non-homoeologous wheat genes were cloned and characterized. The nine PDI and PDI-like sequences of wheat were located in chromosome regions syntenic to those in rice and assigned to eight plant phylogenetic groups. The nine wheat genes differed in their sequences, genomic organization as well as in the domain composition and architecture of their deduced proteins; conversely each of them showed high structural conservation with genes from other plant species in the same phylogenetic group. The extensive quantitative RT-PCR analysis of the nine genes in a set of 23 wheat samples, including tissues and developmental stages, showed their constitutive, even though highly variable expression. Conclusions The nine wheat genes showed high diversity, while the members of each phylogenetic group were highly conserved even between taxonomically distant plant species like the moss Physcomitrella patens. Although constitutively expressed the nine wheat genes were characterized by different expression profiles reflecting their different genomic organization, protein domain architecture and probably promoter sequences; the high conservation among species indicated the ancient origin and diversification of the still evolving gene family. The comprehensive

  17. Protein disulfide isomerase inhibition blocks thrombin generation in humans by interfering with platelet factor V activation

    PubMed Central

    Stopa, Jack D.; Neuberg, Donna; Puligandla, Maneka; Furie, Bruce; Zwicker, Jeffrey I.

    2017-01-01

    BACKGROUND: Protein disulfide isomerase (PDI) is required for thrombus formation. We previously demonstrated that glycosylated quercetin flavonoids such as isoquercetin inhibit PDI activity and thrombus formation in animal models, but whether extracellular PDI represents a viable anticoagulant target in humans and how its inhibition affects blood coagulation remain unknown. METHODS: We evaluated effects of oral administration of isoquercetin on platelet-dependent thrombin generation in healthy subjects and patients with persistently elevated anti-phospholipid antibodies. RESULTS: Following oral administration of 1,000 mg isoquercetin to healthy adults, the measured peak plasma quercetin concentration (9.2 μM) exceeded its IC50 for inhibition of PDI by isoquercetin in vitro (2.5 ± 0.4 μM). Platelet-dependent thrombin generation decreased by 51% in the healthy volunteers compared with baseline (P = 0.0004) and by 64% in the anti-phospholipid antibody cohort (P = 0.015) following isoquercetin ingestion. To understand how PDI affects thrombin generation, we evaluated substrates of PDI identified using an unbiased mechanistic-based substrate trapping approach. These studies identified platelet factor V as a PDI substrate. Isoquercetin blocked both platelet factor Va and thrombin generation with an IC50 of ~5 μM. Inhibition of PDI by isoquercetin ingestion resulted in a 53% decrease in the generation of platelet factor Va (P = 0.001). Isoquercetin-mediated inhibition was reversed with addition of exogenous factor Va. CONCLUSION: These studies show that oral administration of isoquercetin inhibits PDI activity in plasma and diminishes platelet-dependent thrombin generation predominantly by blocking the generation of platelet factor Va. These pharmacodynamic and mechanistic observations represent an important step in the development of a novel class of antithrombotic agents targeting PDI. TRIAL REGISTRATION: Clinicaltrials.gov (NCT01722669) FUNDING: National Heart

  18. Arabidopsis phosphoribosylanthranilate isomerase: molecular genetic analysis of triplicate tryptophan pathway genes.

    PubMed Central

    Li, J; Zhao, J; Rose, A B; Schmidt, R; Last, R L

    1995-01-01

    Phosphoribosylanthranilate isomerase (PAI) catalyzes the third step of the tryptophan biosynthetic pathway. Arabidopsis PAI cDNAs were cloned from a cDNA expression library by complementation of an Escherichia coli trpC- PAI deficiency mutation. Genomic DNA blot hybridization analysis detected three nonallelic genes encoding PAI in the Arabidopsis genome. DNA sequence analysis of cDNA and genomic clones indicated that the PAI1 and PAI2. All three PAI polypeptides possess an N-terminal putative plastid target sequence, suggesting that these enzymes all function in plastids. The PAI1 gene is flanked by nearly identical direct repeats of approximately 350 nucleotides. Our results indicate that, in contrast to most microorganisms, the Arabidopsis PAI protein is not fused with indole-3-glycerolphosphate synthase, which catalyzes the next step in the pathway. Yeast artificial chromosome hybridization studies indicated that the PAI2 gene is tightly linked to the anthranilate synthase alpha subunit 1 (ASA1) gene on chromosome 5. PAI1 was mapped to the top of chromosome 1 using recombinant inbred lines, and PAI3 is loosely linked to PAI1. cDNA restriction mapping and sequencing and RNA gel blot hybridization analysis indicated that all three genes are transcribed in wild-type plants. The expression of antisense PAI1 RNA significantly reduced the immunologically observable PAI protein and enzyme activity in transgenic plants. The plants expressing antisense RNA also showed two phenotypes consistent with a block early in the pathway: blue fluorescence under UV light and resistance to the anthranilate analog 6-methylanthranilate. The extreme nucleotide conservation between the unlinked PAI1 and PAI2 loci suggests that this gene family is actively evolving. PMID:7773017

  19. Cytosolic Triosephosphate Isomerase from Arabidopsis thaliana Is Reversibly Modified by Glutathione on Cysteines 127 and 218

    PubMed Central

    Dumont, Sébastien; Bykova, Natalia V.; Pelletier, Guillaume; Dorion, Sonia; Rivoal, Jean

    2016-01-01

    In plant cells, an increase in cellular oxidants can have multiple effects, including the promotion of mixed disulfide bonds between glutathione and some proteins (S-glutathionylation). The present study focuses on the cytosolic isoform of the glycolytic enzyme triosephosphate isomerase (cTPI) from Arabidopsis thaliana and its reversible modification by glutathione. We used purified recombinant cTPI to demonstrate the enzyme sensitivity to inhibition by N-ethylmaleimide, hydrogen peroxide and diamide. Treatment of cTPI with diamide in the presence of reduced glutathione (GSH) led to a virtually complete inhibition of its enzymatic activity by S-glutathionylation. Recombinant cTPI was also sensitive to the oxidized form of glutathione (GSSG) in the micromolar range. Activity of cTPI was restored after reversion of S-glutathionylation by two purified recombinant A. thaliana cytosolic glutaredoxins (GRXs). GRXs-mediated deglutathionylation of cTPI was dependent on a GSH-regenerating system. Analysis of cTPI by mass spectrometry after S-glutathionylation by GSSG revealed that two Cys residues (Cys127 and Cys218) were modified by glutathione. The role of these two residues was assessed using site-directed mutagenesis. Mutation of Cys127 and Cys218 to Ser separately or together caused different levels of decrease in enzyme activity, loss of stability, as well as alteration of intrinsic fluorescence, underlining the importance of these Cys residues in protein conformation. Comparison of wild-type and mutant proteins modified with biotinyl glutathione ethyl ester (BioGEE) showed partial binding with single mutants and total loss of binding with the double mutant, demonstrating that both Cys residues were significantly S-glutathionylated. cTPI modification with BioGEE was reversed using DTT. Our study provides the first identification of the amino acid residues involved in cTPI S-glutathionylation and supports the hypothesis that this reversible modification could be part

  20. Phycoerythrin-specific bilin lyase-isomerase controls blue-green chromatic acclimation in marine Synechococcus.

    PubMed

    Shukla, Animesh; Biswas, Avijit; Blot, Nicolas; Partensky, Frédéric; Karty, Jonathan A; Hammad, Loubna A; Garczarek, Laurence; Gutu, Andrian; Schluchter, Wendy M; Kehoe, David M

    2012-12-04

    The marine cyanobacterium Synechococcus is the second most abundant phytoplanktonic organism in the world's oceans. The ubiquity of this genus is in large part due to its use of a diverse set of photosynthetic light-harvesting pigments called phycobiliproteins, which allow it to efficiently exploit a wide range of light colors. Here we uncover a pivotal molecular mechanism underpinning a widespread response among marine Synechococcus cells known as "type IV chromatic acclimation" (CA4). During this process, the pigmentation of the two main phycobiliproteins of this organism, phycoerythrins I and II, is reversibly modified to match changes in the ambient light color so as to maximize photon capture for photosynthesis. CA4 involves the replacement of three molecules of the green light-absorbing chromophore phycoerythrobilin with an equivalent number of the blue light-absorbing chromophore phycourobilin when cells are shifted from green to blue light, and the reverse after a shift from blue to green light. We have identified and characterized MpeZ, an enzyme critical for CA4 in marine Synechococcus. MpeZ attaches phycoerythrobilin to cysteine-83 of the α-subunit of phycoerythrin II and isomerizes it to phycourobilin. mpeZ RNA is six times more abundant in blue light, suggesting that its proper regulation is critical for CA4. Furthermore, mpeZ mutants fail to normally acclimate in blue light. These findings provide insights into the molecular mechanisms controlling an ecologically important photosynthetic process and identify a unique class of phycoerythrin lyase/isomerases, which will further expand the already widespread use of phycoerythrin in biotechnology and cell biology applications.

  1. Testing Electrostatic Complementarity in Enzyme Catalysis: Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole

    PubMed Central

    Kraut, Daniel A; Sigala, Paul A; Pybus, Brandon; Liu, Corey W; Ringe, Dagmar; Petsko, Gregory A

    2006-01-01

    A longstanding proposal in enzymology is that enzymes are electrostatically and geometrically complementary to the transition states of the reactions they catalyze and that this complementarity contributes to catalysis. Experimental evaluation of this contribution, however, has been difficult. We have systematically dissected the potential contribution to catalysis from electrostatic complementarity in ketosteroid isomerase. Phenolates, analogs of the transition state and reaction intermediate, bind and accept two hydrogen bonds in an active site oxyanion hole. The binding of substituted phenolates of constant molecular shape but increasing p K a models the charge accumulation in the oxyanion hole during the enzymatic reaction. As charge localization increases, the NMR chemical shifts of protons involved in oxyanion hole hydrogen bonds increase by 0.50–0.76 ppm/p K a unit, suggesting a bond shortening of ˜0.02 Å/p K a unit. Nevertheless, there is little change in binding affinity across a series of substituted phenolates (ΔΔG = −0.2 kcal/mol/p K a unit). The small effect of increased charge localization on affinity occurs despite the shortening of the hydrogen bonds and a large favorable change in binding enthalpy (ΔΔH = −2.0 kcal/mol/p K a unit). This shallow dependence of binding affinity suggests that electrostatic complementarity in the oxyanion hole makes at most a modest contribution to catalysis of ˜300-fold. We propose that geometrical complementarity between the oxyanion hole hydrogen-bond donors and the transition state oxyanion provides a significant catalytic contribution, and suggest that KSI, like other enzymes, achieves its catalytic prowess through a combination of modest contributions from several mechanisms rather than from a single dominant contribution. PMID:16602823

  2. Triosephosphate isomerase deficiency: consequences of an inherited mutation at mRNA, protein and metabolic levels

    PubMed Central

    Oláh, Judit; Orosz, Ferenc; Puskás, László G.; Hackler, Jr, László; Horányi, Margit; Polgár, László; Hollán, Susan; Ovádi, Judit

    2005-01-01

    Triosephosphate isomerase (TPI) deficiency is a unique glycolytic enzymopathy coupled with neurodegeneration. Two Hungarian compound heterozygote brothers inherited the same TPI mutations (F240L and E145Stop), but only the younger one suffers from neurodegeneration. In the present study, we determined the kinetic parameters of key glycolytic enzymes including the mutant TPI for rational modelling of erythrocyte glycolysis. We found that a low TPI activity in the mutant cells (lower than predicted from the protein level and specific activity of the purified recombinant enzyme) is coupled with an increase in the activities of glycolytic kinases. The modelling rendered it possible to establish the steady-state flux of the glycolysis and metabolite concentrations, which was not possible experimentally due to the inactivation of the mutant TPI and other enzymes during the pre-steady state. Our results showed that the flux was 2.5-fold higher and the concentration of DHAP (dihydroxyacetone phosphate) and fructose 1,6-bisphosphate increased 40- and 5-fold respectively in the erythrocytes of the patient compared with the control. Although the rapid equilibration of triosephosphates is not achieved, the energy state of the cells is not ‘sick’ due to the activation of key regulatory enzymes. In lymphocytes of the two brothers, the TPI activity was also lower (20%) than that of controls; however, the remaining activity was high enough to maintain the rapid equilibration of triosephosphates; consequently, no accumulation of DHAP occurs, as judged by our experimental and computational data. Interestingly, we found significant differences in the mRNA levels of the brothers for TPI and some other, apparently unrelated, proteins. One of them is the prolyl oligopeptidase, the activity decrease of which has been reported in well-characterized neurodegenerative diseases. We found that the peptidase activity of the affected brother was reduced by 30% compared with that of his

  3. Structural effects of protein aging: Terminal marking by deamidation in human triosephosphate isomerase

    DOE PAGES

    Torres-Larios, Alfredo; Enríquez-Flores, Sergio; Méndez, Sara -Teresa; ...

    2015-04-17

    Deamidation, the loss of the ammonium group of asparagine and glutamine to form aspartic and glutamic acid, is one of the most commonly occurring post-translational modifications in proteins. Since deamidation rates are encoded in the protein structure, it has been proposed that they can serve as molecular clocks for the timing of biological processes such as protein turnover, development and aging. Despite the importance of this process, there is a lack of detailed structural information explaining the effects of deamidation on the structure of proteins. Here, we studied the effects of deamidation on human triosephosphate isomerase (HsTIM), an enzyme formore » which deamidation of N15 and N71 has been long recognized as the signal for terminal marking of the protein. Deamidation was mimicked by site directed mutagenesis; thus, three mutants of HsTIM (N15D, N71D and N15D/N71D) were characterized. The results show that the N71D mutant resembles, structurally and functionally, the wild type enzyme. In contrast, the N15D mutant displays all the detrimental effects related to deamidation. The N15D/N71D mutant shows only minor additional effects when compared with the N15D mutation, supporting that deamidation of N71 induces negligible effects. The crystal structures show that, in contrast to the N71D mutant, where minimal alterations are observed, the N15D mutation forms new interactions that perturb the structure of loop 1 and loop 3, both critical components of the catalytic site and the interface of HsTIM. Based on a phylogenetic analysis of TIM sequences, we propose the conservation of this mechanism for mammalian TIMs.« less

  4. Structural effects of protein aging: Terminal marking by deamidation in human triosephosphate isomerase

    SciTech Connect

    Torres-Larios, Alfredo; Enríquez-Flores, Sergio; Méndez, Sara -Teresa; Castillo-Villanueva, Adriana; Gómez-Manzo, Saúl; Velázquez, Gabriel López-; Marcial-Quino, Jaime; Torres-Arroyo, Angélica; García-Torres, Itzhel; Reyes-Vivas, Horacio; Oria-Hernández, Jesús; de la Mora-de la Mora, Ignacio

    2015-04-17

    Deamidation, the loss of the ammonium group of asparagine and glutamine to form aspartic and glutamic acid, is one of the most commonly occurring post-translational modifications in proteins. Since deamidation rates are encoded in the protein structure, it has been proposed that they can serve as molecular clocks for the timing of biological processes such as protein turnover, development and aging. Despite the importance of this process, there is a lack of detailed structural information explaining the effects of deamidation on the structure of proteins. Here, we studied the effects of deamidation on human triosephosphate isomerase (HsTIM), an enzyme for which deamidation of N15 and N71 has been long recognized as the signal for terminal marking of the protein. Deamidation was mimicked by site directed mutagenesis; thus, three mutants of HsTIM (N15D, N71D and N15D/N71D) were characterized. The results show that the N71D mutant resembles, structurally and functionally, the wild type enzyme. In contrast, the N15D mutant displays all the detrimental effects related to deamidation. The N15D/N71D mutant shows only minor additional effects when compared with the N15D mutation, supporting that deamidation of N71 induces negligible effects. The crystal structures show that, in contrast to the N71D mutant, where minimal alterations are observed, the N15D mutation forms new interactions that perturb the structure of loop 1 and loop 3, both critical components of the catalytic site and the interface of HsTIM. Based on a phylogenetic analysis of TIM sequences, we propose the conservation of this mechanism for mammalian TIMs.

  5. Relationship of isopentenyl diphosphate (IDP) isomerase activity to isoprene emission of oak leaves.

    PubMed

    Brüggemann, Nicolas; Schnitzler, Jörg-Peter

    2002-10-01

    Oaks emit large amounts of isoprene, a compound that plays an important role in tropospheric chemistry. Isopentenyl diphosphate isomerase (IDI, E.C. 5.3.3.2) catalyzes the isomerization of isopentenyl diphosphate (IDP) to dimethylallyl diphosphate (DMADP), and in isoprene-emitting plants, isoprene synthase (IS) converts the DMADP to isoprene. To study the role of IDI in isoprene biosynthesis of oak leaves, we compared IDI and IS activities in pedunculate oak (Quercus robur L.) and pubescent oak (Quercus pubescens Willd.) with the isoprene emission rates of these species. We developed a non-radioactive enzyme assay to detect IDI activity in crude leaf extracts of Q. robur. The substrate dependency of IDI activity showed biphasic kinetics with Michaelis constants (K(m)(IDP)) of 0.7 +/- 0.2 micro M for a high-affinity phase and 39.5 +/- 6.9 micro M for a low-affinity phase, potentially attributable to different IDI isoforms. Under standard assay conditions, the temperature optimum for IDI activity was about 42 degrees C, but IDI activity was detectable up to 60 degrees C. A sharp pH optimum appeared around pH 7, with 20 mM Mg(2+) also required for IDI activity. Neither IDI activity nor IS activity showed diurnal variation in Q. robur leaves. The sum of IDI activities showed a significant linear correlation with IS activity in both Q. robur and Q. pubescens leaves, and both enzyme activities showed a linear relationship to isoprene emission factors in leaves of these oak species, indicating the possible involvement of IDI in isoprene biosynthesis by oak leaves.

  6. Structural Effects of Protein Aging: Terminal Marking by Deamidation in Human Triosephosphate Isomerase

    PubMed Central

    de la Mora-de la Mora, Ignacio; Torres-Larios, Alfredo; Enríquez-Flores, Sergio; Méndez, Sara-Teresa; Castillo-Villanueva, Adriana; Gómez-Manzo, Saúl; López-Velázquez, Gabriel; Marcial-Quino, Jaime; Torres-Arroyo, Angélica; García-Torres, Itzhel; Reyes-Vivas, Horacio; Oria-Hernández, Jesús

    2015-01-01

    Deamidation, the loss of the ammonium group of asparagine and glutamine to form aspartic and glutamic acid, is one of the most commonly occurring post-translational modifications in proteins. Since deamidation rates are encoded in the protein structure, it has been proposed that they can serve as molecular clocks for the timing of biological processes such as protein turnover, development and aging. Despite the importance of this process, there is a lack of detailed structural information explaining the effects of deamidation on the structure of proteins. Here, we studied the effects of deamidation on human triosephosphate isomerase (HsTIM), an enzyme for which deamidation of N15 and N71 has been long recognized as the signal for terminal marking of the protein. Deamidation was mimicked by site directed mutagenesis; thus, three mutants of HsTIM (N15D, N71D and N15D/N71D) were characterized. The results show that the N71D mutant resembles, structurally and functionally, the wild type enzyme. In contrast, the N15D mutant displays all the detrimental effects related to deamidation. The N15D/N71D mutant shows only minor additional effects when compared with the N15D mutation, supporting that deamidation of N71 induces negligible effects. The crystal structures show that, in contrast to the N71D mutant, where minimal alterations are observed, the N15D mutation forms new interactions that perturb the structure of loop 1 and loop 3, both critical components of the catalytic site and the interface of HsTIM. Based on a phylogenetic analysis of TIM sequences, we propose the conservation of this mechanism for mammalian TIMs. PMID:25884638

  7. Survival of glucose phosphate isomerase null somatic cells and germ cells in adult mouse chimaeras.

    PubMed

    Keighren, Margaret A; Flockhart, Jean H; West, John D

    2016-05-15

    The mouse Gpi1 gene encodes the glycolytic enzyme glucose phosphate isomerase. Homozygous Gpi1(-/-) null mouse embryos die but a previous study showed that some homozygous Gpi1(-/-) null cells survived when combined with wild-type cells in fetal chimaeras. One adult female Gpi1(-/-)↔Gpi1(c/c) chimaera with functional Gpi1(-/-) null oocytes was also identified in a preliminary study. The aims were to characterise the survival of Gpi1(-/-) null cells in adult Gpi1(-/-)↔Gpi1(c/c) chimaeras and determine if Gpi1(-/-) null germ cells are functional. Analysis of adult Gpi1(-/-)↔Gpi1(c/c) chimaeras with pigment and a reiterated transgenic lineage marker showed that low numbers of homozygous Gpi1(-/-) null cells could survive in many tissues of adult chimaeras, including oocytes. Breeding experiments confirmed that Gpi1(-/-) null oocytes in one female Gpi1(-/-)↔Gpi1(c/c) chimaera were functional and provided preliminary evidence that one male putative Gpi1(-/-)↔Gpi1(c/c) chimaera produced functional spermatozoa from homozygous Gpi1(-/-) null germ cells. Although the male chimaera was almost certainly Gpi1(-/-)↔Gpi1(c/c), this part of the study is considered preliminary because only blood was typed for GPI. Gpi1(-/-) null germ cells should survive in a chimaeric testis if they are supported by wild-type Sertoli cells. It is also feasible that spermatozoa could bypass a block at GPI, but not blocks at some later steps in glycolysis, by using fructose, rather than glucose, as the substrate for glycolysis. Although chimaera analysis proved inefficient for studying the fate of Gpi1(-/-) null germ cells, it successfully identified functional Gpi1(-/-) null oocytes and revealed that some Gpi1(-/-) null cells could survive in many adult tissues.

  8. Testing electrostatic complementarity in enzyme catalysis: hydrogen bonding in the ketosteroid isomerase oxyanion hole.

    PubMed

    Kraut, Daniel A; Sigala, Paul A; Pybus, Brandon; Liu, Corey W; Ringe, Dagmar; Petsko, Gregory A; Herschlag, Daniel

    2006-04-01

    A longstanding proposal in enzymology is that enzymes are electrostatically and geometrically complementary to the transition states of the reactions they catalyze and that this complementarity contributes to catalysis. Experimental evaluation of this contribution, however, has been difficult. We have systematically dissected the potential contribution to catalysis from electrostatic complementarity in ketosteroid isomerase. Phenolates, analogs of the transition state and reaction intermediate, bind and accept two hydrogen bonds in an active site oxyanion hole. The binding of substituted phenolates of constant molecular shape but increasing pK(a) models the charge accumulation in the oxyanion hole during the enzymatic reaction. As charge localization increases, the NMR chemical shifts of protons involved in oxyanion hole hydrogen bonds increase by 0.50-0.76 ppm/pK(a) unit, suggesting a bond shortening of 0.02 A/pK(a) unit. Nevertheless, there is little change in binding affinity across a series of substituted phenolates (DeltaDeltaG = -0.2 kcal/mol/pK(a) unit). The small effect of increased charge localization on affinity occurs despite the shortening of the hydrogen bonds and a large favorable change in binding enthalpy (DeltaDeltaH = -2.0 kcal/mol/pK(a) unit). This shallow dependence of binding affinity suggests that electrostatic complementarity in the oxyanion hole makes at most a modest contribution to catalysis of 300-fold. We propose that geometrical complementarity between the oxyanion hole hydrogen-bond donors and the transition state oxyanion provides a significant catalytic contribution, and suggest that KSI, like other enzymes, achieves its catalytic prowess through a combination of modest contributions from several mechanisms rather than from a single dominant contribution.

  9. Peri/Epicellular Protein Disulfide Isomerase Sustains Vascular Lumen Caliber Through an Anticonstrictive Remodeling Effect.

    PubMed

    Tanaka, Leonardo Y; Araújo, Haniel A; Hironaka, Gustavo K; Araujo, Thaís L S; Takimura, Celso K; Rodriguez, Andres I; Casagrande, Annelise S; Gutierrez, Paulo S; Lemos-Neto, Pedro Alves; Laurindo, Francisco R M

    2016-03-01

    Whole-vessel remodeling critically determines lumen caliber in vascular (patho)physiology, and it is reportedly redox-dependent. We hypothesized that the cell-surface pool of the endoplasmic reticulum redox chaperone protein disulfide isomerase-A1 (peri/epicellular=pecPDI), which is known to support thrombosis, also regulates disease-associated vascular architecture. In human coronary atheromas, PDI expression inversely correlated with constrictive remodeling and plaque stability. In a rabbit iliac artery overdistension model, there was unusually high PDI upregulation (≈25-fold versus basal, 14 days postinjury), involving both intracellular and pecPDI. PecPDI neutralization with distinct anti-PDI antibodies did not enhance endoplasmic reticulum stress or apoptosis. In vivo pecPDI neutralization with PDI antibody-containing perivascular gel from days 12 to 14 post injury promoted 25% decrease in the maximally dilated arteriographic vascular caliber. There was corresponding whole-vessel circumference loss using optical coherence tomography without change in neointima, which indicates constrictive remodeling. This was accompanied by decreased hydrogen peroxide generation. Constrictive remodeling was corroborated by marked changes in collagen organization, that is, switching from circumferential to radial fiber orientation and to a more rigid fiber type. The cytoskeleton architecture was also disrupted; there was a loss of stress fiber coherent organization and a switch from thin to medium thickness actin fibers, all leading to impaired viscoelastic ductility. Total and PDI-associated expressions of β1-integrin, and levels of reduced cell-surface β1-integrin, were diminished after PDI antibody treatment, implicating β1-integrin as a likely pecPDI target during vessel repair. Indeed, focal adhesion kinase phosphorylation, a downstream β1-integrin effector, was decreased by PDI antibody. Thus, the upregulated pecPDI pool tunes matrix/cytoskeleton reshaping to

  10. Carotenoid isomerase is key determinant of petal color of Calendula officinalis.

    PubMed

    Kishimoto, Sanae; Ohmiya, Akemi

    2012-01-02

    Orange petals of calendula (Calendula officinalis) accumulate red carotenoids with the cis-configuration at the C-5 or C-5' position (5-cis-carotenoids). We speculated that the orange-flowered calendula is a carotenoid isomerase (crtiso) loss-of-function mutant that impairs the cis-to-trans conversion of 5-cis-carotenoids. We compared the sequences and enzyme activities of CRTISO from orange- and yellow-flowered calendulas. Four types of CRTISO were expressed in calendula petals. The deduced amino acid sequence of one of these genes (CoCRTISO1) was different between orange- and yellow-flowered calendulas, whereas the sequences of the other three CRTISOs were identical between these plants. Analysis of the enzymatic activities of the CoCRTISO homologs showed that CoCRTISO1-Y, which was expressed in yellow petals, converted carotenoids from the cis-to-trans-configuration, whereas both CoCRTISO1-ORa and 1-ORb, which were expressed in orange petals, showed no activity with any of the cis-carotenoids we tested. Moreover, the CoCRTISO1 genotypes of the F2 progeny obtained by crossing orange and yellow lines linked closely to petal color. These data indicate that CoCRTISO1 is a key regulator of the accumulation of 5-cis-carotenoids in calendula petals. Site-directed mutagenesis showed that the deletion of Cys-His-His at positions 462-464 in CoCRTISO1-ORa and a Gly-to-Glu amino acid substitution at position 450 in CoCRTISO1-ORb abolished enzyme activity completely, indicating that these amino acid residues are important for the enzymatic activity of CRTISO.

  11. Small molecule-induced oxidation of protein disulfide isomerase is neuroprotective

    PubMed Central

    Kaplan, Anna; Gaschler, Michael M.; Dunn, Denise E.; Colligan, Ryan; Brown, Lewis M.; Palmer, Arthur G.; Lo, Donald C.; Stockwell, Brent R.

    2015-01-01

    Protein disulfide isomerase (PDI) is a chaperone protein in the endoplasmic reticulum that is up-regulated in mouse models of, and brains of patients with, neurodegenerative diseases involving protein misfolding. PDI’s role in these diseases, however, is not fully understood. Here, we report the discovery of a reversible, neuroprotective lead optimized compound (LOC)14, that acts as a modulator of PDI. LOC14 was identified using a high-throughput screen of ∼10,000 lead-optimized compounds for potent rescue of viability of PC12 cells expressing mutant huntingtin protein, followed by an evaluation of compounds on PDI reductase activity in an in vitro screen. Isothermal titration calorimetry and fluorescence experiments revealed that binding to PDI was reversible with a Kd of 62 nM, suggesting LOC14 to be the most potent PDI inhibitor reported to date. Using 2D heteronuclear single quantum correlation NMR experiments, we were able to map the binding site of LOC14 as being adjacent to the active site and to observe that binding of LOC14 forces PDI to adopt an oxidized conformation. Furthermore, we found that LOC14-induced oxidation of PDI has a neuroprotective effect not only in cell culture, but also in corticostriatal brain slice cultures. LOC14 exhibited high stability in mouse liver microsomes and blood plasma, low intrinsic microsome clearance, and low plasma-protein binding. These results suggest that LOC14 is a promising lead compound to evaluate the potential therapeutic effects of modulating PDI in animal models of disease. PMID:25848045

  12. Carotenoid Isomerase Is Key Determinant of Petal Color of Calendula officinalis*

    PubMed Central

    Kishimoto, Sanae; Ohmiya, Akemi

    2012-01-01

    Orange petals of calendula (Calendula officinalis) accumulate red carotenoids with the cis-configuration at the C-5 or C-5′ position (5-cis-carotenoids). We speculated that the orange-flowered calendula is a carotenoid isomerase (crtiso) loss-of-function mutant that impairs the cis-to-trans conversion of 5-cis-carotenoids. We compared the sequences and enzyme activities of CRTISO from orange- and yellow-flowered calendulas. Four types of CRTISO were expressed in calendula petals. The deduced amino acid sequence of one of these genes (CoCRTISO1) was different between orange- and yellow-flowered calendulas, whereas the sequences of the other three CRTISOs were identical between these plants. Analysis of the enzymatic activities of the CoCRTISO homologs showed that CoCRTISO1-Y, which was expressed in yellow petals, converted carotenoids from the cis-to-trans-configuration, whereas both CoCRTISO1-ORa and 1-ORb, which were expressed in orange petals, showed no activity with any of the cis-carotenoids we tested. Moreover, the CoCRTISO1 genotypes of the F2 progeny obtained by crossing orange and yellow lines linked closely to petal color. These data indicate that CoCRTISO1 is a key regulator of the accumulation of 5-cis-carotenoids in calendula petals. Site-directed mutagenesis showed that the deletion of Cys-His-His at positions 462–464 in CoCRTISO1-ORa and a Gly-to-Glu amino acid substitution at position 450 in CoCRTISO1-ORb abolished enzyme activity completely, indicating that these amino acid residues are important for the enzymatic activity of CRTISO. PMID:22069331

  13. The prolyl isomerase Pin1 increases β-cell proliferation and enhances insulin secretion.

    PubMed

    Nakatsu, Yusuke; Mori, Keiichi; Matsunaga, Yasuka; Yamamotoya, Takeshi; Ueda, Koji; Inoue, Yuki; Mitsuzaki-Miyoshi, Keiko; Sakoda, Hideyuki; Fujishiro, Midori; Yamaguchi, Suguru; Kushiyama, Akifumi; Ono, Hiraku; Ishihara, Hisamitsu; Asano, Tomoichiro

    2017-07-14

    The prolyl isomerase Pin1 binds to the phosphorylated Ser/Thr-Pro motif of target proteins and enhances their cis-trans conversion. This report is the first to show that Pin1 expression in pancreatic β cells is markedly elevated by high-fat diet feeding and in ob/ob mice. To elucidate the role of Pin1 in pancreatic β cells, we generated β-cell-specific Pin1 KO (βPin1 KO) mice. These mutant mice showed exacerbation of glucose intolerance but had normal insulin sensitivity. We identified two independent factors underlying impaired insulin secretion in the βPin1 KO mice. Pin1 enhanced pancreatic β-cell proliferation, as indicated by a reduced β-cell mass in βPin1 KO mice compared with control mice. Moreover, a diet high in fat and sucrose failed to increase pancreatic β-cell growth in the βPin1 KO mice, an observation to which up-regulation of the cell cycle protein cyclin D appeared to contribute. The other role of Pin1 was to activate the insulin-secretory step: Pin1 KO β cells showed impairments in glucose- and KCl-induced elevation of the intracellular Ca(2+) concentration and insulin secretion. We also identified salt-inducible kinase 2 (SIK2) as a Pin1-binding protein that affected the regulation of Ca(2+) influx and found Pin1 to enhance SIK2 kinase activity, resulting in a decrease in p35 protein, a negative regulator of Ca(2+) influx. Taken together, our observations demonstrate critical roles of Pin1 in pancreatic β cells and that Pin1 both promotes β-cell proliferation and activates insulin secretion. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Novel Protein Disulfide Isomerase Inhibitor with Anticancer Activity in Multiple Myeloma.

    PubMed

    Vatolin, Sergei; Phillips, James G; Jha, Babal K; Govindgari, Shravya; Hu, Jennifer; Grabowski, Dale; Parker, Yvonne; Lindner, Daniel J; Zhong, Fei; Distelhorst, Clark W; Smith, Mitchell R; Cotta, Claudiu; Xu, Yan; Chilakala, Sujatha; Kuang, Rebecca R; Tall, Samantha; Reu, Frederic J

    2016-06-01

    Multiple myeloma cells secrete more disulfide bond-rich proteins than any other mammalian cell. Thus, inhibition of protein disulfide isomerases (PDI) required for protein folding in the endoplasmic reticulum (ER) should increase ER stress beyond repair in this incurable cancer. Here, we report the mechanistically unbiased discovery of a novel PDI-inhibiting compound with antimyeloma activity. We screened a 30,355 small-molecule library using a multilayered multiple myeloma cell-based cytotoxicity assay that modeled disease niche, normal liver, kidney, and bone marrow. CCF642, a bone marrow-sparing compound, exhibited a submicromolar IC50 in 10 of 10 multiple myeloma cell lines. An active biotinylated analog of CCF642 defined binding to the PDI isoenzymes A1, A3, and A4 in MM cells. In vitro, CCF642 inhibited PDI reductase activity about 100-fold more potently than the structurally distinct established inhibitors PACMA 31 and LOC14. Computational modeling suggested a novel covalent binding mode in active-site CGHCK motifs. Remarkably, without any further chemistry optimization, CCF642 displayed potent efficacy in an aggressive syngeneic mouse model of multiple myeloma and prolonged the lifespan of C57BL/KaLwRij mice engrafted with 5TGM1-luc myeloma, an effect comparable to the first-line multiple myeloma therapeutic bortezomib. Consistent with PDI inhibition, CCF642 caused acute ER stress in multiple myeloma cells accompanied by apoptosis-inducing calcium release. Overall, our results provide an illustration of the utility of simple in vivo simulations as part of a drug discovery effort, along with a sound preclinical rationale to develop a new small-molecule therapeutic to treat multiple myeloma. Cancer Res; 76(11); 3340-50. ©2016 AACR. ©2016 American Association for Cancer Research.

  15. Post-Streptococcal Auto-Antibodies Inhibit Protein Disulfide Isomerase and Are Associated with Insulin Resistance

    PubMed Central

    Aran, Adi; Weiner, Karin; Lin, Ling; Finn, Laurel Ann; Greco, Mary Ann; Peppard, Paul; Young, Terry; Ofran, Yanay; Mignot, Emmanuel

    2010-01-01

    Post-streptococcal autoimmunity affects millions worldwide, targeting multiple organs including the heart, brain, and kidneys. To explore the post-streptococcal autoimmunity spectrum, we used western blot analyses, to screen 310 sera from healthy subjects with (33%) and without (67%) markers of recent streptococcal infections [anti-Streptolysin O (ASLO) or anti-DNAse B (ADB)]. A 58 KDa protein, reacting strongly with post-streptococcal sera, was identified as Protein Disulfide Isomerase (PDI), an abundant protein with pleiotropic metabolic, immunologic, and thrombotic effects. Anti-PDI autoantibodies, purified from human sera, targeted similar epitopes in Streptolysin O (SLO, P51-61) and PDI (P328-338). The correlation between post-streptococcal status and anti-human PDI auto-immunity was further confirmed in a total of 2987 samples (13.6% in 530 ASLO positive versus 5.6% in 2457 ASLO negative samples, p<0.0001). Finally, anti-PDI auto-antibodies inhibited PDI-mediated insulin degradation in vitro (n = 90, p<0.001), and correlated with higher serum insulin (14.1 iu/ml vs. 12.2 iu/ml, n = 1215, p = 0.039) and insulin resistance (Homeostatic Model Assessment (HOMA) 4.1 vs. 3.1, n = 1215, p = 0.004), in a population-based cohort. These results identify PDI as a major target of post-streptococcal autoimmunity, and establish a new link between infection, autoimmunity, and metabolic disturbances. PMID:20886095

  16. Structural characterization of a ribose-5-phosphate isomerase B from the pathogenic fungus Coccidioides immitis

    PubMed Central

    2011-01-01

    Background Ribose-5-phosphate isomerase is an enzyme that catalyzes the interconversion of ribose-5-phosphate and ribulose-5-phosphate. This family of enzymes naturally occurs in two distinct classes, RpiA and RpiB, which play an important role in the pentose phosphate pathway and nucleotide and co-factor biogenesis. Results Although RpiB occurs predominantly in bacteria, here we report crystal structures of a putative RpiB from the pathogenic fungus Coccidioides immitis. A 1.9 Å resolution apo structure was solved by combined molecular replacement and single wavelength anomalous dispersion (SAD) phasing using a crystal soaked briefly in a solution containing a high concentration of iodide ions. RpiB from C. immitis contains modest sequence and high structural homology to other known RpiB structures. A 1.8 Å resolution phosphate-bound structure demonstrates phosphate recognition and charge stabilization by a single positively charged residue whereas other members of this family use up to five positively charged residues to contact the phosphate of ribose-5-phosphate. A 1.7 Å resolution structure was obtained in which the catalytic base of C. immitis RpiB, Cys76, appears to form a weakly covalent bond with the central carbon of malonic acid with a bond distance of 2.2 Å. This interaction may mimic that formed by the suicide inhibitor iodoacetic acid with RpiB. Conclusion The C. immitis RpiB contains the same fold and similar features as other members of this class of enzymes such as a highly reactive active site cysteine residue, but utilizes a divergent phosphate recognition strategy and may recognize a different substrate altogether. PMID:21995815

  17. Endothelial cells microparticle-associated protein disulfide isomerase promotes platelet activation in metabolic syndrome

    PubMed Central

    Li, Yi-hui; Song, Dai-jun; Chen, Tong-shuai; Zhang, Wei; Zhong, Ming; Zhang, Yun; Xing, Yan-qiu; Wang, Zhi-hao

    2016-01-01

    Background Metabolic syndrome (MetS) is a common challenge in the world, and the platelet activation is enhanced in MetS patients. However, the fundamental mechanism that underlies platelet activation in MetS remains incompletely understood. Endothelial cells are damaged seriously in MetS patients, then they release more endothelial microparticles (EMPs). After all, whether the EMPs participate in platelet activation is still obscure. If they were, how did they work? Results We demonstrated that the levels of EMPs, PMPs (platelet derived microparticles) and microparticle-carried-PDI activity increased in MetS patients. IR endothelial cells released more EMPs, the EMP-PDI was more activated. EMPs can enhance the activation of CD62P, GPIIb/IIIa and platelet aggregation and this process can be partly inhibited by PDI inhibitor such as RL90 and rutin. Activated platelets stimulated by EMPs expressed more PDI on cytoplasm and released more PMPs. Materials and Methods We obtained plasma from 23 MetS patients and 8 normal healthy controls. First we built insulin resistance (IR) model of human umbilical vein endothelial cells (HUVECs), and then we separated EMPs from HUVECs culture medium and used these EMPs to stimulate platelets. Levels of microparticles, P-selectin(CD62P), Glycoprotein IIb/IIIa (GPIIb/IIIa) were detected by flow cytometry and levels of EMPs were detected by enzyme-linked immunosorbent assay (ELISA). The protein disulfide isomerase (PDI) activity was detected by insulin transhydrogenase assay. Platelet aggregation was assessed by turbidimetry. Conclusion EMPs can promote the activation of GPIIb/IIIa in platelets and platelet aggregation by the PDI which is carried on the surface of EMPs. PMID:27825126

  18. Substrate-Induced Dimerization of Engineered Monomeric Variants of Triosephosphate Isomerase from Trichomonas vaginalis.

    PubMed

    Lara-Gonzalez, Samuel; Estrella, Priscilla; Portillo, Carmen; Cruces, María E; Jimenez-Sandoval, Pedro; Fattori, Juliana; Migliorini-Figueira, Ana C; Lopez-Hidalgo, Marisol; Diaz-Quezada, Corina; Lopez-Castillo, Margarita; Trasviña-Arenas, Carlos H; Sanchez-Sandoval, Eugenia; Gómez-Puyou, Armando; Ortega-Lopez, Jaime; Arroyo, Rossana; Benítez-Cardoza, Claudia G; Brieba, Luis G

    2015-01-01

    The dimeric nature of triosephosphate isomerases (TIMs) is maintained by an extensive surface area interface of more than 1600 Å2. TIMs from Trichomonas vaginalis (TvTIM) are held in their dimeric state by two mechanisms: a ball and socket interaction of residue 45 of one subunit that fits into the hydrophobic pocket of the complementary subunit and by swapping of loop 3 between subunits. TvTIMs differ from other TIMs in their unfolding energetics. In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer. Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model. In contrast to other monomeric TIMs, monomeric variants of TvTIM1 are stable and unexpectedly one of them (I45A) is only 29-fold less active than wild-type TvTIM1. The high enzymatic activity of monomeric TvTIMs contrast with the marginal catalytic activity of diverse monomeric TIMs variants. The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.

  19. Nitric oxide mediates selective degeneration of hypothalamic orexin neurons through dysfunction of protein disulfide isomerase.

    PubMed

    Obukuro, Kanae; Nobunaga, Mizuki; Takigawa, Moeko; Morioka, Hiroshi; Hisatsune, Akinori; Isohama, Yoichiro; Shimokawa, Hiroaki; Tsutsui, Masato; Katsuki, Hiroshi

    2013-07-31

    We addressed the role of nitric oxide (NO) in orexin neuron degeneration that has been observed under various pathological conditions. Administration of an NO donor NOC18 (50 nmol) into the third ventricle of mice resulted in a significant decrease of orexin-immunoreactive (-IR) neurons, in contrast to a modest change in melanin-concentrating hormone-IR neurons. In addition, NOC18 promoted formation of orexin-A-IR aggregates within orexin neurons. An endoplasmic reticulum stress inducer tunicamycin replicated the effect of NOC18 with regard to decrease of orexin-IR neurons and formation of aggregates. We also found that NOC18 caused an increase in S-nitrosation of protein disulfide isomerase (PDI) and a decrease in PDI activity in hypothalamic tissues. Moreover, PDI inhibitors, such as cystamine and securinine, caused a selective decrease of orexin neurons and promoted formation of orexin-A-IR aggregates. Aggregate formation in orexin-IR neurons was also induced by local injection of small interfering RNA targeting PDI. Interestingly, sleep deprivation for 7 consecutive days induced a selective decrease of orexin-IR neurons, which was preceded by aggregate formation in orexin-IR neurons and an increase in S-nitrosated PDI in the hypothalamus. Activity of neuronal NO synthase (nNOS)-positive neurons in the lateral hypothalamus as assessed by c-Fos expression was elevated in response to sleep deprivation. Finally, sleep deprivation-induced decrease of orexin-IR neurons, formation of aggregates, and S-nitrosation of PDI were not observed in nNOS knock-out mice. These results indicate that nNOS-derived NO may mediate specific pathological events in orexin neurons, including neuropeptide misfolding via S-nitrosation and inactivation of PDI.

  20. Overexpression and characterization of a recombinant l-ribose isomerase from Actinotalea fermentans ATCC 43279.

    PubMed

    Tseng, Wen-Chi; Wu, Tai-Jeng; Chang, Ya-Ju; Cheng, Hung-Wen; Fang, Tsuei-Yun

    2017-10-10

    A putative l-ribose isomerase (EC 5.3.1.B3, l-RI) gene of Actinotalea fermentans ATCC 43279 was chemically synthesized, subcloned into pET-21b vector, and then overexpressed in Escherichia coli. After 0.5mM IPTG induction at 20°C for 20h, the recombinant l-RI was highly expressed with up to 50% of the total proteins. About 70% of the expressed l-RI appeared in the cell-free extract as a soluble form, and a high yield of active l-RI, 23,800U/L or 952U/g of wet cells, was achieved. The purified recombinant l-RI demonstrated its optimal activity at 45°C and pH 8 (in tricine-NaOH buffer). Metal ions are not required for l-RI activity, but Hg(2+) inhibits its activity completely. The enzyme has a half-life of 74min at 50°C and an equilibrium ratio of 30:70 between l-ribulose and l-ribose at 45°C. The Vmax, kcat, KM, and catalytic efficiency (kcat/KM) of the recombinant l-RI against l-ribose are 232U/mg, 6700min(-1), 31.3mM, and 214min(-1)mM(-1), respectively. The high expression yield of the active recombinant A. fermentansl-RI and its highest Vmax, kcat, and catalytic efficiency among the characterized recombinant l-RIs suggest that this recombinant enzyme shows a potential application to produce l-ribose in industry. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Hydrogen Bonding in the Active Site of Ketosteroid Isomerase: Electronic Inductive Effects and Hydrogen Bond Coupling

    PubMed Central

    Hanoian, Philip; Sigala, Paul A.; Herschlag, Daniel; Hammes-Schiffer, Sharon

    2010-01-01

    Computational studies are performed to analyze the physical properties of hydrogen bonds donated by Tyr16 and Asp103 to a series of substituted phenolate inhibitors bound in the active site of ketosteroid isomerase (KSI). As the solution pKa of the phenolate increases, these hydrogen bond distances decrease, the associated NMR chemical shifts increase, and the fraction of protonated inhibitor increases, in agreement with prior experiments. The quantum mechanical/molecular mechanical calculations provide insight into the electronic inductive effects along the hydrogen-bonding network that includes Tyr16, Tyr57, and Tyr32, as well as insight into hydrogen bond coupling in the active site. The calculations predict that the most-downfield NMR chemical shift observed experimentally corresponds to the Tyr16-phenolate hydrogen bond and that Tyr16 is the proton donor when a bound naphtholate inhibitor is observed to be protonated in electronic absorption experiments. According to these calculations, the electronic inductive effects along the hydrogen-bonding network of tyrosines cause the Tyr16 hydroxyl to be more acidic than the Asp103 carboxylic acid moiety, which is immersed in a relatively nonpolar environment. When one of the distal tyrosine residues in the network is mutated to phenylalanine, thereby diminishing this inductive effect, the Tyr16-phenolate hydrogen bond lengthens, and the Asp103-phenolate hydrogen bond shortens, as observed in NMR experiments. Furthermore, the calculations suggest that the differences in the experimental NMR data and electronic absorption spectra for pKSI and tKSI, two homologous bacterial forms of the enzyme, are due predominantly to the third tyrosine that is present in the hydrogen-bonding network of pKSI but not tKSI. These studies also provide experimentally testable predictions about the impact of mutating the distal tyrosine residues in this hydrogen-bonding network on the NMR chemical shifts and electronic absorption spectra

  2. Patagonfibrase modifies protein expression of tissue factor and protein disulfide isomerase in rat skin.

    PubMed

    Peichoto, María Elisa; Santoro, Marcelo Larami

    2016-09-01

    Patagonfibrase is a hemorrhagic metalloproteinase isolated from the venom of the South American rear-fanged snake Philodryas patagoniensis, and is an important contributor to local lesions inflicted by this species. The tissue factor (TF)-factor VIIa complex, besides triggering the coagulation cascade, has been demonstrated to be involved in inflammatory events. Our aim was to determine whether patagonfibrase affects the expression of TF and protein disulfide isomerase (PDI), an enzyme that controls TF biological activity, at the site of patagonfibrase injection, and thus if they may play a role in hemostatic and inflammatory events induced by snake venoms. Patagonfibrase (60 μg/kg) was administered s.c. to rats, and after 3 h blood was collected to evaluate hemostasis parameters, and skin fragments close to the site of injection were taken to assess TF and PDI expression. Patagonfibrase did not alter blood cell counts, plasma fibrinogen levels, or levels of TF activity in plasma. However, by semiquantitative Western blotting, patagonfibrase increased TF expression by 2-fold, and decreased PDI expression by 3-fold in skin samples. In agreement, by immunohistochemical analyses, prominent TF expression was observed in the subcutaneous tissue. Thus, patagonfibrase affects the local expression of TF and PDI without inducing any systemic hemostatic disturbance, although that they may be involved in the local inflammatory events induced by hemorrhagic metalloproteinases. Once antivenom therapy is not totally effective to treat the local injury induced by snake venoms, modulation of the activity and expression of TF and/or PDI might become a strategy for treating snake envenomation.

  3. Iron Binding Properties of Recombinant Class A Protein Disulfide Isomerase from Arabidopsis thaliana.

    PubMed

    Remelli, William; Santabarbara, Stefano; Carbonera, Donatella; Bonomi, Francesco; Ceriotti, Aldo; Casazza, Anna Paola

    2017-04-07

    The protein disulfide isomerase (PDI) family comprises a wide set of enzymes mainly involved in thiol-disulfide exchange reactions in the endoplasmic reticulum. Class A PDIs (PDI-A) constitute the smallest members of the family, consisting of a single thioredoxin (TRX) module without any additional domains. To date, their