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Sample records for peptidyl prolyl cis-trans

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. The intriguing Cyclophilin A-HIV-1 Vpr interaction: prolyl cis/trans isomerisation catalysis and specific binding

    PubMed Central

    2010-01-01

    Background Cyclophilin A (CypA) represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication, although the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The interaction of HIV-1 viral protein R (Vpr) with the human peptidyl prolyl isomerase CypA is known to occur in vitro and in vivo. However, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined. Results Characterization of the interactions of human CypA with N-terminal peptides of HIV-1 Vpr has been achieved using a combination of nuclear magnetic resonace (NMR) exchange spectroscopy and surface plasmon resonance spectroscopy (SPR). NMR data at atomic resolution indicate prolyl cis/trans isomerisation of the highly conserved proline residues Pro-5, -10, -14 and -35 of Vpr are catalyzed by human CypA and require only very low concentrations of the isomerase relative to that of the peptide substrates. Of the N-terminal peptides of Vpr only those containing Pro-35 bind to CypA in a biosensor assay. SPR studies of specific N-terminal peptides with decreasing numbers of residues revealed that a seven-residue motif centred at Pro-35 consisting of RHFPRIW, which under membrane-like solution conditions comprises the loop region connecting helix 1 and 2 of Vpr and the two terminal residues of helix 1, is sufficient to maintain strong specific binding. Conclusions Only N-terminal peptides of Vpr containing Pro-35, which appears to be vital for manifold functions of Vpr, bind to CypA in a biosensor assay. This indicates that Pro-35 is essential for a specific CypA-Vpr binding interaction, in contrast to the general prolyl cis/trans isomerisation observed for all proline residues of Vpr, which only involve transient enzyme-substrate interactions. Previously suggested models depicting CypA as a chaperone that plays a role in HIV-1 virulence are now supported by our data

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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.

  5. Analysis of peptidyl-propyl-cis/trans isomerase 1 (PIN1) gene -842(G > C) and -667(T > C) polymorphic variants in relation to breast cancer risk and clinico-pathological parameters.

    PubMed

    Naidu, Rakesh; Har, Yip C; Taib, Nur A M

    2011-10-01

    The purpose of this study was to investigate the association between the peptidyl-propyl-cis/trans isomerase 1 (PIN1) -842(G > C) and -667(T > C) polymorphic variants and breast cancer risk among Malaysian ethnic groups namely the Malays, Chinese and Indians, as well as clinico-pathological characteristics of the patients. The polymerase chain reaction-restriction fragment length polymorphism was used to genotype 387 breast cancer patients and 252 normal and healthy women who had no history of any malignancy. The distribution of -842(G > C) and -667(T > C) genotypes and alleles frequencies between breast cancer cases and normal individuals showed lack of statistical significance among the Malays (p > 0.05), Chinese (p > 0.05) and Indians (p > 0.05), respectively. Multivariate logistic regression analysis showed that the Malay, Chinese and Indian women who were -842CC homozygotes (p = 0.198, 0.089, 0.620), -842GC heterozygotes (p = 0.492, 0.176, 0.377) and -842C allele carriers (P = 0.226, 0.059, 0.669), respectively, were not associated with breast cancer risk. Furthermore Malay, Chinese and Indian women who were heterozygous (p = 0.777, 0.319, 0.710) and homozygous (p = 0.864, 0.986, 0.954) for -667C allele or carriers of -667C allele (p = 0.977, 0.915, 0.880), respectively, were not associated with an increased risk of breast cancer. None of the -842C and -667C allele genotypes were significantly associated with the clinico-pathological characteristics. Our findings suggest that the polymorphic variants of -842(G > C) and -667(T > C) genes may not appear to have an influence on breast cancer risk among Malaysian Malay, Chinese and Indian women.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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.

  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. 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

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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

  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. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. Randomized Subspace Learning for Proline Cis-Trans Isomerization Prediction.

    PubMed

    Al-Jarrah, Omar Y; Yoo, Paul D; Taha, Kamal; Muhaidat, Sami; Shami, Abdallah; Zaki, Nazar

    2015-01-01

    Proline residues are common source of kinetic complications during folding. The X-Pro peptide bond is the only peptide bond for which the stability of the cis and trans conformations is comparable. The cis-trans isomerization (CTI) of X-Pro peptide bonds is a widely recognized rate-limiting factor, which can not only induces additional slow phases in protein folding but also modifies the millisecond and sub-millisecond dynamics of the protein. An accurate computational prediction of proline CTI is of great importance for the understanding of protein folding, splicing, cell signaling, and transmembrane active transport in both the human body and animals. In our earlier work, we successfully developed a biophysically motivated proline CTI predictor utilizing a novel tree-based consensus model with a powerful metalearning technique and achieved 86.58 percent Q2 accuracy and 0.74 Mcc, which is a better result than the results (70-73 percent Q2 accuracies) reported in the literature on the well-referenced benchmark dataset. In this paper, we describe experiments with novel randomized subspace learning and bootstrap seeding techniques as an extension to our earlier work, the consensus models as well as entropy-based learning methods, to obtain better accuracy through a precise and robust learning scheme for proline CTI prediction.

  18. 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.

  19. IRIS Toxicological Review of Cis-& Trans-1,2-Dichloroethylene (Final Report)

    EPA Science Inventory

    EPA has finalized the Toxicological Review of cis- & trans-1,2-Dichloroethylene: in support of the Integrated Risk Information System (IRIS). Now final, this assessment may be used by EPA’s program and regional offices to inform decisions to protect human health.

  20. IRIS Toxicological Review of Cis-& Trans-1,2-Dichloroethylene (Final Report)

    EPA Science Inventory

    EPA has finalized the Toxicological Review of cis- & trans-1,2-Dichloroethylene: in support of the Integrated Risk Information System (IRIS). Now final, this assessment may be used by EPA’s program and regional offices to inform decisions to protect human health.

  1. 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.

  2. 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

  3. 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.

  4. Refolding additive, dimethylbenzylammonium propane sulfonate (NDSB- 256), accelerates gly-pro cis-trans isomerization.

    PubMed

    Wang, Haimei; Hosoda, Kazuo; Terawaki, Shin-Ichi; Wakamatsu, Kaori

    2014-01-01

    Proline cis-trans isomerization plays a key role in the rate-determining steps of protein folding, and many different peptide-proline cis-trans isomerases (PPIases) catalyze this reaction. The acceleration of isomerization would be beneficial for in vitro refolding of protein preparations for industrial and research purposes. So we analyzed whether low-molecular-weight compounds that have been reported to enhance protein refolding have the activity to accelerate the isomerization. To evaluate the effects of chemicals on the isomerization rate, we set up a new NMR (EXSY) method that is invulnerable to their inhibitory activity, if any, and to their large NMR signals. With this method, we found that dimethylbenzylammonium propane sulfonate (NDSB-256) increase the isomerization rate in a concentration-dependent manner for the first time. Acceleration by imidazole (suggested but not experimentally confirmed) was also demonstrated. Arginine, a most popular refolding additive, did not show any significant effects on the isomerization reaction as expected.

  5. Characteristic structural features of indolicidin: effects of the cis-trans isomerism on its conformation.

    PubMed

    Leitgeb, Balázs

    2014-01-01

    Indolicidin is an antimicrobial peptide showing a broad spectrum of antibacterial and antifungal activities, and according to the cis-trans isomerism of three Xaa-Pro peptide bonds, eight different stereoisomers could be distinguished for this peptide. As the cis-trans isomerism about the Xaa-Pro peptide bonds was not considered in previous studies, the structural features of distinct stereoisomeric forms were not characterized in detail, so far. In this theoretical study, the influences of cis-trans isomerism on the conformation of indolicidin were investigated, as well as the typical structural properties of each stereoisomer were determined, focusing on the secondary structures and intramolecular interactions. Based on the results derived from the molecular dynamics simulations, it could be concluded that the eight different stereoisomeric forms of indolicidin adopted characteristic conformational features. Nevertheless, the appearance of various turn structures and intramolecular interactions proved to be dependent on the cis or trans nature of Xaa-Pro peptide bonds, indicating the relevant role of Pro amino acids in determining the three-dimensional structure of this peptide.

  6. Structural Elucidation of cis/trans Dicaffeoylquinic Acid Photoisomerization Using Ion Mobility Spectrometry-Mass Spectrometry.

    PubMed

    Zheng, Xueyun; Renslow, Ryan S; Makola, Mpho M; Webb, Ian K; Deng, Liulin; Thomas, Dennis G; Govind, Niranjan; Ibrahim, Yehia M; Kabanda, Mwadham M; Dubery, Ian A; Heyman, Heino M; Smith, Richard D; Madala, Ntakadzeni E; Baker, Erin S

    2017-04-06

    Due to the recently uncovered health benefits and anti-HIV activities of dicaffeoylquinic acids (diCQAs), understanding their structures and functions is of great interest for drug discovery efforts. DiCQAs are analytically challenging to identify and quantify since they commonly exist as a diverse mixture of positional and geometric (cis/trans) isomers. In this work, we utilized ion mobility spectrometry coupled with mass spectrometry to separate the various isomers before and after UV irradiation. The experimental collision cross sections were then compared with theoretical structures to differentiate and identify the diCQA isomers. Our analyses found that naturally the diCQAs existed predominantly as trans/trans isomers, but after 3 h of UV irradiation, cis/cis, cis/trans, trans/cis, and trans/trans isomers were all present in the mixture. This is the first report of successful differentiation of cis/trans diCQA isomers individually, which shows the great promise of IMS coupled with theoretical calculations for determining the structure and activity relationships of different isomers in drug discovery studies.

  7. Time-dependent inhibition of peptidylprolyl cis-trans-isomerases by FK506 is probably due to cis-trans isomerization of the inhibitor's imide bond.

    PubMed Central

    Zarnt, T; Lang, K; Burtscher, H; Fischer, G

    1995-01-01

    Free in solution, the immunosuppressive compounds cyclosporin A (CsA), FK506, ascomycin and rapamycin are present in many solvents in various slowly interconverting conformations. Together with their cellular receptor proteins, cyclophilin (CyP) and FK506-binding protein (FKBP), however, these inhibitors have been shown to have a homogeneous conformation. The existence of a slow cis-trans interconversion of an imidic bond in the inhibitor molecule during the course of the formation of the CsA-CyP18cy complex (where CyP18cy is human 18 kDa cytosolic CyP) prompted us to investigate the reaction of the peptidomacrolides FK506, ascomycin and rapamycin with two specific binding-proteins in more detail. Since formation of the FK506-FKBP complex results in the inhibition of the peptidylprolyl cis-trans-isomerase activity of the binding protein, we used the enzyme's decrease in enzymic activity to monitor binding of the inhibitors to their enzyme targets. For FK506, the kinetics of inhibition of human 12 kDa cytosolic FKBP (FKBP12cy) were clearly dependent on time. Subsequent to a rapid inactivation reaction, not resolved in its kinetics due to manual mixing, a slow dominant first-order inactivation process with a relaxation time of 1163 s at 10 degrees C was observed. Concomitantly the Ki value of the slow phase dropped 2.6-fold within the first 60 min of incubation. Using the FKBP12cy homologue 25 kDa membrane FKBP (FKBP25mem), a bacterial peptidylprolyl cis-trans-isomerase, the rate and amplitudes of the inhibition reactions were very similar to FKBP12cy. On the other hand, the kinetics and amplitudes of the inhibition of FKBP12cy varied significantly if rapamycin was used as an inhibitor instead of FK 506. Owing to reduced conformation transition in rapamycin upon binding to FKBP12cy, the slow phase during inhibition was significantly decreased in amplitude. A likely reason for this became apparent when the activation-enthalpy and the pH-dependence of the rate

  8. Time-dependent inhibition of peptidylprolyl cis-trans-isomerases by FK506 is probably due to cis-trans isomerization of the inhibitor's imide bond.

    PubMed

    Zarnt, T; Lang, K; Burtscher, H; Fischer, G

    1995-01-01

    Free in solution, the immunosuppressive compounds cyclosporin A (CsA), FK506, ascomycin and rapamycin are present in many solvents in various slowly interconverting conformations. Together with their cellular receptor proteins, cyclophilin (CyP) and FK506-binding protein (FKBP), however, these inhibitors have been shown to have a homogeneous conformation. The existence of a slow cis-trans interconversion of an imidic bond in the inhibitor molecule during the course of the formation of the CsA-CyP18cy complex (where CyP18cy is human 18 kDa cytosolic CyP) prompted us to investigate the reaction of the peptidomacrolides FK506, ascomycin and rapamycin with two specific binding-proteins in more detail. Since formation of the FK506-FKBP complex results in the inhibition of the peptidylprolyl cis-trans-isomerase activity of the binding protein, we used the enzyme's decrease in enzymic activity to monitor binding of the inhibitors to their enzyme targets. For FK506, the kinetics of inhibition of human 12 kDa cytosolic FKBP (FKBP12cy) were clearly dependent on time. Subsequent to a rapid inactivation reaction, not resolved in its kinetics due to manual mixing, a slow dominant first-order inactivation process with a relaxation time of 1163 s at 10 degrees C was observed. Concomitantly the Ki value of the slow phase dropped 2.6-fold within the first 60 min of incubation. Using the FKBP12cy homologue 25 kDa membrane FKBP (FKBP25mem), a bacterial peptidylprolyl cis-trans-isomerase, the rate and amplitudes of the inhibition reactions were very similar to FKBP12cy. On the other hand, the kinetics and amplitudes of the inhibition of FKBP12cy varied significantly if rapamycin was used as an inhibitor instead of FK 506. Owing to reduced conformation transition in rapamycin upon binding to FKBP12cy, the slow phase during inhibition was significantly decreased in amplitude. A likely reason for this became apparent when the activation-enthalpy and the pH-dependence of the rate

  9. Communication: An accurate calculation of the S1 C2H2 cis-trans isomerization barrier height.

    PubMed

    Baraban, Joshua H; Matthews, Devin A; Stanton, John F

    2016-03-21

    A high level ab initio calculation of the cis-trans isomerization barrier height in the first excited singlet electronic state of acetylene is found to agree very well with a recent experimental determination.

  10. cis-trans-Isomerization of unsaturated fatty acids during /γ-irradiation of barley grains

    NASA Astrophysics Data System (ADS)

    Geißler, Christian; Brede, Ortwin; Reinhardt, Jürgen

    2003-06-01

    Gamma-irradiating barley grains with doses of 10-100 kGy, a dose dependent isomerization of the naturally occurring cis-unsaturated fatty acids such as oleic, cis-vaccenic, linoleic and also of linolenic acid was found. Whereas the effect was negligible up to 10 kGy, at 50 kGy the trans-fatty acid level became comparable to that of other natural products like butter fat which means that there is no essential nutrition danger. The cis-trans-isomerization found in barley grains is explained mainly by a thiyl radical driven process rather than direct isomerization.

  11. Solvent isotope effects on retinal cis-trans isomerization in the dark adaptation of bacteriorhodopsin

    SciTech Connect

    Seltzer, S.

    1992-04-22

    The solvent isotope effect on the first-order rate constant for dark adaptation of bacteriorhodopsin, near neutral pH (pD), is inverse; k{sub D}/k{sub H} = 1.24. The fit of the variation of isotope effect with the atom fraction of deuterium in the solvent to the Gross-Butler equation leads to the conclusion that the proton(s) in motion is (are) less tightly bound in the reactant than in a reactive intermediate, formed prior to the rate-controlling step of cis-trans isomerization. The near-unity isotope effect on the equilibrium between the bound 13-cis- and all-trans-retinals in the dark-adapted state mixture indicates that the isotope effects on the forward and reverse rate constants for isomerization are approximately equal to each other and equal to the isotope effect on the observed rate constant for dark adaptation. The results support a previously proposed mechanism of rate-controlling Asp-212 nucleophilic catalysis of retinal cis-trans isomerication in the dark adaptation process. 27 refs., 2 figs., 1 tab.

  12. Classical description of the dynamics and time-resolved spectroscopy of nonadiabatic cis- trans photoisomerization

    NASA Astrophysics Data System (ADS)

    Uspenskiy, Igor; Strodel, Birgit; Stock, Gerhard

    2006-10-01

    The mapping formulation of nonadiabatic quantum dynamics is applied to obtain a classical description of the ultrafast dynamics and time-resolved spectroscopy of a photochemical reaction. Adopting a previously studied dissipative two-state two-mode model of nonadiabatic cis-trans photoisomerization, classical mapping simulations are compared to quantum-mechanical reduced density matrix calculations. Overall, the simple classical method is found to reproduce the quantum reference calculations quite well. In particular, it is studied if the classical approach yields the correct long-time cis/trans localization of the wave packet and therefore the correct quantum yield of the photoreaction. As the long-time behavior of the classical mapping formulation suffers from the well-known zero point energy problem of classical mechanics, a new practical method is proposed to determine a zero point energy correction. Employing a second-order Franck-Condon-type approximation, the capability of the classical method to simulate time- and frequency-resolved pump-probe spectra of the nonadiabatic photoreaction is studied. The potential of the classical approach as a practical method to describe condensed-phase photoreactions is discussed.

  13. Cis/trans Fluorescent Recognition by Naphthalimide Dyes ⊂ CB [7] Assembly.

    PubMed

    Li, Junyong; Gu, Xiaomin; Yuan, Xiaosheng; Qiu, Qiqi; Sun, Jie; Wang, Haibo

    2016-07-01

    A novel method to recognize cis/trans isomers was studied here. The naphthalimide dye as guest could bind with host cucurbit [7]uril (CB [7]) and 1:1 naphthalimide dye ⊂ CB [7] assembly was formed. Moreover, this assembly was used as a fluorescent probe to recognized Fumaric acid (FA) and maleic acid (MA) via fluorescence titration. Two carboxyls in MA are in the same side, they could form stable interaction with the assembly and the fluorescence intensity decreased obviously when naphthalimide dye ⊂ CB [7] was titrated by MA (nearly quenched in 1.5 equiv). But two carboxyls in FA are in opposite sides, the interaction between FA and the assembly was weak and not stable, and the fluorescence intensity changed inconspicuously when the assembly was titrated by FA.

  14. Holographic recording materials development. [development of cis-trans isomerization for holographic memory

    NASA Technical Reports Server (NTRS)

    1974-01-01

    Developments in the area of organic cis-trans isomerization systems for holographic memory applications are reported. The chemical research effort consisted of photochemical studies leading to the selection of a stilbene derivative and a polymer matrix system which have greatly improved refractive index differences between the cis and trans isomers as well as demonstrated efficiency of the photoisomerization process. In work on lithium niobate effects of sample stoichiometry and of read and write beam polarizations on recording efficiency were investigated. LiNbO3 was used for a study of angular sensitivity and of capability for simultaneous recording of extended objects without interference. The current status of LiNbO3 as a holographic recording material is summarized.

  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. Flavylium based dual photochromism: addressing cis-trans isomerization and ring opening-closure by different light inputs.

    PubMed

    Gago, Sandra; Basílio, Nuno; Moro, Artur J; Pina, Fernando

    2015-04-30

    The multistate system of 4',7-dihydroxy-3-methoxyflavylium is constituted by a multiequilibrium involving trans-chalcone, cis-chalcone, hemiketal, flavylium cation and quinoidal base. This system possesses two independently addressable inter-connected photochromic systems based on the cis-trans isomerization and ring opening-closure of the hemiketal.

  17. Multiple gas-phase conformations of proline-containing peptides: Is it always cis/trans isomerization?

    PubMed Central

    Lietz, Christopher B.; Chen, Zhengwei; Son, Chang Yun; Pang, Xueqin; Cui, Qiang; Li, Lingjun

    2016-01-01

    Ion mobility-mass spectrometry (IM-MS) is often employed to look at the secondary, tertiary, and quaternary structure of naked peptides and proteins in the gas-phase. Recently, it has offered a unique glimpse into proline-containing peptides and their cis/trans Xxx-Pro isomers. An experimental “signature” has been identified wherein a proline-containing peptide has its Pro residues substituted with another amino acid and the presence or absence of conformations in the IM-MS spectra are observed. Despite high probability that one could attribute these conformations to cis/trans isomers, it is also possible that cis/trans isomers are not the cause of the additional conformations in proline-containing peptides. However, the experimental evidence of such a system has not been demonstrated or reported. Herein, we present the IM-MS analysis of Neuropeptide Y’s wild-type (WT) signal sequence and Leu7Pro (L7P) mutant. Although comparison of arrival times and collision cross sections of [M+4H]4+ ions yield the cis/trans “signature”, molecular dynamics indicates that a cis-Pro7 is not very stable and that trans-Pro7 conformations of the same cross section arise with equal frequency. We believe this work further underscores the importance of theoretical calculations in IM-MS structural assignments. PMID:27434776

  18. Multiple gas-phase conformations of proline-containing peptides: is it always cis/trans isomerization?

    PubMed

    Lietz, Christopher B; Chen, Zhengwei; Yun Son, Chang; Pang, Xueqin; Cui, Qiang; Li, Lingjun

    2016-08-02

    Ion mobility-mass spectrometry (IM-MS) is often employed to look at the secondary, tertiary, and quaternary structures of naked peptides and proteins in the gas-phase. Recently, it has offered a unique glimpse into proline-containing peptides and their cis/trans Xxx-Pro isomers. An experimental "signature" has been identified wherein a proline-containing peptide has its Pro residues substituted with another amino acid and the presence or absence of conformations in the IM-MS spectra is observed. Despite the high probability that one could attribute these conformations to cis/trans isomers, it is also possible that cis/trans isomers are not the cause of the additional conformations in proline-containing peptides. However, the experimental evidence of such a system has not been demonstrated or reported. Herein, we present the IM-MS analysis of Neuropeptide Y's wild-type (WT) signal sequence and Leu7Pro (L7P) mutant. Although comparison of arrival times and collision cross-sections of [M + 4H](4+) ions yields the cis/trans "signature", molecular dynamics indicates that a cis-Pro7 is not very stable and that trans-Pro7 conformations of the same cross-section arise with equal frequency. We believe that this work further underscores the importance of theoretical calculations in IM-MS structural assignments.

  19. 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.

  20. Structure of the conical intersections driving the cis-trans photoisomerization of conjugated molecules.

    PubMed

    Ruiz, Diego Sampedro; Cembran, Alessandro; Garavelli, Marco; Olivucci, Massimo; Fuss, Werner

    2002-12-01

    High-level ab initio calculations show that the singlet photochemical cis-trans isomerization of organic molecules under isolated conditions can occur according to two distinct mechanisms. These mechanisms are characterized by the different structures of the conical intersection funnels controlling photoproduct formation. In nonpolar (e.g. hydrocarbon) polyenes the lowest-lying funnel corresponds to a (CH)3 kink with both double and adjacent single bonds twisted, which may initiate hula-twist (HT) isomerization. On the other hand, in polar conjugated systems such as protonated Schiff bases (PSB) the funnel shows a structure with just one twisted double bond. The ground-state relaxation paths departing from the funnels indicate that the HT motion may take place in nonpolar conjugated systems but also that the single-bond twist may be turned back, whereas in free conjugated polar molecules such as PSB a one-bond flip mechanism dominates from the beginning. The available experimental evidence either supports these predictions or is at least consistent with them.

  1. Identification of Conus Peptidylprolyl Cis-Trans Isomerases (PPIases) and Assessment of Their Role in the Oxidative Folding of Conotoxins

    PubMed Central

    Safavi-Hemami, Helena; Bulaj, Grzegorz; Olivera, Baldomero M.; Williamson, Nicholas A.; Purcell, Anthony W.

    2010-01-01

    Peptidylprolyl cis-trans isomerases (PPIases) are ubiquitous proteins that catalyze the cis-trans isomerization of prolines. A number of proteins, such as Drosophila rhodopsin and the human immunodeficiency viral protein HIV-1 Gag, have been identified as endogenous substrates for PPIases. However, very little is known about the interaction of PPIases with small, disulfide-rich peptides. Marine cone snails synthesize a wide array of cysteine-rich peptides, called conotoxins, many of which contain one or more prolines or hydroxyprolines. To identify whether PPIase-associated cis-trans isomerization of these residues affects the oxidative folding of conotoxins, we identified, sequenced, and expressed three functionally active isoforms of PPIase from the venom gland of Conus novaehollandiae, and we characterized their ability to facilitate oxidative folding of conotoxins in vitro. Three conotoxins, namely μ-GIIIA, μ-SIIIA, and ω-MVIIC, derived from two distinct toxin gene families were assayed. Conus PPIase significantly increased the rate of appearance of the native form of μ-GIIIA, a peptide containing three hydroxyprolines. In contrast, the presence of PPIase had no effect on the folding of μ-SIIIA and ω-MVIIC, peptides containing no or one proline residue, respectively. We further showed that an endoplasmic reticulum-resident PPIase isoform facilitated folding of μ-GIIIA more efficiently than two cytosolic isoforms. This is the first study to demonstrate PPIase-assisted folding of conotoxins, small disulfide-rich peptides with unique structural properties. PMID:20147296

  2. Cyclophilin A (CyPA) Induces Chemotaxis Independent of Its Peptidylprolyl Cis-Trans Isomerase Activity

    PubMed Central

    Song, Fei; Zhang, Xin; Ren, Xiao-Bai; Zhu, Ping; Xu, Jing; Wang, Li; Li, Yi-Fei; Zhong, Nan; Ru, Qiang; Zhang, Da-Wei; Jiang, Jian-Li; Xia, Bin; Chen, Zhi-Nan

    2011-01-01

    Cyclophilin A (CyPA) is a ubiquitously distributed peptidylprolyl cis-trans isomerase (PPIase) that possesses diverse biological functions. Extracellular CyPA is a potent chemokine, which can directly induce leukocyte chemotaxis and contribute to the pathogenesis of inflammation-mediated diseases. Although it has been identified that the chemotaxis activity of CyPA is mediated through its cell surface signaling receptor CD147, the role of CyPA PPIase activity in this process is disputable, and the underlying molecular mechanism is still poorly understood. In this study, we present the first evidence that CyPA induces leukocyte chemotaxis through a direct binding with the ectodomain of CD147 (CD147ECT), independent of its PPIase activity. Although NMR study indicates that the CD147ECT binding site on CyPA overlaps with the PPIase active site, the PPIase inactive mutant CyPAR55A exhibits similar CD147ECT binding ability and chemotaxis activity to those of CyPAWT. Furthermore, we have identified three key residues of CyPA involved in CD147ECT binding and found that mutations H70A, T107A, and R69A result in similar levels of reduction in CD147ECT binding ability and chemotaxis activity for CyPA, without affecting the PPIase activity. Our findings indicate that there exists a novel mechanism for CyPA to regulate cellular signaling processes, shedding new light on its applications in drug development and providing a new targeting site for drug design. PMID:21245143

  3. Energy resolved tandem mass spectrometry experiments for resolution of isobaric compounds: a case of cis/trans isomerism.

    PubMed

    Menicatti, Marta; Guandalini, Luca; Dei, Silvia; Floriddia, Elisa; Teodori, Elisabetta; Traldi, Pietro; Bartolucci, Gianluca

    A series of N-alkanol-N-cyclohexanol amine aryl esters cis/trans isomers that showed high efficacy to reverse the acquired resistance of cancer cells during chemotherapeutic therapy (MDR mechanism) was studied. These compounds were two 1,4 cyclohexane cis/trans derivatives (named ELF26A and ELF26B, respectively), and their positional isomers (named ELF34A and ELF34B, respectively) where the aryl-moieties were exchanged. In order to evaluate the behaviour of these compounds during biological tests, a method based on liquid chromatography coupled with mass spectrometry (LC-MS), operating in tandem mass spectrometry (MS/MS) mode, was developed. A unique chromatographic method suitable to separate the two pairs of cis/trans isomers was not achieved and the MS/MS experiments of the different compounds was not always able to characterise the different isomers. Therefore, a system of linear equations of deconvolution analysis (LEDA) tool was proposed to determine the relative proportions of individual cis/trans isomers in the sample. Considering the pharmaceutical interest of the compounds under investigation, the analytical method developed was tested to be effective at the active concentration levels, corresponding to a concentration of ng mL(-1) of compound in a processed sample. Precision and accuracy of the LEDA algorithm at three levels of relative concentrations of analytes were checked, i.e. low-level (about 25% in the mixture), mid-level (about 50% in the mixture) and high-level (about 70% in the mixture). Evaluation of performances of the algorithm proved that the accuracy (between 88.3% and 99.9%) and precision (between 2.0% and 3.7%) for simultaneous analysis of the mixtures of the four isomers is feasible. It is worth highlighting that the choice of characteristic product ions and optimal abundance ratios plays an important role in the application of the LEDA approach. Therefore, performing an investigation on the energetics of fragmentation pathway allowed

  4. 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

  5. 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

  6. cis-trans photoisomerization of 1,3,5,7-octatetraene in n-hexane at 4.2 K

    PubMed Central

    Granville, Mark F.; Holtom, Gary R.; Kohler, Bryan E.

    1980-01-01

    Photoisomerization of the linear polyene 1,3,5,7-octatetraene has been observed in an n-hexane matrix maintained at the boiling point of helium. To a good approximation, only the trans,trans and cis,trans isomers participate in the photochemistry. These compounds have been unambiguously identified by comparing the observed high-resolution fluorescence spectra to those of chromatographically purified reference compounds. Although the quantum yield of this process is probably low, its microscopic rate seems to compete favorably with vibrational deactivation. PMID:16592751

  7. Prolyl endopeptidases.

    PubMed

    Gass, J; Khosla, C

    2007-02-01

    This review describes the structure and function of prolyl endopeptidase (PEP) enzymes and how they are being evaluated as drug targets and therapeutic agents. The most well studied PEP family has a two-domain structure whose unique seven-blade beta-propeller domain works with the catalytic domain to hydrolyze the peptide bond on the carboxyl side of internal proline residues of an oligopeptide substrate. Structural and functional studies on this protease family have elucidated the mechanism for peptide entry between the two domains. Other structurally unrelated PEPs have been identified, but have not been studied in detail. Human PEP has been evaluated as a pharmacological target for neurological diseases due to its high brain concentration and ability to cleave neuropeptides in vitro. Recently, microbial PEPs have been studied as potential therapeutics for celiac sprue, an inflammatory disease of the small intestine triggered by proline-rich gluten.

  8. Structural and dynamic implications of an effector-induced backbone amide cis-trans isomerization in cytochrome P450cam

    PubMed Central

    Asciutto, Eliana K.; Madura, Jeffry D.; Pochapsky, Susan Sondej; OuYang, Bo; Pochapsky, Thomas C.

    2009-01-01

    Experimental evidence has been provided for a functionally relevant cis-trans isomerization of the Ile 88-Pro 89 peptide bond in cytochrome P450cam (CYP101). The isomerization is proposed to be a key element of the structural reorganization leading to the catalytically competent form of CYP101 upon binding of the effector protein putidaredoxin (Pdx). A detailed comparison of the results of molecular dynamics simulations on the cis and trans conformations of substrate- and carbonmonoxy-bound ferrous CYP101 with sequence-specific Pdx-induced structural perturbations identified by nuclear magnetic resonance is presented, providing insight into the structural and dynamic consequences of the isomerization. The mechanical coupling between the Pdx binding site on the proximal face of CYP101 and the site of isomerization is described. PMID:19327368

  9. Mechanism elucidation of the cis-trans isomerization of an azole ruthenium-nitrosyl complex and its osmium counterpart.

    PubMed

    Gavriluta, Anatolie; Büchel, Gabriel E; Freitag, Leon; Novitchi, Ghenadie; Tommasino, Jean Bernard; Jeanneau, Erwann; Kuhn, Paul-Steffen; González, Leticia; Arion, Vladimir B; Luneau, Dominique

    2013-06-03

    Synthesis and X-ray diffraction structures of cis and trans isomers of ruthenium and osmium metal complexes of general formulas (nBu4N)[cis-MCl4(NO)(Hind)], where M = Ru (1) and Os (3), and (nBu4N)[trans-MCl4(NO)(Hind)], where M = Ru (2) and Os (4) and Hind = 1H-indazole are reported. Interconversion between cis and trans isomers at high temperatures (80-130 °C) has been observed and studied by NMR spectroscopy. Kinetic data indicate that isomerizations correspond to reversible first order reactions. The rates of isomerization reactions even at 110 °C are very low with rate constants of 10(-5) s(-1) and 10(-6) s(-1) for ruthenium and osmium complexes, respectively, and the estimated rate constants of isomerization at room temperature are of ca. 10(-10) s(-1). The activation parameters, which have been obtained from fitting the reaction rates at different temperatures to the Eyring equation for ruthenium [ΔH(cis-trans)‡ = 122.8 ± 1.3; ΔH(trans-cis)‡ = 138.8 ± 1.0 kJ/mol; ΔS(cis-trans)‡ = -18.7 ± 3.6; ΔS(trans-cis)‡ = 31.8 ± 2.7 J/(mol·K)] and osmium [ΔH(cis-trans)‡ = 200.7 ± 0.7; ΔH(trans-cis)‡ = 168.2 ± 0.6 kJ/mol; ΔS(cis-trans)‡ = 142.7 ± 8.9; ΔS(trans-cis)‡ = 85.9 ± 3.9 J/(mol·K)] reflect the inertness of these systems. The entropy of activation for the osmium complexes is highly positive and suggests the dissociative mechanism of isomerization. In the case of ruthenium, the activation entropy for the cis to trans isomerization is negative [-18.6 J/(mol·K)], while being positive [31.0 J/(mol·K)] for the trans to cis conversion. The thermodynamic parameters for cis to trans isomerization of [RuCl4(NO)(Hind)]-, viz. ΔH° = 13.5 ± 1.5 kJ/mol and ΔS° = -5.2 ± 3.4 J/(mol·K) indicate the low difference between the energies of cis and trans isomers. The theoretical calculation has been carried out on isomerization of ruthenium complexes with DFT methods. The dissociative, associative, and intramolecular twist isomerization

  10. Pump-Probe Spectroscopy of Thermal Cis-Trans Isomerization Process of Methyl Red in 1-Octanol

    NASA Astrophysics Data System (ADS)

    Joukai, Tatsuya; Kita, Kenji; Yano, Ryuzi

    Thermal cis-trans isomerization process of Methyl Red in 1-Octanol at room temperature was studied by pump-probe spectroscopy. The isomerization rate increased as the dye concentration was increased. However, at concentrations above ~ 3 × 10-5 mol/L, the isomerization rate became almost constant regardless of the dye concentration. We interpreted that the concentration dependence of the thermal isomerization rate was caused by the creation of hydrogen-bonded dimer molecules. When a dimer is formed, one of the dye molecules which form the dimer easily changes its form from the cis isomer to the trans isomer. As the dye concentration is increased, the ratio of dyes which form dimer molecules will increase. When the dye concentration is high enough, most dye molecules form dimer molecules. Thus the isomerization rate averaged over the dye molecules is constant.

  11. A semiclassical study of cis-trans isomerization in HONO using an interpolating moving least-squares potential

    NASA Astrophysics Data System (ADS)

    Pham, Phong; Guo, Yin

    2013-04-01

    The interpolating moving least-squares (IMLS) approach for constructing potential energy surfaces has been developed and employed in standard classical trajectory simulations in the past few years. We extend the approach to the tunneling regime by combining the IMLS fitting method and the semiclassical scheme that incorporates tunneling into classical trajectory calculations. Dynamics of cis-trans isomerization in nitrous acid (HONO) is studied as a test case to investigate various aspects of the approach such as the strategy for growing the surface, the basis set employed, the scaling of the IMLS fits, and the accuracy of the surface required for obtaining converged rate coefficients. The validity of the approach is demonstrated through comparison with other semiclassical and quantum mechanical studies on HONO.

  12. Prolyl isomerization as a molecular timer in phage infection.

    PubMed

    Eckert, Barbara; Martin, Andreas; Balbach, Jochen; Schmid, Franz X

    2005-07-01

    Prolyl cis-trans isomerizations are intrinsically slow reactions and known to be rate-limiting in many protein folding reactions. Here we report that a proline is used as a molecular timer in the infection of Escherichia coli cells by the filamentous phage fd. The phage is activated for infection by the disassembly of the two N-terminal domains, N1 and N2, of its gene-3-protein, which is located at the phage tip. Pro213, in the hinge between N1 and N2, sets a timer for the infective state. The timer is switched on by cis-to-trans and switched off by the unusually slow trans-to-cis isomerization of the Gln212-Pro213 peptide bond. The switching rate and thus the infectivity of the phage are determined by the local sequence around Pro213, and can be tuned by mutagenesis.

  13. 40 CFR 180.545 - Prallethrin (RS)-2-methyl-4-oxo-3-(2-propynyl)cyclopent-2-enyl (1RS)-cis, trans-chrysanthemate...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.545 Prallethrin (RS)-2-methyl-4...-methyl-4-oxo-3-(2-propynyl)cyclopent-2-enyl (1RS)-cis, trans-chrysanthemate as follows: (2) In or on food commodities in food handling establishments where food and food products are held, processed, prepared and/or...

  14. Transient-Absorption Spectroscopy of Cis-Trans Isomerization of N,N-dimethyl-4,4'-Azodianiline with 3D-Printed Temperature-Controlled Sample Holder

    ERIC Educational Resources Information Center

    Kosenkov, Dmytro; Shaw, James; Zuczek, Jennifer; Kholod, Yana

    2016-01-01

    The laboratory unit demonstrates a project based approach to teaching physical chemistry laboratory where upper-division undergraduates carry out a transient-absorption experiment investigating the kinetics of cis-trans isomerization of N,N-dimethyl-4,4'-azodianiline. Students participate in modification of a standard flash-photolysis spectrometer…

  15. 1H-13C HSQC NMR spectroscopy for estimating procyanidin/prodelphinidin and cis/trans flavan-3-ol ratios of condensed tannin samples: correlation with thiolysis

    USDA-ARS?s Scientific Manuscript database

    Studies with a diverse array of 22 condensed tannin (CT) fractions from 9 plant species demonstrated that procyanidin/prodelphinidin (PC/PD) and cis/trans flavan-3-ol ratios can be appraised by 1H-13C HSQC NMR. The method was developed from fractions containing 44 to ~100% CT, PC/PD ratios ranging f...

  16. Transient-Absorption Spectroscopy of Cis-Trans Isomerization of N,N-dimethyl-4,4'-Azodianiline with 3D-Printed Temperature-Controlled Sample Holder

    ERIC Educational Resources Information Center

    Kosenkov, Dmytro; Shaw, James; Zuczek, Jennifer; Kholod, Yana

    2016-01-01

    The laboratory unit demonstrates a project based approach to teaching physical chemistry laboratory where upper-division undergraduates carry out a transient-absorption experiment investigating the kinetics of cis-trans isomerization of N,N-dimethyl-4,4'-azodianiline. Students participate in modification of a standard flash-photolysis spectrometer…

  17. Unexpectedly fast cis/trans isomerization of Xaa-Pro peptide bonds in disulfide-constrained cyclic peptides.

    PubMed

    Shi, Tiesheng; Spain, Stephen M; Rabenstein, Dallas L

    2004-01-28

    Acyclic dithiol and cyclic disulfide forms of the peptides Ac-Cys-Pro-Xaa-Cys-NH2 (Xaa = Phe, His, Tyr, Gly, and Thr) and Ac-Cys-Gly-Pro-Cys-NH2 and the peptide Ac-Ala-Gly-Pro-Ala-NH2 were synthesized and characterized by mass spectrometry and NMR spectroscopy. Rate constants kct and ktc for cis-to-trans and trans-to-cis isomerization, respectively, across the Cys-Pro or Gly-Pro peptide bonds were determined by magnetization transfer NMR techniques over a range of temperatures, and activation parameters were derived from the temperature dependence of the rate constants. It was found that constraints imposed by the disulfide bond confer an unexpected rate enhancement for cis/trans isomerization, ranging from a factor of 2 to 13. It is proposed that the rate enhancements are a result of an intramolecular catalysis mechanism in which the NH proton of the Pro-Xaa peptide bond hydrogen bonds to the proline nitrogen in the transition state. The peptides Ac-Cys-Pro-Xaa-Cys-NH2 and Ac-Cys-Gly-Pro-Cys-NH2 are model compounds for proline-containing active sites of the thioredoxin superfamily of oxidoreductase enzymes; the results suggest that the backbones of the active sites of the oxidized form of these enzymes may have unusual conformational flexibility.

  18. Does the cis/trans configuration of peptide bonds in bioactive tripeptides play a role in ACE-1 enzyme inhibition?

    PubMed Central

    Siltari, Aino; Viitanen, Riikka; Kukkurainen, Sampo; Vapaatalo, Heikki; Valjakka, Jarkko

    2014-01-01

    Background The milk casein-derived bioactive tripeptides isoleucine-proline-proline (IPP) and valine-proline-proline (VPP) have been shown to prevent development of hypertension in animal models and to lower blood pressure in moderately hypertensive subjects in most but not all clinical trials. Inhibition of angiotensin-converting enzyme 1 (ACE-1) has been suggested as the explanation for these antihypertensive and beneficial vascular effects. Previously, human umbilical vein endothelial cells (HUVEC) have not been used to test ACE-1 inhibiting properties of casein derived tripeptides in vasculature. Purpose We focused on the cis/trans configurations of the peptide bonds in proline-containing tripeptides in order to discover whether the different structural properties of these peptides influence their activity in ACE-1 inhibition. We hypothesized that the configuration of proline-containing peptides plays a significant role in enzyme inhibition. Methods AutoDock 4.2 docking software was used to predict suitable peptide bond configurations of the tripeptides. Besides modeling studies, we completed ACE-1 activity measurements in vitro using HUVEC cultures. Results In HUVEC cells, both IPP and VPP inhibited ACE-1. Based on molecular docking studies, we propose that in ACE-1 inhibition IPP and VPP share a similar cis configuration between the first aliphatic (isoleucine or valine) and the second (proline) amino acid residues and more different configurations between two proline residues. In vivo experiments are needed to validate the significance of the present findings. PMID:24596454

  19. Semiautomated microtiter plate assay for monitoring peptidylprolyl cis/trans isomerase activity in normal and pathological human sera.

    PubMed

    Küllertz, G; Lüthe, S; Fischer, G

    1998-03-01

    An UV/VIS spectrophotometric assay technique was developed that was able to routinely monitor peptidylprolyl cis/trans isomerase (PPIase) activity of biological fluids in 96-well microtiter plates. The assay, based on monitoring the cis-to-trans isomerization of succinyl-Phe-cisPro-Phe-4-nitroanilide as substrate in a chymotrypsin-coupled reaction, yields a throughput of 96 samples per 30 min. The assay's capacity was exemplified by dealing with the PPIase activity in several normal and pathological human sera. Reference values of 151 healthy subjects (83 females, 69 males, 17 to 60 years old) were found to possess significant sex-specific differences. PPIase activity factor K of the sera was significantly greater in males (5th, 50th, 95th percentiles: 17, 36, 55 K) than females (14, 30, 48 K). PPIase activities of sera from healthy donors (n = 151) were significantly higher (Mann-Whitney rank-sum test P < 0.0001) than those of patients (n = 47). PPIase activity in serum samples stored at 4 degrees C was stable for at least 20 h.

  20. Synthesis, DFT and antimicrobial activity assays in vitro for novel cis/trans-but-2-enedioic acid esters

    NASA Astrophysics Data System (ADS)

    Ma, Yan-Long; Zhou, Ru-Jin; Zeng, Xing-Ye; An, Ya-Xiong; Qiu, Song-Shan; Nie, Li-Jun

    2014-04-01

    Six novel cis/trans-but-2-enedioic acid esters had been synthesized to discover the new bioactive molecules that could kill food-related bacteria and fungi. Their structures were analyzed by melting point, LC-MS, 1H NMR and 13C NMR. 4-(Methoxycarbonyl) phenyl ethyl fumarate (6b) was also characterized by single-crystal X-ray diffraction. Their antimicrobial activities were evaluated in vitro by measuring the minimal inhibitory concentration (MIC). Compared with the single monomethyl fumarate and methyl 4-hydroxybenzoate, these compounds had stronger antimicrobial activity against all the eight microorganisms. Among the antibacterial and antifungal compounds, 4-(methoxycarbonyl) phenyl methyl fumarate (6a) showed the best antimicrobial activity. The electronic properties of these compounds were calculated by the density functional theory (DFT) method with 6-31G (d, p) basis set. DFT studies indicated that molecular electrostatic potential (MEP) map, ELUMO, energy gap, electronegativity and electrophilicity index could be helpful to understand the various antimicrobial activities among these compounds. The antimicrobial activity of compound 6a was evaluated in vitro against Salmonellacholeraesuis subsp. choleraesuis, Lactococcus lactis subsp. lactis and Saccharomyces cerevisiae by time-kill, and it was found that compound 6a exhibited significant microbiocidal activity against the three microorganisms.

  1. Quantum coherence effects in natural light-induced processes: cis-trans photoisomerization of model retinal under incoherent excitation.

    PubMed

    Tscherbul, Timur V; Brumer, Paul

    2015-12-14

    We present a theoretical study of quantum coherence effects in the primary cis-trans photoisomerization of retinal in rhodopsin induced by incoherent solar light. Using the partial secular Bloch-Redfield quantum master equation approach based on a two-state two-mode linear vibronic coupling model of the retinal chromophore [S. Hahn and G. Stock, J. Phys. Chem. B, 2000, 104, 1146-1149], we show that a sudden turn-on of incoherent pumping can generate substantial Fano coherences among the excited states of retinal. These coherences are the most pronounced in the regime where the matrix elements of the transition dipole moment between the ground and excited eigenstates are parallel to one another. We show that even when the transition dipole moments are perpendicular (implying the absence of light-induced Fano coherence) a small amount of excited-state coherence is still generated due to the coupling to intramolecular vibrational modes and the protein environment, causing depopulation of the excited eigenstates. The overall effect of the coherences on the steady-state population and on the photoproduct quantum yield is shown to be small; however we observe a significant transient effect on the formation of the trans photoproduct, enhancing the photoreaction quantum yield by ∼11% at 200 fs. These calculations suggest that coupling to intramolecular vibrational modes and the protein environment play an important role in photoreaction dynamics, suppressing oscillations in the quantum yield associated with Fano interference.

  2. NMR spectroscopic studies of a TAT-derived model peptide in imidazolium-based ILs: influence on chemical shifts and the cis/trans equilibrium state.

    PubMed

    Wiedemann, Christoph; Ohlenschläger, Oliver; Mrestani-Klaus, Carmen; Bordusa, Frank

    2017-09-13

    NMR spectroscopy was used to study systematically the impact of imidazolium-based ionic liquid (IL) solutions on a TAT-derived model peptide containing Xaa-Pro peptide bonds. The selected IL anions cover a wide range of the Hofmeister series of ions. Based on highly resolved one- and two-dimensional NMR spectra individual (1)H and (13)C peptide chemical shift differences were analysed and a classification of IL anions according to the Hofmeister series was derived. The observed chemical shift changes indicate significant interactions between the peptide and the ILs. In addition, we examined the impact of different ILs towards the cis/trans equilibrium state of the Xaa-Pro peptide bonds. In this context, the IL cations appear to be of exceptional importance for inducing an alteration of the native cis/trans equilibrium state of Xaa-Pro bonds in favour of the trans-isomers.

  3. Au nanoparticle scaffolds modulating intermolecular interactions among the conjugated azobenzenes chemisorbed on curved surfaces: tuning the kinetics of cis-trans isomerisation

    NASA Astrophysics Data System (ADS)

    Raimondo, Corinna; Kenens, Bart; Reinders, Federica; Mayor, Marcel; Uji-I, Hiroshi; Samorì, Paolo

    2015-08-01

    π-π Intermolecular interactions among adjacent conjugated azobenzenes chemisorbed on (non-)flat Au surfaces can be tuned by varying the curvature of the Au nanoparticles. Here we show that such interactions rule the thermal cis-trans isomerization kinetics, towards a better control on the azobenzene bistability for its optimal integration as a responsive material.π-π Intermolecular interactions among adjacent conjugated azobenzenes chemisorbed on (non-)flat Au surfaces can be tuned by varying the curvature of the Au nanoparticles. Here we show that such interactions rule the thermal cis-trans isomerization kinetics, towards a better control on the azobenzene bistability for its optimal integration as a responsive material. Electronic supplementary information (ESI) available: Physico-chemical characterization of the different sizes of nanoparticles, UV-Vis, Surface Enhanced Raman Spectroscopy (SERS), materials and methods. See DOI: 10.1039/c5nr03688g

  4. Asymmetric epoxidation of cis/trans-β-methylstyrene catalysed by immobilised Mn(salen) with different linkages: heterogenisation of homogeneous asymmetric catalysis.

    PubMed

    Zhang, Haidong; Zou, Yu; Wang, Yi-Meng; Shen, Yu; Zheng, Xuxu

    2014-06-16

    Immobilised Mn(salen) catalysts with two different linkages were studied in the asymmetric epoxidation of cis/trans-β-methylstyrene using NaClO as oxidant. The immobilised Mn(salen) complexes inside nanopores can lead to different catalytic behaviour compared with that of homogeneous Jacobsen catalyst. The rigidity of the linkage was found to be a key factor affecting the catalytic performance of immobilised catalysts. The immobilised catalyst with a rigid linkage exhibited comparable chemical selectivity, enantioselectivity and cis/trans ratio of product formation to that obtained with homogeneous Jacobsen catalysts. In contrast, the immobilised catalyst with a flexible linkage gave remarkably lower chemical selectivity, enantioselectivity and inverted cis/trans ratio compared with the results obtained with the homogeneous Jacobsen catalyst and the immobilised catalyst with rigid linkage. Thus, for immobilised Mn(salen) catalysts, a rigid linkage connecting active centres to the support is essential to obtain activity and enantioselectivity as high as those obtained in homogeneous systems.

  5. Differential loss of prolyl isomerase or chaperone activity of Ran-binding protein 2 (Ranbp2) unveils distinct physiological roles of its cyclophilin domain in proteostasis.

    PubMed

    Cho, Kyoung-in; Patil, Hemangi; Senda, Eugene; Wang, Jessica; Yi, Haiqing; Qiu, Sunny; Yoon, Dosuk; Yu, Minzhong; Orry, Andrew; Peachey, Neal S; Ferreira, Paulo A

    2014-02-21

    The immunophilins, cyclophilins, catalyze peptidyl cis-trans prolyl-isomerization (PPIase), a rate-limiting step in protein folding and a conformational switch in protein function. Cyclophilins are also chaperones. Noncatalytic mutations affecting the only cyclophilins with known but distinct physiological substrates, the Drosophila NinaA and its mammalian homolog, cyclophilin-B, impair opsin biogenesis and cause osteogenesis imperfecta, respectively. However, the physiological roles and substrates of most cyclophilins remain unknown. It is also unclear if PPIase and chaperone activities reflect distinct cyclophilin properties. To elucidate the physiological idiosyncrasy stemming from potential cyclophilin functions, we generated mice lacking endogenous Ran-binding protein-2 (Ranbp2) and expressing bacterial artificial chromosomes of Ranbp2 with impaired C-terminal chaperone and with (Tg-Ranbp2(WT-HA)) or without PPIase activities (Tg-Ranbp2(R2944A-HA)). The transgenic lines exhibit unique effects in proteostasis. Either line presents selective deficits in M-opsin biogenesis with its accumulation and aggregation in cone photoreceptors but without proteostatic impairment of two novel Ranbp2 cyclophilin partners, the cytokine-responsive effectors, STAT3/STAT5. Stress-induced STAT3 activation is also unaffected in Tg-Ranbp2(R2944A-HA)::Ranbp2(-/-). Conversely, proteomic analyses found that the multisystem proteinopathy/amyotrophic lateral sclerosis proteins, heterogeneous nuclear ribonucleoproteins A2/B1, are down-regulated post-transcriptionally only in Tg-Ranbp2(R2944A-HA)::Ranbp2(-/-). This is accompanied by the age- and tissue-dependent reductions of diubiquitin and ubiquitylated proteins, increased deubiquitylation activity, and accumulation of the 26 S proteasome subunits S1 and S5b. These manifestations are absent in another line, Tg-Ranbp2(CLDm-HA)::Ranbp2(-/-), harboring SUMO-1 and S1-binding mutations in the Ranbp2 cyclophilin-like domain. These results

  6. Differential Loss of Prolyl Isomerase or Chaperone Activity of Ran-binding Protein 2 (Ranbp2) Unveils Distinct Physiological Roles of Its Cyclophilin Domain in Proteostasis*

    PubMed Central

    Cho, Kyoung-in; Patil, Hemangi; Senda, Eugene; Wang, Jessica; Yi, Haiqing; Qiu, Sunny; Yoon, Dosuk; Yu, Minzhong; Orry, Andrew; Peachey, Neal S.; Ferreira, Paulo A.

    2014-01-01

    The immunophilins, cyclophilins, catalyze peptidyl cis-trans prolyl-isomerization (PPIase), a rate-limiting step in protein folding and a conformational switch in protein function. Cyclophilins are also chaperones. Noncatalytic mutations affecting the only cyclophilins with known but distinct physiological substrates, the Drosophila NinaA and its mammalian homolog, cyclophilin-B, impair opsin biogenesis and cause osteogenesis imperfecta, respectively. However, the physiological roles and substrates of most cyclophilins remain unknown. It is also unclear if PPIase and chaperone activities reflect distinct cyclophilin properties. To elucidate the physiological idiosyncrasy stemming from potential cyclophilin functions, we generated mice lacking endogenous Ran-binding protein-2 (Ranbp2) and expressing bacterial artificial chromosomes of Ranbp2 with impaired C-terminal chaperone and with (Tg-Ranbp2WT-HA) or without PPIase activities (Tg-Ranbp2R2944A-HA). The transgenic lines exhibit unique effects in proteostasis. Either line presents selective deficits in M-opsin biogenesis with its accumulation and aggregation in cone photoreceptors but without proteostatic impairment of two novel Ranbp2 cyclophilin partners, the cytokine-responsive effectors, STAT3/STAT5. Stress-induced STAT3 activation is also unaffected in Tg-Ranbp2R2944A-HA::Ranbp2−/−. Conversely, proteomic analyses found that the multisystem proteinopathy/amyotrophic lateral sclerosis proteins, heterogeneous nuclear ribonucleoproteins A2/B1, are down-regulated post-transcriptionally only in Tg-Ranbp2R2944A-HA::Ranbp2−/−. This is accompanied by the age- and tissue-dependent reductions of diubiquitin and ubiquitylated proteins, increased deubiquitylation activity, and accumulation of the 26 S proteasome subunits S1 and S5b. These manifestations are absent in another line, Tg-Ranbp2CLDm-HA::Ranbp2−/−, harboring SUMO-1 and S1-binding mutations in the Ranbp2 cyclophilin-like domain. These results unveil

  7. Conformations of heterochiral and homochiral proline-pseudoproline segments in peptides: context dependent cis-trans peptide bond isomerization.

    PubMed

    Raghothama, Srinivasarao; Raghavender, Upadhyayula Surya; Aravinda, Subrayashastry; Shamala, Narayanaswamy; Balaram, Padmanabhan

    2009-01-01

    The pseudoproline residue (Psi Pro, L-2,2-dimethyl-1,3-thiazolidine-4-carboxylic acid) has been introduced into heterochiral diproline segments that have been previously shown to facilitate the formation of beta-hairpins, containing central two and three residue turns. NMR studies of the octapeptide Boc-Leu-Phe-Val-(D)Pro-Psi Pro-Leu-Phe-Val-OMe (1), Boc-Leu-Val-Val-(D)Pro-Psi Pro-Leu-Val-Val-OMe (2), and the nonapeptide sequence Boc-Leu-Phe-Val-(D)Pro-Psi Pro-(D)Ala-Leu-Phe-Val-OMe (3) established well-registered beta-hairpin structures in chloroform solution, with the almost exclusive population of the trans conformation for the peptide bond preceding the Psi Pro residue. The beta-hairpin conformation of 1 is confirmed by single crystal X-ray diffraction. Truncation of the strand length in Boc-Val-(D)Pro-Psi Pro-Leu-OMe (4) results in an increase in the population of the cis conformer, with a cis/trans ratio of 3.65. Replacement of Psi Pro in 4 by (L)Pro in 5, results in almost exclusive population of the trans form, resulting in an incipient beta-hairpin conformation, stabilized by two intramolecular hydrogen bonds. Further truncation of the sequence gives an appreciable rise in the population of cis conformers in the tripeptide Piv-(D)Pro-Psi Pro-Leu-OMe (6). In the homochiral segment Piv-Pro-Psi Pro-Leu-OMe (7) only the cis form is observed with the NMR evidence strongly supporting a type VIa beta-turn conformation, stabilized by a 4-->1 hydrogen bond between the Piv (CO) and Leu (3) NH groups. The crystal structure of the analog peptide 7a (Piv-Pro-Psi(H,CH3)Pro-Leu-NHMe) confirms the cis peptide bond geometry for the Pro-Psi(H,CH3)Pro peptide bond, resulting in a type VIa beta-turn conformation.

  8. Cis-trans isomerization in the S1 state of acetylene: identification of cis-well vibrational levels.

    PubMed

    Merer, Anthony J; Steeves, Adam H; Baraban, Joshua H; Bechtel, Hans A; Field, Robert W

    2011-06-28

    acetylene that was previously thought to be unobservable, as well as the first high resolution spectroscopic results describing cis-trans isomerization.

  9. Theoretical study on the torsional direction of simple ethylenoids after electronic relaxation at the conical intersection in the cis-trans photoisomerization

    NASA Astrophysics Data System (ADS)

    Amatatsu, Yoshiaki

    2015-07-01

    The conical intersections (CIXs) for the cis-trans photoisomerization of simple ethylenoids, such as ethylene, styrene and stilbene, have been calculated by complete active space self-consistent-field method. This is because we check if a simple relationship which is found in the case of fluorene-based ethylenoids is also true for simple ethylenoids. Thereby, the four CIXs, which are distinguished by the directions of wagging and rocking motions of the anionic part against the ethylenic bond, are found to be related with the torsional direction of the ethylenic bond after electronic relaxation at each CIX.

  10. Cis-trans isomerization of the (5-nitro-2-furyl)acrylamide, AF-2, initiated by ascorbate, glutathione, Fe(II) and OH-.

    PubMed

    Clarke, E D; Wardman, P; Wilson, I

    1984-01-01

    The cis-trans isomerization of the (5-nitro-2-furyl)acrylamide, AF-2, has been investigated using some important biological reducing agents to initiate reaction. Physiological concentrations of L-ascorbic acid, glutathione and iron(II) all accomplish isomerization in a catalytic manner over a period of minutes. Base-catalysed isomerization has also been observed. In all cases, the presence of oxygen severely inhibits isomerization. It is proposed that the mechanism involves a free-radical chain process; AF-2 or analogues are thus extremely sensitive probes for the generation of nitro radicals in biochemical reducing systems because of the high efficiency of isomerization.

  11. MNDO barrier heights for catalyzed bicycle-pedal, hula-twist, and ordinary cis-trans isomerizations of protonated retinal Schiff base

    SciTech Connect

    Seltzer, S.

    1987-03-18

    Energy barriers to dark cis-trans isomerization in a protonated retinal Schiff base model in the presence and absence of electrostatic and nucleophilic catalysts have been calculated by the MNDO method. Three general processes - ordinary double bond isomerization, concerted isomerization about two double bonds by bicycle-pedal motion, and one-step double bond and adjacent single bond isomerization by hula-twist motion - are considered. Point negative charges or negatively charged nucleophiles near the protonated nitrogen substantially increase the barrier to cis-trans isomerization over what they would be in the absence of these agents. Negative charge or a nucleophile near C13 lowers the barrier to bicycle-pedal isomerization. Dark isomerization by a hula-twist motion required greater energy and is not substantially aided by the placement of a negative charge or nucleophile near any of the skeletal atoms in the isomerizing system. The importance of this to the mechanism of dark-light adaptation of bacteriorhodopsin is discussed.

  12. Prolyl 4-hydroxylase

    PubMed Central

    Gorres, Kelly L.; Raines, Ronald T.

    2010-01-01

    Posttranslational modifications can cause profound changes in protein function. Typically, these modifications are reversible, and thus provide a biochemical on–off switch. In contrast, proline residues are the substrates for an irreversible reaction that is the most common posttranslational modification in humans. This reaction, which is catalyzed by prolyl 4-hydroxylase (P4H), yields (2S,4R)-4-hydroxyproline (Hyp). The protein substrates for P4Hs are diverse. Likewise, the biological consequences of prolyl hydroxylation vary widely, and include altering protein conformation and protein–protein interactions, and enabling further modification. The best known role for Hyp is in stabilizing the collagen triple helix. Hyp is also found in proteins with collagen-like domains, as well as elastin, conotoxins, and argonaute 2. A prolyl hydroxylase domain protein acts on the hypoxia inducible factor α, which plays a key role in sensing molecular oxygen, and could act on inhibitory κB kinase and RNA polymerase II. P4Hs are not unique to animals, being found in plants and microbes as well. Here, we review the enzymic catalysts of prolyl hydroxylation, along with the chemical and biochemical consequences of this subtle but abundant posttranslational modification. PMID:20199358

  13. A cis/trans Test of the Effect of the First Enzyme for Histidine Biosynthesis on Regulation of the Histidine Operon

    PubMed Central

    Kovach, John S.; Ballesteros, Antonio O.; Meyers, Marilyn; Soria, Marco; Goldberger, Robert F.

    1973-01-01

    Previous studies showed that when triazolalanine was added to a derepressed culture of a histidine auxotroph, repression of the histidine operon occurred as though histidine had been added (6). However, when triazolalanine was added to a derepressed culture of a strain with a mutation in the first gene of the histidine operon which rendered the first enzyme for histidine biosynthesis resistant to inhibition by histidine, repression did not occur. The studies reported here represent a cis/trans test of this effect of mutations to feedback resistance. Using specially constructed merodiploid strains, we were able to show that the wild-type allele is dominant to the mutant (feedback resistant) allele and that the effect operates in trans. We conclude that the enzyme encoded by the first gene of the histidine operon exerts its regulatory effect on the operon not by acting locally at its site of synthesis, but by acting as a freely diffusible protein. PMID:4572718

  14. Structural Insights into Substrate Binding by PvFKBP35, a Peptidylprolyl cis-trans Isomerase from the Human Malarial Parasite Plasmodium vivax

    PubMed Central

    Alag, Reema; Balakrishna, Asha Manikkoth; Rajan, Sreekanth; Qureshi, Insaf A.; Shin, Joon; Lescar, Julien; Grüber, Gerhard

    2013-01-01

    The immunosuppressive drug FK506 binding proteins (FKBPs), an immunophilin family with the immunosuppressive drug FK506 binding property, exhibit peptidylprolyl cis-trans isomerase (PPIase) activity. While the cyclophilin-catalyzed peptidylprolyl isomerization of X-Pro peptide bonds has been extensively studied, the mechanism of the FKBP-mediated peptidylprolyl isomerization remains uncharacterized. Thus, to investigate the binding of FKBP with its substrate and the underlying catalytic mechanism of the FKBP-mediated proline isomerization, here we employed the FK506 binding domain (FKBD) of the human malarial parasite Plasmodium vivax FK506 binding protein 35 (PvFKBP35) and examined the details of the molecular interaction between the isomerase and a peptide substrate. The crystallographic structures of apo PvFKBD35 and its complex with the tetrapeptide substrate succinyl-Ala-Leu-Pro-Phe-p-nitroanilide (sALPFp) determined at 1.4 Å and 1.65 Å resolutions, respectively, showed that the substrate binds to PvFKBD35 in a cis conformation. Nuclear magnetic resonance (NMR) studies demonstrated the chemical shift perturbations of D55, H67, V73, and I74 residues upon the substrate binding. In addition, the X-ray crystal structure, along with the mutational studies, shows that Y100 is a key residue for the catalytic activity. Taken together, our results provide insights into the catalytic mechanism of PvFKBP35-mediated cis-trans isomerization of substrate and ultimately might aid designing substrate mimetic inhibitors targeting the malarial parasite FKBPs. PMID:23435727

  15. 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.

  16. 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

  17. Chicken FK506-binding protein, FKBP65, a member of the FKBP family of peptidylprolyl cis-trans isomerases, is only partially inhibited by FK506.

    PubMed Central

    Zeng, B; MacDonald, J R; Bann, J G; Beck, K; Gambee, J E; Boswell, B A; Bächinger, H P

    1998-01-01

    The chicken FK506-binding protein FKBP65, a peptidylprolyl cis-trans isomerase, is a rough endoplasmic reticulum protein that contains four domains homologous to FKBP13, another rough endoplasmic reticulum PPIase. Analytical ultracentrifugation suggests that in FKBP65 these four domains are arranged in a linear extended structure with a length of about 26 nm and a diameter of about 3 nm. All four domains are therefore expected to be accessible to substrates. The specificity of FKBP65 towards a number of peptide substrates was determined. The specific activity of FKBP65 is generally lower than that of FKBP12 when expressed as a per domain activity. The substrate specificity of FKBP65 also differs from that of FKBP12. Inhibition studies show that only one of the four domains can be inhibited by FK506, a powerful inhibitor of all other known FKBPs. Furthermore, the same domain seems to be susceptible to inhibition by cyclosporin A. No other FKBPs were shown to be inhibited by cyclosporin A. It is also shown that FKBP65 can catalyse the re-folding of type III collagen in vitro with a kcat/Km = 4.3 x 10(3) M-1.s-1. PMID:9461498

  18. Homodimerization of the G Protein Srbeta in the Nucleotide-Free State Involves Proline cis/trans Isomerication in the Switch II Region

    SciTech Connect

    Schwartz,T.; Schmidt, D.; Brohawn, S.; Blobel, G.

    2006-01-01

    Protein translocation across and insertion into membranes is essential to all life forms. Signal peptide-bearing nascent polypeptide chains emerging from the ribosome are first sampled by the signal-recognition particle (SRP), then targeted to the membrane via the SRP receptor (SR), and, finally, transferred to the protein-conducting channel. In eukaryotes, this process is tightly controlled by the concerted action of three G proteins, the 54-kD subunit of SRP and the {alpha}- and {beta}-subunits of SR. We have determined the 2.2-Angstroms crystal structure of the nucleotide-free SR{beta} domain. Unexpectedly, the structure is a homodimer with a highly intertwined interface made up of residues from the switch regions of the G domain. The remodeling of the switch regions does not resemble any of the known G protein switch mechanisms. Biochemical analysis confirms homodimerization in vitro, which is incompatible with SR{alpha} binding. The switch mechanism involves cis/trans isomerization of a strictly conserved proline, potentially implying a new layer of regulation of cotranslational transport.

  19. Fatty acids attached to all-trans-astaxanthin alter its cis-trans equilibrium, and consequently its stability, upon light-accelerated autoxidation.

    PubMed

    de Bruijn, Wouter J C; Weesepoel, Yannick; Vincken, Jean-Paul; Gruppen, Harry

    2016-03-01

    Fatty acid esterification, common in naturally occurring astaxanthin, has been suggested to influence both colour stability and degradation of all-trans-astaxanthin. Therefore, astaxanthin stability was studied as influenced by monoesterification and diesterification with palmitate. Increased esterification decelerated degradation of all-trans-astaxanthin (RP-UHPLC-PDA), whereas, it had no influence on colour loss over time (spectrophotometry). This difference might be explained by the observation that palmitate esterification influenced the cis-trans equilibrium. Free astaxanthin produced larger amounts of 9-cis isomer whereas monopalmitate esterification resulted in increased 13-cis isomerization. The molar ratios of 9-cis:13-cis after 60min were 1:1.7 (free), 1:4.8 (monopalmitate) and 1:2.6 (dipalmitate). The formation of 9-cis astaxanthin, with its higher molar extinction coefficient than that of all-trans-astaxanthin, might compensate for colour loss induced by conjugated double bond cleavage. As such, it was concluded that spectrophotometry is not an accurate measure of the degradation of the all-trans-astaxanthin molecule. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Effect of the medium on intramolecular H-atom tunneling: cis-trans conversion of formic acid in solid matrixes of noble gases.

    PubMed

    Trakhtenberg, Leonid I; Fokeyev, Anatoly A; Zyubin, Alexander S; Mebel, Alexander M; Lin, S H

    2010-12-30

    Intramolecular tunneling of a hydrogen atom in formic acid at low temperatures has been studied theoretically on the basis of quantum-chemical modeling of HCOOH@Nb(12) clusters. Three noble matrixes (Ar, Kr, and Xe) are considered. Energetic and geometric parameters as well as vibrational frequencies for the formic acid in cis and trans configurations surrounded by 12 Nb atoms are calculated within the frame of the MP2 approach with extended basis sets. The rate constant of HCOOH cis-trans conversion is analyzed by taking into account matrix reorganization and the change of HCOOH position in the cluster. The matrix reorganization is considered within the Debye model of lattice vibrations, whereas the external motion of HCOOH in the cluster is treated using the Einstein model of solids. It has been shown that the literature experimental data on the cis to trans tunneling reaction in the formic acid can be accounted for within the proposed mechanism, which describes the matrix reorganization and the change of the HCOOH position in the noble gas matrix, with fitting parameters of the suggested theoretical model attaining reasonable values.

  1. Effect of processing conditions on the content of cis/trans carotene isomers as provitamin A carotenoids in Korean sweet potato varieties.

    PubMed

    Kim, Heon Woong; Kim, Jung Bong; Poovan, Shanmugavelan; Chung, Mi Nam; Cho, Soo Muk; Lee, Young Min; Cho, Young Sook; Kim, Jae Hyun; Kim, Haeng Ran

    2014-11-01

    The present investigation intends to evaluate the changes in the content of cis/trans carotene isomers as provitamin A carotenoids by steaming and roasting processes in the roots of four Korean sweet potato varieties viz. Shinzami, Younwhangmi, Chuwhangmi and Jinhongmi using a liquid chromatography with diode array detection and the negative ion atmospheric pressure chemical ionization mass spectrometric (LC-DAD-APCI/MS) method and UV spectral pattern library created from several reference data. Except Shinzami, the content of all trans β-carotenes was found to slightly decreased or remained constant when steamed or roasted. The content of cis α-/β-carotenes was potentially increased about 2-fold or greater when raw or steamed and the content was slightly decreased while roasted. In Chuwhangmi, the content of 13-cis α-carotene and all trans α-carotenes were rapidly increased when steamed and slightly decreased when roasted. Chuwhangmi exhibited 27.2 mg/100 g DW content of all trans β-carotenes when roasted and thus, it was considered as a relatively superior cultivar.

  2. Homodimerization of the G protein SRβ in the nucleotide-free state involves proline cis/trans isomerization in the switch II region

    PubMed Central

    Schwartz, Thomas U.; Schmidt, Daniel; Brohawn, Stephen G.; Blobel, Günter

    2006-01-01

    Protein translocation across and insertion into membranes is essential to all life forms. Signal peptide-bearing nascent polypeptide chains emerging from the ribosome are first sampled by the signal-recognition particle (SRP), then targeted to the membrane via the SRP receptor (SR), and, finally, transferred to the protein-conducting channel. In eukaryotes, this process is tightly controlled by the concerted action of three G proteins, the 54-kD subunit of SRP and the α- and β-subunits of SR. We have determined the 2.2-Å crystal structure of the nucleotide-free SRβ domain. Unexpectedly, the structure is a homodimer with a highly intertwined interface made up of residues from the switch regions of the G domain. The remodeling of the switch regions does not resemble any of the known G protein switch mechanisms. Biochemical analysis confirms homodimerization in vitro, which is incompatible with SRα binding. The switch mechanism involves cis/trans isomerization of a strictly conserved proline, potentially implying a new layer of regulation of cotranslational transport. PMID:16627619

  3. 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.

  4. Complementary DNA encoding the human T-cell FK506-binding protein, a peptidylprolyl cis-trans isomerase distinct from cyclophilin

    SciTech Connect

    Maki, Noboru; Sekiguchi, Fumiko; Nishimaki, Junichi; Miwa, Keiko; Hayano, Toshiya; Takahashi, Nobuhiro; Suzuki, Masanori )

    1990-07-01

    The recently discovered macrolide FK506 has been demonstrated to have potent immunosuppressive activity at concentrations 100-fold lower than cyclosporin A, a cyclic undecapeptide that is used to prevent rejection after transplantation of bone marrow and organs, such as kidney, heart, and liver. After the recent discovery that the cylcosporin A-binding protein cyclophilin is identical to peptidylprolyl cis-trans isomerase, a cellular binding protein for FK506 was found to be distinct from cyclophilin but to have the same enzymatic activity. In this study, the authors isolated a cDNA coding for FK506-binding protein (FKBP) from human peripheral blood T cells by using mixed 20-mer oligonucleotide probes synthesized on the basis of the sequence, Glu-Asp-Gly-Lys-Lys-Phe-Asp, reported for bovine FKBP. The DNA isolated contained an open reading frame encoding 108 amino acid residues. The first 40 residues of the deduced amino acid sequence were identical to those of the reported amino-terminal sequence of bovine FKBP, indicating that the DNA sequence isolated represents the gene coding for FKBP. This result suggests that two catalytically similar proteins, cyclophilin and FKBP, evolved independently. In Northern blot analysis, mRNA species of {approx}1.8 kilobases that hybridized with human FKBP cDNA were detected in poly(A){sup +} RNAs from brain, lung, liver, and placental cells and leukocytes. Induction of Jurkat leukemic T cells with phorbol 12-myristate 13-acetate and ionomycin did not affect the level of FKBP mRNA.

  5. Chiral Cyclobutane β-Amino Acid-Based Amphiphiles: Influence of Cis/Trans Stereochemistry on Condensed Phase and Monolayer Structure.

    PubMed

    Sorrenti, Alessandro; Illa, Ona; Ortuño, Rosa M; Pons, Ramon

    2016-07-12

    New diastereomeric nonionic amphiphiles, cis- and trans-1, based on an optically pure cyclobutane β-amino ester moiety have been investigated to gain insight into the influence exerted by cis/trans stereochemistry and stereochemical constraints on the physicochemical behavior, molecular organization, and morphology of their Langmuir monolayers and dry solid states. All these features are relevant to the rational design of functional materials. trans-1 showed a higher thermal stability than cis-1. For the latter, a higher fluidity of its monolayers was observed when compared with the films formed by trans-1 whose BAM images revealed the formation of condensed phase domains with a dendritic shape, which are chiral, and all of them feature the same chiral sign. Although the formation of LC phase domains was not observed by BAM for cis-1, compact dendritic crystals floating on a fluid subphase were observed beyond the collapse, which are attributable to multilayered 3D structures. These differences can be explained by the formation of hydrogen bonds between the amide groups of consecutive molecules allowing the formation of extended chains for trans-1 giving ordered arrangements. However, for cis-1, this alignment coexists with another one that allows the simultaneous formation of two hydrogen bonds between the amide and the ester groups of adjacent molecules. In addition, the propensity to form intramolecular hydrogen bonds must be considered to justify the formation of different patterns of hydrogen bonding and, consequently, the formation of less ordered phases. Those characteristics are congruent also with the results obtained from SAXS-WAXS experiments which suggest a more bent configuration for cis-1 than for trans-1.

  6. Chiral Cyclobutane β-Amino Acid-Based Amphiphiles: Influence of Cis/Trans Stereochemistry on Solution Self-Aggregation and Recognition.

    PubMed

    Sorrenti, Alessandro; Illa, Ona; Pons, Ramon; Ortuño, Rosa M

    2015-09-08

    Novel diastereomeric anionic amphiphiles based on the rigid cyclobutane β-amino acid scaffold have been synthesized and deeply investigated with the aim of generating new functional supramolecular architectures on the basis of the rational design of original amphiphilic molecules and the control of their self-assembly. The main interest has been focused on the effect that cis/trans stereochemistry exerts on their molecular organization and recognition. In diluted solutions, the relative stereochemistry mainly influences the headgroup solvation and anionic-charge stabilization, i.e., better stabilized in the cis diastereoisomer due to intramolecular hydrogen-bonding and/or charge-dipole interactions. This provokes differences in their physicochemical behavior (pKa, cmc, conductivity) as well as in the structural parameters of the spherical micelles formed. Although both diastereoisomers form fibers that evolve with time from the spherical micelles, they display markedly different morphology and kinetics of formation. In the lyotropic liquid crystal domain, the greatest differences are observed at the highest concentrations and can be ascribed to different hydrogen-bonding and molecular packing imposed by the stereochemical constraints. Remarkably, the spherical micelles of the two anionic surfactants show dramatically diverse enantioselection ability for bilirubin enantiomers. In addition, both the surfactants form heteroaggregates with bilirubin at submicellar concentrations but with a different expression of supramolecular chirality. This points out that the unlike relative configuration of the two surfactants influences their chiral recognition ability as well as the fashion in which chirality is expressed at the supramolecular level by controlling the molecular organization in both micellar aggregates and surfactant/bilirubin heteroaggregates. All these differential features can be appropriate and useful for the design and development of new soft materials with

  7. A Crystallographic Study of Bright Far-Red Fluorescent Protein mKate Reveals pH-induced cis-trans Isomerization of the Chromophore

    SciTech Connect

    Pletnev, Sergei; Shcherbo, Dmitry; Chudakov, Dmitry M.; Pletneva, Nadezhda; Merzlyak, Ekaterina M.; Wlodawer, Alexander; Dauter, Zbigniew; Pletnev, Vladimir

    2008-11-03

    The far-red fluorescent protein mKate {lambda}{sup ex}, 588 nm; {lambda}{sub em}, 635 nm; chromophore-forming triad Met{sup 63}-Tyr{sup 64}-Gly{sup 65}, originating from wild-type red fluorescent progenitor eqFP578 (sea anemone Entacmaea quadricolor), is monomeric and characterized by the pronounced pH dependence of fluorescence, relatively high brightness, and high photostability. The protein has been crystallized at a pH ranging from 2 to 9 in three space groups, and four structures have been determined by x-ray crystallography at the resolution of 1.75--2.6 {angstrom}. The pH-dependent fluorescence of mKate has been shown to be due to reversible cis-trans isomerization of the chromophore phenolic ring. In the non-fluorescent state at pH 2.0, the chromophore of mKate is in the trans-isomeric form. The weakly fluorescent state of the protein at pH 4.2 is characterized by a mixture of trans and cis isomers. The chromophore in a highly fluorescent state at pH 7.0/9.0 adopts the cis form. Three key residues, Ser{sup 143}, Leu{sup 174}, and Arg{sup 197} residing in the vicinity of the chromophore, have been identified as being primarily responsible for the far-red shift in the spectra. A group of residues consisting of Val{sup 93}, Arg{sup 122}, Glu{sup 155}, Arg{sup 157}, Asp{sup 159}, His{sup 169}, Ile{sup 171}, Asn{sup 173}, Val{sup 192}, Tyr{sup 194}, and Val{sup 216}, are most likely responsible for the observed monomeric state of the protein in solution.

  8. Cyclophilin A (CyPA) induces chemotaxis independent of its peptidylprolyl cis-trans isomerase activity: direct binding between CyPA and the ectodomain of CD147.

    PubMed

    Song, Fei; Zhang, Xin; Ren, Xiao-Bai; Zhu, Ping; Xu, Jing; Wang, Li; Li, Yi-Fei; Zhong, Nan; Ru, Qiang; Zhang, Da-Wei; Jiang, Jian-Li; Xia, Bin; Chen, Zhi-Nan

    2011-03-11

    Cyclophilin A (CyPA) is a ubiquitously distributed peptidylprolyl cis-trans isomerase (PPIase) that possesses diverse biological functions. Extracellular CyPA is a potent chemokine, which can directly induce leukocyte chemotaxis and contribute to the pathogenesis of inflammation-mediated diseases. Although it has been identified that the chemotaxis activity of CyPA is mediated through its cell surface signaling receptor CD147, the role of CyPA PPIase activity in this process is disputable, and the underlying molecular mechanism is still poorly understood. In this study, we present the first evidence that CyPA induces leukocyte chemotaxis through a direct binding with the ectodomain of CD147 (CD147(ECT)), independent of its PPIase activity. Although NMR study indicates that the CD147(ECT) binding site on CyPA overlaps with the PPIase active site, the PPIase inactive mutant CyPA(R55A) exhibits similar CD147(ECT) binding ability and chemotaxis activity to those of CyPA(WT). Furthermore, we have identified three key residues of CyPA involved in CD147(ECT) binding and found that mutations H70A, T107A, and R69A result in similar levels of reduction in CD147(ECT) binding ability and chemotaxis activity for CyPA, without affecting the PPIase activity. Our findings indicate that there exists a novel mechanism for CyPA to regulate cellular signaling processes, shedding new light on its applications in drug development and providing a new targeting site for drug design.

  9. Synthesis of 7-azabicyclo[2.2.1]heptane and 2-oxa-4-azabicyclo[3.3.1]non-3-ene derivatives by base-promoted heterocyclization of alkyl N-(cis(trans)-3,trans(cis)-4-dibromocyclohex-1-yl)carbamates and N-(cis(trans)-3,trans(cis)-4-dibromocyclohex-1-yl)-2,2,2-trifluoroacetamides.

    PubMed

    Gómez-Sanchez, Elena; Soriano, Elena; Marco-Contelles, José

    2007-11-09

    We have studied the base-promoted heterocyclization of alkyl N-(cis(trans)-3,trans(cis)-4-dibromocyclohex-1-yl)carbamates and N-(cis(trans)-3,trans(cis)-4-dibromocyclohex-1-yl)-2,2,2-trifluoroacetamides, investigating the effect of the nitrogen protecting group and the relative configuration of the leaving group at C3 and C4 on the outcome of this reaction. We have observed that the sodium hydride-promoted heterocyclization of alkyl N-(cis-3,trans-4-dibromocyclohex-1-yl)carbamates (10, 12, 14, 16, 18) is a convenient method for the synthesis of 7-azabicyclo[2.2.1]heptane derivatives. For instance, the reaction of tert-butyl N-(cis-3,trans-4-dibromocyclohex-1-yl)carbamate (10) with sodium hydride in DMF at room temperature provides 2-bromo-7-[(tert-butoxy)carbonyl]-7-azabicyclo[2.2.1]heptane (2) (52% yield), whose t-BuOK-promoted hydrogen bromide elimination affords 7-[(tert-butoxy)carbonyl]-7-azabicyclo[2.2.1]hept-2-ene (31) in 78% yield, an intermediate in the total synthesis of epibatidine (1). However, the NaH/DMF-mediated heterocyclization of alkyl N-(trans-3,cis-4-dibromocyclohex-1-yl)carbamates (11, 13) is a more structure dependent reaction, where the nucleophilic attack of the oxygen atom of the protecting group controls the outcome of the reaction, giving rise to benzooxazolone and 2-oxa-4-azabicyclo[3.3.1]non-3-ene derivatives, respectively, from low to moderate yields, in complex reaction mixtures. Conversely, the NaH/DMF heterocyclizations of N-(cis-3,trans-4-dibromocyclohex-1-yl)-2,2,2-trifluoroacetamide (40) or N-(trans-3,cis-4-dibromocyclohex-1-yl)-2,2,2-trifluoroacetamide (42) are very clean reactions giving 7-azabicyclo[2.2.1]heptane or 2-oxa-4-azabicyclo[3.3.1]non-3-ene derivatives, respectively, in good yields. Finally, a mechanistic investigation, based on DFT calculations, has been carried out to rationalize the formation of the different adducts.

  10. An on-line SPE-HPLC method for effective sample preconcentration and determination of fenoxycarb and cis, trans-permethrin in surface waters.

    PubMed

    Šatínský, Dalibor; Naibrtová, Linda; Fernández-Ramos, Carolina; Solich, Petr

    2015-09-01

    A new on-line SPE-HPLC method using fused-core columns for on-line solid phase extraction and large volume sample injection for increasing the sensitivity of detection was developed for the determination of insecticides fenoxycarb and cis-, trans-permethrin in surface waters. The separation was carried out on fused-core column Phenyl-Hexyl (100×4.6 mm), particle size 2.7 µm with mobile phase acetonitrile:water in gradient mode at flow rate 1.0 mL min(-1), column temperature 45°C. Large volume sample injection (1500 µL) to the extraction dimension using short precolumn Ascentis Express RP C-18 (5×4.6 mm); fused-core particle size 2.7 µm allowed effective sample preconcentration and efficient ballast sample matrix removal. The washing mobile phase consisting of a mixture of acetonitrile:water; 30:70, (v/v) was pumped at flow rate of 0.5 mL min(-1) through the extraction precolumn to the waste. Time of the valve switch for transferring the preconcentrated sample zone from the extraction to the separation column was set at 3rd min. Elution of preconcentrated insecticides from the extraction precolumn and separation on the analytical column was performed in gradient mode. Linear gradient elution started from 40% of acetonitrile at time of valve switch from SPE column (3rd min) to 95% of acetonitrile at 7th min. Synthetic dye sudan I was chosen as an internal standard. UV detection at wavelength 225 nm was used and the method reached the limits of detection (LOD) at ng mL(-1) levels for both insecticides. The method showing on-line sample pretreatment and preconcentration with highly sensitive determination of insecticides was applied for monitoring of fenoxycarb and both permethrin isomers in different surface water samples in Czech Republic. The time of whole analysis including on-line extraction, interferences removal, chromatography separation and system equilibration was less than 8 min. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Synthesis and characterization of fac-[M(CO)3(P)(OO)] and cis-trans-[M(CO)2(P)2(OO)] complexes (M = Re, (99m)Tc) with acetylacetone and curcumin as OO donor bidentate ligands.

    PubMed

    Triantis, Charalampos; Tsotakos, Theodoros; Tsoukalas, Charalampos; Sagnou, Marina; Raptopoulou, Catherine; Terzis, Aris; Psycharis, Vassilis; Pelecanou, Maria; Pirmettis, Ioannis; Papadopoulos, Minas

    2013-11-18

    The synthesis and characterization of neutral mixed ligand complexes fac-[M(CO)3(P)(OO)] and cis-trans-[M(CO)2(P)2(OO)] (M = Re, (99m)Tc), with deprotonated acetylacetone or curcumin as the OO donor bidentate ligands and a phosphine (triphenylphosphine or methyldiphenylphosphine) as the monodentate P ligand, is described. The complexes were synthesized through the corresponding fac-[M(CO)3(H2O)(OO)] (M = Re, (99m)Tc) intermediate aqua complex. In the presence of phosphine, replacement of the H2O molecule of the intermediate complex at room temperature generates the neutral tricarbonyl monophosphine fac-[Re(CO)3(P)(OO)] complex, while under reflux conditions further replacement of the trans to the phosphine carbonyl generates the new stable dicarbonyl bisphosphine complex cis-trans-[Re(CO)2(P)2(OO)]. The Re complexes were fully characterized by elemental analysis, spectroscopic methods, and X-ray crystallography showing a distorted octahedral geometry around Re. Both the monophosphine and the bisphosphine complexes of curcumin show selective binding to β-amyloid plaques of Alzheimer's disease. At the (99m)Tc tracer level, the same type of complexes, fac-[(99m)Tc(CO)3(P)(OO)] and cis-trans-[(99m)Tc(CO)2(P)2(OO)], are formed introducing new donor combinations for (99m)Tc(I). Overall, β-diketonate and phosphine constitute a versatile ligand combination for Re(I) and (99m)Tc(I), and the successful employment of the multipotent curcumin as β-diketone provides a solid example of the pharmacological potential of this system.

  12. 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

  13. 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.

  14. 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

  15. 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

  16. Bacillus anthracis Prolyl 4-Hydroxylase Modifies Collagen-like Substrates in Asymmetric Patterns*

    PubMed Central

    Schnicker, Nicholas J.; Dey, Mishtu

    2016-01-01

    Proline hydroxylation is the most prevalent post-translational modification in collagen. The resulting product trans-4-hydroxyproline (Hyp) is of critical importance for the stability and thus function of collagen, with defects leading to several diseases. Prolyl 4-hydroxylases (P4Hs) are mononuclear non-heme iron α-ketoglutarate (αKG)-dependent dioxygenases that catalyze Hyp formation. Although animal and plant P4Hs target peptidyl proline, prokaryotes have been known to use free l-proline as a precursor to form Hyp. The P4H from Bacillus anthracis (BaP4H) has been postulated to act on peptidyl proline in collagen peptides, making it unusual within the bacterial clade, but its true physiological substrate remains enigmatic. Here we use mass spectrometry, fluorescence binding, x-ray crystallography, and docking experiments to confirm that BaP4H recognizes and acts on peptidyl substrates but not free l-proline, using elements characteristic of an Fe(II)/αKG-dependent dioxygenases. We further show that BaP4H can hydroxylate unique peptidyl proline sites in collagen-derived peptides with asymmetric hydroxylation patterns. The cofactor-bound crystal structures of BaP4H reveal active site conformational changes that define open and closed forms and mimic “ready” and “product-released” states of the enzyme in the catalytic cycle. These results help to clarify the role of BaP4H as well as provide broader insights into human collagen P4H and proteins with poly-l-proline type II helices. PMID:27129244

  17. Bacillus anthracis Prolyl 4-Hydroxylase Modifies Collagen-like Substrates in Asymmetric Patterns.

    PubMed

    Schnicker, Nicholas J; Dey, Mishtu

    2016-06-17

    Proline hydroxylation is the most prevalent post-translational modification in collagen. The resulting product trans-4-hydroxyproline (Hyp) is of critical importance for the stability and thus function of collagen, with defects leading to several diseases. Prolyl 4-hydroxylases (P4Hs) are mononuclear non-heme iron α-ketoglutarate (αKG)-dependent dioxygenases that catalyze Hyp formation. Although animal and plant P4Hs target peptidyl proline, prokaryotes have been known to use free l-proline as a precursor to form Hyp. The P4H from Bacillus anthracis (BaP4H) has been postulated to act on peptidyl proline in collagen peptides, making it unusual within the bacterial clade, but its true physiological substrate remains enigmatic. Here we use mass spectrometry, fluorescence binding, x-ray crystallography, and docking experiments to confirm that BaP4H recognizes and acts on peptidyl substrates but not free l-proline, using elements characteristic of an Fe(II)/αKG-dependent dioxygenases. We further show that BaP4H can hydroxylate unique peptidyl proline sites in collagen-derived peptides with asymmetric hydroxylation patterns. The cofactor-bound crystal structures of BaP4H reveal active site conformational changes that define open and closed forms and mimic "ready" and "product-released" states of the enzyme in the catalytic cycle. These results help to clarify the role of BaP4H as well as provide broader insights into human collagen P4H and proteins with poly-l-proline type II helices. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Complex regulation of prolyl-4-hydroxylases impacts root hair expansion.

    PubMed

    Velasquez, Silvia M; Ricardi, Martiniano M; Poulsen, Christian Peter; Oikawa, Ai; Dilokpimol, Adiphol; Halim, Adnan; Mangano, Silvina; Denita Juarez, Silvina Paola; Marzol, Eliana; Salgado Salter, Juan D; Dorosz, Javier Gloazzo; Borassi, Cecilia; Möller, Svenning Rune; Buono, Rafael; Ohsawa, Yukiko; Matsuoka, Ken; Otegui, Marisa S; Scheller, Henrik V; Geshi, Naomi; Petersen, Bent Larsen; Iusem, Norberto D; Estevez, José M

    2015-05-01

    Root hairs are single cells that develop by tip growth, a process shared with pollen tubes, axons, and fungal hyphae. However, structural plant cell walls impose constraints to accomplish tip growth. In addition to polysaccharides, plant cell walls are composed of hydroxyproline-rich glycoproteins (HRGPs), which include several groups of O-glycoproteins, including extensins (EXTs). Proline hydroxylation, an early post-translational modification (PTM) of HRGPs catalyzed by prolyl 4-hydroxylases (P4Hs), defines their subsequent O-glycosylation sites. In this work, our genetic analyses prove that P4H5, and to a lesser extent P4H2 and P4H13, are pivotal for root hair tip growth. Second, we demonstrate that P4H5 has in vitro preferred specificity for EXT substrates rather than for other HRGPs. Third, by P4H promoter and protein swapping approaches, we show that P4H2 and P4H13 have interchangeable functions but cannot replace P4H5. These three P4Hs are shown to be targeted to the secretory pathway, where P4H5 forms dimers with P4H2 and P4H13. Finally, we explore the impact of deficient proline hydroxylation on the cell wall architecture. Taken together, our results support a model in which correct peptidyl-proline hydroxylation on EXTs, and possibly in other HRGPs, is required for proper cell wall self-assembly and hence root hair elongation in Arabidopsis thaliana.

  19. SPPS of protected peptidyl aminoalkyl amides.

    PubMed

    Karavoltsos, Manolis; Mourtas, Spyros; Gatos, Dimitrios; Barlos, Kleomenis

    2002-11-01

    Monophthaloyl diamines derived from naturally occurring amino acids were attached through their free amino functions to resins of the trityl type. The phthaloyl groups were removed by hydrazinolysis, and peptide chains were assembled using Fmoc/tBu-amino acids on the liberated amino functions. The peptidyl aminoalkyl amides obtained were cleaved from the resins by mild acidolysis, with the tBu-side chain protection remaining intact.

  20. Determination of the cis-trans isomerization barriers of L-alanyl-L-proline in aqueous solutions and at water/hydrophobic interfaces by on-line temperature-jump relaxation HPLC and dynamic on-column reaction HPLC.

    PubMed

    Shibukawa, Masami; Miyake, Ayaka; Eda, Sayaka; Saito, Shingo

    2015-09-15

    Proline cis-trans isomerization is known to play a key role in the rate-determining steps of protein folding. It is thus very important to understand the influence of environments, not only bulk solutions but also microenvironments such as interfaces, on the isomerization reaction of proline peptides. Here we present two HPLC methods for measurements of kinetic and equilibrium parameters for the isomerization reactions in bulk solutions and at liquid/solid interfaces. On-line temperature-jump relaxation HPLC (T-jump HPLC) allows the determination of forward and reverse rate constants of the isomerization in a bulk solution by monitoring the whole time course of conversion of pure isomers from both sides of the reaction, in contrast to other HPLC and capillary zone electrophoresis as well as spectrometric and calorimetric methods, which use a mixture of the isomers. We can then determine cis-trans isomerization barriers of the peptide at liquid/solid interfaces from the kinetic data obtained by dynamic on-column reaction HPLC and T-jump HPLC. We observed that the interconversion around the peptide bond for l-alanyl-l-proline (Ala-Pro) in water is accelerated at the surfaces of an alkyl-bonded silica and a poly(styrene-divinylbenzene) copolymer resin, and this is caused by a remarkable decrease in the enthalpy of activation. The molecular structures of the cis and trans forms of Ala-Pro estimated by quantum mechanics calculation reveal that an equilibrium shift toward the cis form as well as the rapid isomerization of Ala-Pro at the water/hydrophobic interfaces can be attributed to the lower polarity of the interfacial water at the surfaces of the hydrophobic materials compared to that of bulk water.

  1. 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.

  2. 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.

  3. Mitochondrial targeting of radioprotectants using peptidyl conjugates

    PubMed Central

    Zabbarova, Irina; Amoscato, Andrew; Epperly, Michael; Xiao, Jingbo; Wipf, Peter

    2011-01-01

    Ionizing radiation activates a mitochondrial nitric oxide synthase, leading to inhibition of the respiratory chain, generation of excess superoxide, peroxynitrite production and nitrosative damage. We have measured the radioprotective effects of a nitric oxide synthase antagonist (AMT) versus a free radical scavenger (4-amino-TEMPO) using electrochemical detection of nitric oxide and peroxynitrite. To enhance their efficacy, we have conjugated these compounds to peptides and peptide isosteres—derived from the antibiotic gramicidin S—that target the mitochondria. The targeting ability of these peptidyl conjugates was measured using quantitative mass spectrometry. PMID:17205174

  4. Prolyl hydroxylation in elastin is not random.

    PubMed

    Schmelzer, Christian E H; Nagel, Marcus B M; Dziomba, Szymon; Merkher, Yulia; Sivan, Sarit S; Heinz, Andrea

    2016-10-01

    This study aimed to investigate the prolyl and lysine hydroxylation in elastin from different species and tissues. Enzymatic digests of elastin samples from human, cattle, pig and chicken were analyzed using mass spectrometry and bioinformatics tools. It was confirmed at the protein level that elastin does not contain hydroxylated lysine residues regardless of the species. In contrast, prolyl hydroxylation sites were identified in all elastin samples. Moreover, the analysis of the residues adjacent to prolines allowed the determination of the substrate site preferences of prolyl 4-hydroxylase. It was found that elastins from all analyzed species contain hydroxyproline and that at least 20%-24% of all proline residues were partially hydroxylated. Determination of the hydroxylation degrees of specific proline residues revealed that prolyl hydroxylation depends on both the species and the tissue, however, is independent of age. The fact that the highest hydroxylation degrees of proline residues were found for elastin from the intervertebral disc and knowledge of elastin arrangement in this tissue suggest that hydroxylation plays a biomechanical role. Interestingly, a proline-rich domain of tropoelastin (domain 24), which contains several repeats of bioactive motifs, does not show any hydroxyproline residues in the mammals studied. The results show that prolyl hydroxylation is not a coincidental feature and may contribute to the adaptation of the properties of elastin to meet the functional requirements of different tissues. The study for the first time shows that prolyl hydroxylation is highly regulated in elastin. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Probing cis-trans isomerization in the S{sub 1} state of C{sub 2}H{sub 2} via H-atom action and hot band-pumped IR-UV double resonance spectroscopies

    SciTech Connect

    Changala, P. Bryan; Baraban, Joshua H.; Field, Robert W.; Merer, Anthony J.

    2015-08-28

    We report novel experimental strategies that should prove instrumental in extending the vibrational and rotational assignments of the S{sub 1} state of acetylene, C{sub 2}H{sub 2}, in the region of the cis-trans isomerization barrier. At present, the assignments are essentially complete up to ∼500 cm{sup −1} below the barrier. Two difficulties arise when the assignments are continued to higher energies. One is that predissociation into C{sub 2}H + H sets in roughly 1100 cm{sup −1} below the barrier; the resulting quenching of laser-induced fluorescence (LIF) reduces its value for recording spectra in this region. The other difficulty is that tunneling through the barrier causes a staggering in the K-rotational structure of isomerizing vibrational levels. The assignment of these levels requires data for K values up to at least 3. Given the rotational selection rule K′ − ℓ{sup ′′} = ± 1, such data must be obtained via excited vibrational levels of the ground state with ℓ{sup ′′} > 0. In this paper, high resolution H-atom resonance-enhanced multiphoton ionization spectra are demonstrated to contain predissociated bands which are almost invisible in LIF spectra, while preliminary data using a hyperthermal pulsed nozzle show that ℓ{sup ′′} = 2 states can be selectively populated in a jet, giving access to K′ = 3 states in IR-UV double resonance.

  6. Mutation in Cyclophilin B That Causes Hyperelastosis Cutis in American Quarter Horse Does Not Affect Peptidylprolyl cis-trans Isomerase Activity but Shows Altered Cyclophilin B-Protein Interactions and Affects Collagen Folding*

    PubMed Central

    Ishikawa, Yoshihiro; Vranka, Janice A.; Boudko, Sergei P.; Pokidysheva, Elena; Mizuno, Kazunori; Zientek, Keith; Keene, Douglas R.; Rashmir-Raven, Ann M.; Nagata, Kazuhiro; Winand, Nena J.; Bächinger, Hans Peter

    2012-01-01

    The rate-limiting step of folding of the collagen triple helix is catalyzed by cyclophilin B (CypB). The G6R mutation in cyclophilin B found in the American Quarter Horse leads to autosomal recessive hyperelastosis cutis, also known as hereditary equine regional dermal asthenia. The mutant protein shows small structural changes in the region of the mutation at the side opposite the catalytic domain of CypB. The peptidylprolyl cis-trans isomerase activity of the mutant CypB is normal when analyzed in vitro. However, the biosynthesis of type I collagen in affected horse fibroblasts shows a delay in folding and secretion and a decrease in hydroxylysine and glucosyl-galactosyl hydroxylysine. This leads to changes in the structure of collagen fibrils in tendon, similar to those observed in P3H1 null mice. In contrast to cyclophilin B null mice, where little 3-hydroxylation was found in type I collagen, 3-hydroxylation of type I collagen in affected horses is normal. The mutation disrupts the interaction of cyclophilin B with the P-domain of calreticulin, with lysyl hydroxylase 1, and probably other proteins, such as the formation of the P3H1·CypB·cartilage-associated protein complex, resulting in less effective catalysis of the rate-limiting step in collagen folding in the rough endoplasmic reticulum. PMID:22556420

  7. Zero-point Energy is Needed in Molecular Dynamics Calculations to Access the Saddle Point for H+HCN→H2CN* and cis/trans-HCNH* on a New Potential Energy Surface.

    PubMed

    Wang, Xiaohong; Bowman, Joel M

    2013-02-12

    We calculate the probabilities for the association reactions H+HCN→H2CN* and cis/trans-HCNH*, using quasiclassical trajectory (QCT) and classical trajectory (CT) calculations, on a new global ab initio potential energy surface (PES) for H2CN including the reaction channels. The surface is a linear least-squares fit of roughly 60 000 CCSD(T)-F12b/aug-cc-pVDZ electronic energies, using a permutationally invariant basis with Morse-type variables. The reaction probabilities are obtained at a variety of collision energies and impact parameters. Large differences in the threshold energies in the two types of dynamics calculations are traced to the absence of zero-point energy in the CT calculations. We argue that the QCT threshold energy is the realistic one. In addition, trajectories find a direct pathway to trans-HCNH, even though there is no obvious transition state (TS) for this pathway. Instead the saddle point (SP) for the addition to cis-HCNH is evidently also the TS for direct formation of trans-HCNH.

  8. Structure of Francisella tularensis peptidyl-tRNA hydrolase

    PubMed Central

    Clarke, Teresa E.; Romanov, Vladimir; Lam, Robert; Gothe, Scott A.; Peddi, Srinivasa R.; Razumova, Ekaterina B.; Lipman, Richard S. A.; Branstrom, Arthur A.; Chirgadze, Nickolay Y.

    2011-01-01

    The rational design of novel antibiotics for bacteria involves the identification of inhibitors for enzymes involved in essential biochemical pathways in cells. In this study, the cloning, expression, purification, crystallization and structure of the enzyme peptidyl-tRNA hydrolase from Francisella tularensis, the causative agent of tularemia, was performed. The structure of F. tularensis peptidyl-tRNA hydrolase is comparable to those of other bacterial peptidyl-tRNA hydrolases, with most residues in the active site conserved amongst the family. The resultant reagents, structural data and analyses provide essential information for the structure-based design of novel inhibitors for this class of proteins. PMID:21505237

  9. 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.

  10. Evidence of Sulfur Mustard Exposure in Human Plasma by LC-ESI-MS-MS Detection of the Albumin-Derived Alkylated HETE-CP Dipeptide and Chromatographic Investigation of Its Cis/Trans Isomerism.

    PubMed

    Gandor, Felix; Gawlik, Michael; Thiermann, Horst; John, Harald

    2015-05-01

    Sulfur mustard (SM) is a chemical warfare agent that causes painful blisters and chemically modifies endogenous biomacromolecules by alkylation to hydroxyethylthioethyl (HETE) adducts representing valuable long-term markers for post-exposure analysis. The albumin adduct formed in human plasma in vitro (HETE bound to the side chain of cysteine 34) was isolated and cleaved by current lots of pronase primarily generating the internal modified dipeptide (HETE-cysteine-proline, HETE-CP) instead of the formerly reported HETE-CPF tripeptide. The analyte was detected by liquid chromatography-electrospray ionization tandem-mass spectrometry (LC-ESI-MS-MS). In principle, HETE-CP undergoes a dynamic on-column equilibrium of cis-trans isomerism thus requiring separation at 50°C to obtain one narrow peak. Accordingly, we developed both a novel longer lasting but more sensitive microbore (1 mm i.d., flow 30 µL/min, cycle time 60 min, LOD 50 nM) and a faster, less sensitive narrowbore (2.1 mm i.d., 200 µL/min, cycle time 16 min, LOD 100 nM, both on Atlantis T3 material at 50°C) LC-ESI-MS-MS method suitable for verification analysis. The corresponding tri- and tetrapeptide, Q(HETE)-CPF were monitored simultaneously. HETE-CP peak areas were directly proportional to SM concentrations added to plasma in vitro (0.05-100 µM). Albumin adducts formed by deuterated SM (d8-SM) served as internal standard.

  11. Unusual temperature dependence in the cis/trans-oxetane formation discloses competitive syn versus anti attack for the Paternò-Büchi reaction of triplet-excited ketones with cis- and trans-cylooctenes. Conformational control of diastereoselectivity in the cyclization and cleavage of preoxetane diradicals.

    PubMed

    Adam, Waldemar; Stegmann, Veit R

    2002-04-10

    Toluene-d(8) solutions of cis- and trans-cyclooctene (cis- and trans-1a) as well as (Z)- and (E)-1-methylcyclooctene (cis- and trans-1b) have been irradiated at temperatures between -95 and +110 degrees C in the presence of benzophenone (BP) to afford mixtures of the cis- and trans-configured oxetanes 2a,b and the regioisomeric 2b'. Correspondingly, benzoquinone (BQ) gave with cis- and trans-1a the cycloadducts cis- and trans-3a. The cis/trans diastereomeric ratios of the [2 + 2]-cycloadducts 2 and 3 display a strong temperature dependence; with cis- and trans-1a or cis-1b as starting materials, the diastereoselectivity of the oxetane formation is high at low temperature, under preservation of the initial cyclooctene configuration. With increasing temperature, the cis diastereoselectivity decreases continuously for the cis-cyclooctenes; in the case of the cis-1a, the diastereoselectivity is even switched to trans (cis/trans ca. 20:80) at very high temperatures. For the strained trans-1a, the trans-oxetanes are strongly preferred over the entire temperature range, with only minor leakage (up to 10%) to the cis-oxetanes at very high temperatures. Oxetane formation is accompanied by nonthermal trans-to-cis isomerization of the cyclooctene. The methyl-substituted trans-1b constitutes an exceptional substrate; it displays cis diastereoselectivity in the [2 + 2] photocycloaddition at low temperatures for both regioisomers 2b and 2b', and the trans selectivity increases at moderate temperature (cis/trans = 4:96), to decrease again at high temperature, especially for the minor regioisomer 2b'. This complex temperature behavior of the cis/trans diastereoselectivity may be rationalized in terms of the triplet-diradical mechanism of the Paternò-Büchi reaction. We propose that the cyclooctene may be competitively attacked by the triplet-excited ketone from the higher (syn) or the less (anti) substituted side; such syn and anti trajectories have hitherto not been considered

  12. The prolyl isomerase Pin1 acts as a novel molecular switch for TNF-α–induced priming of the NADPH oxidase in human neutrophils

    PubMed Central

    Boussetta, Tarek; Gougerot-Pocidalo, Marie-Anne; Hayem, Gilles; Ciappelloni, Silvia; Raad, Houssam; Arabi Derkawi, Riad; Bournier, Odile; Kroviarski, Yolande; Zhou, Xiao Zhen; Malter, James S.; Lu, Ping K.; Bartegi, Aghleb; Dang, Pham My-Chan

    2010-01-01

    Neutrophils play a key role in host defense by releasing reactive oxygen species (ROS). However, excessive ROS production by neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can damage bystander tissues, thereby contributing to inflammatory diseases. Tumor necrosis factor-α (TNF-α), a major mediator of inflammation, does not activate NADPH oxidase but induces a state of hyperresponsiveness to subsequent stimuli, an action known as priming. The molecular mechanisms by which TNF-α primes the NADPH oxidase are unknown. Here we show that Pin1, a unique cis-trans prolyl isomerase, is a previously unrecognized regulator of TNF-α–induced NADPH oxidase hyperactivation. We first showed that Pin1 is expressed in neutrophil cytosol and that its activity is markedly enhanced by TNF-α. Inhibition of Pin1 activity with juglone or with a specific peptide inhibitor abrogated TNF-α–induced priming of neutrophil ROS production induced by N-formyl-methionyl-leucyl-phenylalanine peptide (fMLF). TNF-α enhanced fMLF-induced Pin1 and p47phox translocation to the membranes and juglone inhibited this process. Pin1 binds to p47phox via phosphorylated Ser345, thereby inducing conformational changes that facilitate p47phox phosphorylation on other sites by protein kinase C. These findings indicate that Pin1 is critical for TNF-α–induced priming of NADPH oxidase and for excessive ROS production. Pin1 inhibition could potentially represent a novel anti-inflammatory strategy. PMID:20956805

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. Induced-fit Mechanism for Prolyl Endopeptidase

    SciTech Connect

    Li, Min; Chen, Changqing; Davies, David R.; Chiu, Thang K.

    2010-11-15

    Prolyl peptidases cleave proteins at proline residues and are of importance for cancer, neurological function, and type II diabetes. Prolyl endopeptidase (PEP) cleaves neuropeptides and is a drug target for neuropsychiatric diseases such as post-traumatic stress disorder, depression, and schizophrenia. Previous structural analyses showing little differences between native and substrate-bound structures have suggested a lock-and-key catalytic mechanism. We now directly demonstrate from seven structures of Aeromonus punctata PEP that the mechanism is instead induced fit: the native enzyme exists in a conformationally flexible opened state with a large interdomain opening between the {beta}-propeller and {alpha}/{beta}-hydrolase domains; addition of substrate to preformed native crystals induces a large scale conformational change into a closed state with induced-fit adjustments of the active site, and inhibition of this conformational change prevents substrate binding. Absolute sequence conservation among 28 orthologs of residues at the active site and critical residues at the interdomain interface indicates that this mechanism is conserved in all PEPs. This finding has immediate implications for the use of conformationally targeted drug design to improve specificity of inhibition against this family of proline-specific serine proteases.

  1. Complete thermodynamic and kinetic characterization of the isomer-specific interaction between Pin1-WW domain and the amyloid precursor protein cytoplasmic tail phosphorylated at Thr668.

    PubMed

    De, Soumya; Greenwood, Alexander I; Rogals, Monique J; Kovrigin, Evgenii L; Lu, Kun Ping; Nicholson, Linda K

    2012-10-30

    Peptidyl prolyl cis-trans isomerization acts as an effective molecular timer that plays significant roles in biological and pathological processes. Enzymes such as Pin1 catalyze cis-trans isomerization, accelerating the otherwise slow isomerization rate into time scales relevant for cellular signaling. Here we have combined NMR line shape analysis, fluorescence spectroscopy, and isothermal titration calorimetry to determine the kinetic and thermodynamic parameters describing the trans-specific interaction between the binding domain of Pin1 (WW domain) and a key cis-trans molecular switch in the amyloid precursor protein cytoplasmic tail. A three-state model, in which the cis-trans isomerization equilibrium is coupled to the binding equilibrium through the trans isomer, was found to fit the data well. The trans isomer binds the WW domain with ∼22 μM affinity via very fast association (approaching the diffusion limit) and dissociation rates. The common structural and electrostatic characteristics of Pin1 substrates, which contain a phosphorylated serine/threonine-proline motif, suggest that very rapid binding kinetics are a general feature of Pin1 interactions with other substrates. The fast binding kinetics of the WW domain allows rapid response of Pin1 to the dynamic events of phosphorylation and dephosphorylation in the cell that alter the relative populations of diverse Pin1 substrates. Furthermore, our results also highlight the vastly different rates at which slow uncatalyzed cis-trans isomerization and fast isomer-specific binding events occur. These results, along with the experimental methods presented herein, should guide future experiments aimed at the thermodynamic and kinetic characterization of cis-trans molecular switches and isomer-specific interactions involved in various biological processes.

  2. Complete thermodynamic and kinetic characterization of the isomer-specific interaction between Pin1-WW domain and the amyloid precursor protein cytoplasmic tail phosphorylated at threonine668

    PubMed Central

    De, Soumya; Greenwood, Alexander I.; Rogals, Monique J.; Kovrigin, Evgenii L.; Lu, Kun Ping; Nicholson, Linda K.

    2012-01-01

    Peptidyl prolyl cis-trans isomerization acts as an effective molecular timer that plays significant roles in biological and pathological processes. Enzymes such as Pin1 catalyze cis-trans isomerization, accelerating the otherwise slow isomerization rate into timescales relevant for cellular signaling. Here we have combined NMR lineshape analysis, fluorescence spectroscopy, and isothermal titration calorimetry to determine the kinetic and thermodynamic parameters describing the trans-specific interaction between the binding domain of Pin1 (WW domain) and a key cis-trans molecular switch in the amyloid precursor protein cytoplasmic tail. A three-state model, in which the cis-trans isomerization equilibrium is coupled to the binding equilibrium through the trans isomer, was found to fit the data well. The trans isomer binds the WW domain with ~22 μM affinity via very fast association (approaching the diffusion limit) and dissociation rates. The common structural and electrostatic characteristics of Pin1 substrates, which contain a phosphorylated serine/threonine-proline motif, suggest that very rapid binding kinetics are a general feature of Pin1 interactions with other substrates. The fast binding kinetics of the WW domain allows rapid response of Pin1 to the dynamic events of phosphorylation and dephosphorylation in the cell that alter the relative populations of diverse Pin1 substrates. Furthermore, our results also highlight the vastly different rates at which slow uncatalyzed cis-trans isomerization and fast isomer-specific binding events occur. These results, along with the experimental methods presented herein, should guide future experiments aimed at the thermodynamic and kinetic characterization of cis-trans molecular switches and isomer-specific interactions involved in various biological processes. PMID:23025283

  3. Collagen Prolyl Hydroxylases are Essential for Breast Cancer Metastasis

    PubMed Central

    Gilkes, Daniele M.; Chaturvedi, Pallavi; Bajpai, Saumendra; Wong, Carmen Chak-Lui; Wei, Hong; Pitcairn, Stephen; Hubbi, Maimon E.; Wirtz, Denis; Semenza, Gregg L.

    2013-01-01

    Metastasis is the leading cause of death among patients with breast cancer. Understanding the role of the extracellular matrix in the metastatic process may lead to the development of improved therapies for cancer patients. Intratumoral hypoxia is found in the majority of breast cancers and is associated with an increased risk of metastasis and patient mortality. Here we demonstrate that hypoxia-inducible factor 1 activates the transcription of genes encoding collagen prolyl hydroxylases that are critical for collagen deposition by breast cancer cells. We show that expression of collagen prolyl hydroxylases promotes cancer cell alignment along collagen fibers, resulting in enhanced invasion and metastasis to lymph nodes and lungs. Lastly, we establish the prognostic significance of collagen prolyl hydroxylase mRNA expression in human breast cancer biopsies, and demonstrate that ethyl 3,4-dihydroxybenzoate, a prolyl hydroxylase inhibitor, decreases tumor fibrosis and metastasis in a mouse model of breast cancer. PMID:23539444

  4. Interaction of pleuromutilin derivatives with the ribosomal peptidyl transferase center.

    PubMed

    Long, Katherine S; Hansen, Lykke H; Jakobsen, Lene; Vester, Birte

    2006-04-01

    Tiamulin is a pleuromutilin antibiotic that is used in veterinary medicine. The recently published crystal structure of a tiamulin-50S ribosomal subunit complex provides detailed information about how this drug targets the peptidyl transferase center of the ribosome. To promote rational design of pleuromutilin-based drugs, the binding of the antibiotic pleuromutilin and three semisynthetic derivatives with different side chain extensions has been investigated using chemical footprinting. The nucleotides A2058, A2059, G2505, and U2506 are affected in all of the footprints, suggesting that the drugs are similarly anchored in the binding pocket by the common tricyclic mutilin core. However, varying effects are observed at U2584 and U2585, indicating that the side chain extensions adopt distinct conformations within the cavity and thereby affect the rRNA conformation differently. An Escherichia coli L3 mutant strain is resistant to tiamulin and pleuromutilin, but not valnemulin, implying that valnemulin is better able to withstand an altered rRNA binding surface around the mutilin core. This is likely due to additional interactions made between the valnemulin side chain extension and the rRNA binding site. The data suggest that pleuromutilin drugs with enhanced antimicrobial activity may be obtained by maximizing the number of interactions between the side chain moiety and the peptidyl transferase cavity.

  5. Interaction of Pleuromutilin Derivatives with the Ribosomal Peptidyl Transferase Center

    PubMed Central

    Long, Katherine S.; Hansen, Lykke H.; Jakobsen, Lene; Vester, Birte

    2006-01-01

    Tiamulin is a pleuromutilin antibiotic that is used in veterinary medicine. The recently published crystal structure of a tiamulin-50S ribosomal subunit complex provides detailed information about how this drug targets the peptidyl transferase center of the ribosome. To promote rational design of pleuromutilin-based drugs, the binding of the antibiotic pleuromutilin and three semisynthetic derivatives with different side chain extensions has been investigated using chemical footprinting. The nucleotides A2058, A2059, G2505, and U2506 are affected in all of the footprints, suggesting that the drugs are similarly anchored in the binding pocket by the common tricyclic mutilin core. However, varying effects are observed at U2584 and U2585, indicating that the side chain extensions adopt distinct conformations within the cavity and thereby affect the rRNA conformation differently. An Escherichia coli L3 mutant strain is resistant to tiamulin and pleuromutilin, but not valnemulin, implying that valnemulin is better able to withstand an altered rRNA binding surface around the mutilin core. This is likely due to additional interactions made between the valnemulin side chain extension and the rRNA binding site. The data suggest that pleuromutilin drugs with enhanced antimicrobial activity may be obtained by maximizing the number of interactions between the side chain moiety and the peptidyl transferase cavity. PMID:16569865

  6. Prolyl 4-Hydroxylase: Substrate Isosteres in Which an (E)- or (Z)-Alkene Replaces the Prolyl Peptide Bond.

    PubMed

    Vasta, James D; Choudhary, Amit; Jensen, Katrina H; McGrath, Nicholas A; Raines, Ronald T

    2017-01-10

    Collagen prolyl 4-hydroxylases (CP4Hs) catalyze a prevalent posttranslational modification, the hydroxylation of (2S)-proline residues in protocollagen strands. The ensuing (2S,4R)-4-hydroxyproline residues are necessary for the conformational stability of the collagen triple helix. Prolyl peptide bonds isomerize between cis and trans isomers, and the preference of the enzyme is unknown. We synthesized alkene isosteres of the cis and trans isomers to probe the conformational preferences of human CP4H1. We discovered that the presence of a prolyl peptide bond is necessary for catalysis. The cis isostere is, however, an inhibitor with a potency greater than that of the trans isostere, suggesting that the cis conformation of a prolyl peptide bond is recognized preferentially. Comparative studies with a Chlamydomonas reinhardtii P4H, which has a similar catalytic domain but lacks an N-terminal substrate-binding domain, showed a similar preference for the cis isostere. These findings support the hypothesis that the catalytic domain of CP4Hs recognizes the cis conformation of the prolyl peptide bond and inform the use of alkenes as isosteres for peptide bonds.

  7. 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.

  8. Pushing the Limits of a Molecular Mechanics Force Field To Probe Weak CH···π Interactions in Proteins.

    PubMed

    Barman, Arghya; Batiste, Bruce; Hamelberg, Donald

    2015-04-14

    The relationship among biomolecular structure, dynamics, and function is far from being understood, and the role of subtle, weak interactions in stabilizing different conformational states is even less well-known. The cumulative effect of these interactions has broad implications for biomolecular stability and recognition and determines the equilibrium distribution of the ensemble of conformations that are critical for function. Here, we accurately capture the stabilizing effects of weak CH···π interaction using an empirical molecular mechanics force field in excellent agreement with experiments. We show that the side chain of flanking C-terminal aromatic residues preferentially stabilize the cis isomer of the peptidyl-prolyl bond of the protein backbone through this weak interaction. Cis-trans isomerization of peptidyl-prolyl protein bond plays a pivotal role in many cellular processes, including signal transduction, substrate recognition, and many diseases. Although the cis isomer is relatively less stable than the trans isomer, aromatic side chains of neighboring residues can play a significant role in stabilizing the cis relative to the trans isomer. We carry out extensive regular and accelerated molecular dynamics simulations and establish an approach to simulate the pH profile of the cis/trans ratio in order to probe the stabilizing role of the CH···π interaction. The results agree very well with NMR experiments, provide detailed atomistic description of this crucial biomolecular interaction, and underscore the importance of weak stabilizing interactions in protein function.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. Chemical logic and enzymatic machinery for biological assembly of peptidyl nucleoside antibiotics.

    PubMed

    Walsh, Christopher T; Zhang, Wenjun

    2011-10-21

    Peptidyl nucleoside antibiotics are a group of natural products targeting MraY, a bacterial translocase involved in the lipid-linked cycle in peptidoglycan biosynthesis. In this Perspective, we explore how Nature builds complex peptidyl nucleoside antibiotics scaffolds from simple nucleoside and amino acid building blocks. We discuss the current stage of research on biosynthetic pathways for peptidyl nucleoside antibiotics, primarily focusing on chemical logic and enzymatic machinery for uridine transformation and coupling to peptides. We further survey the nonribosomal biosynthetic paradigm for a subgroup of uridyl peptide antibiotics represented by pacidamycins, concluded by diversification opportunities for antibiotic optimization.

  15. Chemical Logic and Enzymatic Machinery for Biological Assembly of Peptidyl Nucleoside Antibiotics

    PubMed Central

    Walsh, Christopher T.; Zhang, Wenjun

    2011-01-01

    Peptidyl nucleoside antibiotics are a group of natural products targeting MraY, a bacterial translocase involved in the lipid-linked cycle in peptidoglycan biosynthesis. In this Perspective, we explore how Nature builds complex peptidyl nucleoside antibiotics scaffolds from simple nucleoside and amino acid building blocks. We discuss the current stage of research on biosynthetic pathways for peptidyl nucleoside antibiotics, primarily focusing on chemical logic and enzymatic machinery for uridine transformation and coupling to peptides. We further survey the nonribosomal biosynthetic paradigm for a subgroup of uridyl peptide antibiotics represented by pacidamycins, concluded by diversification opportunities for antibiotic optimization. PMID:21851099

  16. Inhibition of serine proteases by peptidyl fluoromethyl ketones

    SciTech Connect

    Imperiali, B.; Abeles, R.H.

    1986-07-01

    Peptidyl fluoromethyl ketones that are specific inhibitors of the serine proteases ..cap alpha..-chymotrypsin and porcine pancreatic elastase were synthesized. By analogy with the corresponding aldehydes it is assumed that the fluoromethyl ketones react with the ..gamma..-OH group of the active site serine to form a stable hemiacetal. /sup 19/F NMR studies of the chymotrypsin-bound trifluoromethyl ketone inhibitors Ac-Leu-ambo-Phe-CF/sub 3//sup 1/ and Ac-ambo-Phe-CF/sub 3/ clearly indicate that the carbonyl carbon is tetrahedral at the active site of the enzyme. The inhibitor is bound as either the stable hydrat or the hemiacetal, involving the active site serine. The effect of varying the number of amino acid residues in the peptidyl portion of the inhibitor and the number of fluorines in the fluoromethyl ketone moiety is examined. In the series of trifluoromethyl ketone elastase inhibitors, the lowering of K/sub i/ concomitant with the change from a dipeptide analogue to a tetrapeptide analogue correlates well with the variation in V/K for hydrolysis of the corresponding amide substrates. This trend is indicative of the inhibitors acting as transition-state analogues. In addition to chain length, the number of fluorine substituents also affects the K/sub i/. In the case of chymotrypsin, the K/sub i/ for Ac-Leu-ambo-Phe-CF/sub 3/ is 30-fold lower than that for Ac-Leu-ambo-Phe-CF/sub 2/H. With elastase this trend is not as profound. In all cases, however, the difluoro- and trifluoromethyl ketones are better inhibitors than the monofluoromethyl and nonfluorinated analogues. This improvement must be associated with both the degree of hydration of the fluoromethyl ketones and the significant effect that fluorine substitution has on lowering the first pK/sub a/ of the hemiacetal hydroxyl. The monofluoromethyl ketone inhibitor of chymotrypsin, Ac-Leu-ambo-Phe-CFH/sub 2/, is a weak competitive inhibitor.

  17. Synthesis and characterization of the novel fluorescent prolyl oligopeptidase inhibitor 4-fluoresceinthiocarbamoyl-6-aminocaproyl-L-prolyl-2(S)-(hydroxyacetyl)pyrrolidine.

    PubMed

    Venäläinen, Jarkko I; Wallén, Erik A A; Poso, Antti; García-Horsman, J Arturo; Männistö, Pekka T

    2005-11-17

    The synthesis and characterization of the first fluorescent prolyl oligopeptidase inhibitor 4-fluoresceinthiocarbamoyl-6-aminocaproyl-L-prolyl-2(S)-(hydroxyacetyl)pyrrolidine is described. This compound has an IC50 value of 0.83 nM and a dissociation half-life of 160 min, and its fluorescence signal is detectable using standard filters for fluorescein. These properties make this compound a suitable probe for visualizing prolyl oligopeptidase in various applications.

  18. A digestive prolyl carboxypeptidase in Tenebrio molitor larvae

    USDA-ARS?s Scientific Manuscript database

    Prolyl carboxypeptidase (PRCP) was purified from the anterior midgut of larvae of a stored product pest, Tenebrio molitor. The cDNA of PRCP was cloned, and the predicted protein was identical to the proteomic sequences of the purified enzyme. The substrate specificity of the enzyme was studied, and ...

  19. Prolyl-specific peptidases for applications in food protein hydrolysis.

    PubMed

    Mika, Nicole; Zorn, Holger; Rühl, Martin

    2015-10-01

    Various food proteins including, e.g. gluten, collagen and casein are rich in L-proline residues. Due to the cyclic structure of proline, these proteins are well protected from enzymatic degradation by typical digestive enzymes. Proline-specific peptidases (PsP) belong to different families of hydrolases acting on peptide bonds (EC 3.4.x.x). They occur in various organisms including bacteria, fungi, plants and insects. Based on their biochemical characteristics, PsP type enzymes are further grouped into different subclasses of which prolyl aminopeptidases (EC 3.4.11.5, PAP), prolyl carboxypeptidases (EC 3.4.17.16, PCP) and prolyl oligopeptidases/prolyl endopeptidases (EC 3.4.21.26, POP/PEP) are of major interest for applications in food biotechnology. This mini review summarises the biochemical assays employed for these subclasses of PsP and their structural properties and the reaction mechanisms. A special focus was set on PsP derived from fungi and insects and important industrial applications in the field of food biotechnology. The degradation of gluten and collagen as well as the hydrolysis of bitter peptides are discussed.

  20. Translational bypassing without peptidyl-tRNA anticodon scanning of coding gap mRNA

    PubMed Central

    Wills, Norma M; O'Connor, Michelle; Nelson, Chad C; Rettberg, Charles C; Huang, Wai Mun; Gesteland, Raymond F; Atkins, John F

    2008-01-01

    Half the ribosomes translating the mRNA for phage T4 gene 60 topoisomerase subunit bypass a 50 nucleotide coding gap between codons 46 and 47. The pairing of codon 46 with its cognate peptidyl-tRNA anticodon dissociates, and following mRNA slippage, peptidyl-tRNA re-pairs to mRNA at a matched triplet 5′ adjacent to codon 47, where translation resumes. Here, in studies with gene 60 cassettes, it is shown that the peptidyl-tRNA anticodon does not scan the intervening sequence for potential complementarity. However, certain coding gap mutants allow peptidyl-tRNA to scan sequences in the bypassed segment. A model is proposed in which the coding gap mRNA enters the ribosomal A-site and forms a structure that precludes peptidyl-tRNA scanning of its sequence. Dissipation of this RNA structure, together with the contribution of 16S rRNA anti-Shine–Dalgarno sequence pairing with GAG, facilitates peptidyl-tRNA re-pairing to mRNA. PMID:18772887

  1. 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.

  2. 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.

  3. Loss of intramolecular electrostatic interactions and limited conformational ensemble may promote self-association of cis-tau peptide.

    PubMed

    Barman, Arghya; Hamelberg, Donald

    2015-03-01

    Self-association of proteins can be triggered by a change in the distribution of the conformational ensemble. Posttranslational modification, such as phosphorylation, can induce a shift in the ensemble of conformations. In the brain of Alzheimer's disease patients, the formation of intra-cellular neurofibrillary tangles deposition is a result of self-aggregation of hyper-phosphorylated tau protein. Biochemical and NMR studies suggest that the cis peptidyl prolyl conformation of a phosphorylated threonine-proline motif in the tau protein renders tau more prone to aggregation than the trans isomer. However, little is known about the role of peptidyl prolyl cis/trans isomerization in tau aggregation. Here, we show that intra-molecular electrostatic interactions are better formed in the trans isomer. We explore the conformational landscape of the tau segment containing the phosphorylated-Thr(231)-Pro(232) motif using accelerated molecular dynamics and show that intra-molecular electrostatic interactions are coupled to the isomeric state of the peptidyl prolyl bond. Our results suggest that the loss of intra-molecular interactions and the more restricted conformational ensemble of the cis isomer could favor self-aggregation. The results are consistent with experiments, providing valuable complementary atomistic insights and a hypothetical model for isomer specific aggregation of the tau protein.

  4. Analysis of aminoacyl- and peptidyl-tRNAs by gel electrophoresis

    PubMed Central

    Janssen, Brian D.; Diner, Elie J.; Hayes, Christopher S.

    2013-01-01

    During protein synthesis, the ribosome translates the genetic information encoded within messenger RNAs into defined amino acid sequences. Transfer RNAs (tRNAs) are crucial adaptor molecules in this process, delivering amino acid residues to the ribosome and holding the nascent peptide chain as it is assembled. Here, we present methods for the analysis of aminoacyl- and peptidyl-tRNA species isolated from Escherichia coli. These approaches utilize denaturing gel electrophoresis at acidic pH to preserve the labile ester bonds that link amino acids to tRNA. Specific aminoacyl- and peptidyl-tRNAs are detected by Northern blot hybridization using probes for tRNA isoacceptors. Small peptidyl-tRNAs can be differentiated from aminoacyl-tRNA through selective deacylation of the latter with copper sulfate. Additionally, peptidyl-tRNAs can be detected through metabolic labeling of the nascent peptide. This latter approach is amenable to pulse-chase analysis to examine peptidyl-tRNA turnover in vivo. We have applied these methods to study programmed translational arrests and the kinetics of paused ribosome turnover. PMID:22736012

  5. Identification of proteins at the peptidyl-tRNA binding site of rat liver ribosomes.

    PubMed

    Fabijanski, S; Pellegrini, M

    1981-01-01

    We have identified proteins involved in the peptidyl-tRNA-binding site of rat liver ribosomes, using an affinity label designed specifically to probe the P-site in eukaryotic peptidyl transferase. The label is a 3'-terminal pentanucleotide fragment of N-acetylleucyl-tRNA in which mercury atoms have been added at the C-5 position of the three cytosine residues. This mercurated fragment can bind to rat liver peptidyl transferase and function as a donor of N-acetylleucine to puromycin. Concomitant with this binding, the mercury atoms present in the fragment can form a covalent linkage with a small number of ribosomal proteins. The major proteins labeled by this reagent are L5 and L36A. Four protein spots are found labeled to a lesser extent: L10, L7/7a, L3/4 and L25/31. Each of these proteins, therefore, is implicated in the binding of the 3'-terminus of peptidyl-tRNA. The results presented here are correlated with other investigations of the structure-function aspects of rat liver peptidyl transferase. Using these data, we have constructed a model for the arrangement of proteins within this active site.

  6. Giving anemia a boost with inhibitors of prolyl hydroxylase.

    PubMed

    Denny, William A

    2012-04-12

    There is much current interest in the development of inhibitors of the prolyl hydroxylase (PHD) enzymes that regulate the hypoxia-inducible transcription factor (HIF), which in turn stimulates the production of erythropoietin and ultimately red blood cells, as a treatment for anemia. A recent paper reports the synthesis and evaluation of a novel class of potent spirohydantoin-based pan-PHD inhibitors for this purpose. The paper is an exemplar of drug development from high-throughput screen to clinical candidate.

  7. Targeting Prolyl Peptidases in Triple-Negative Breast Cancer

    DTIC Science & Technology

    2017-02-01

    cell survival. We identified a protein called PRCP (prolylcarboxypeptidase) that promotes metastasis and survival in breast cancer cells . We found...PRCP/PREP inhibition reduces IRS1 and IRS2 protein levels, blocks proliferation, and induces death in multiple TNBC cell lines of different sub-types...SUBJECT TERMS Triple negative breast cancer, Prolyl peptidases, Breast cancer treatment, Animal model 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF

  8. 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.

  9. 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.

  10. Prolyl hydroxylase domain enzymes: important regulators of cancer metabolism

    PubMed Central

    Yang, Ming; Su, Huizhong; Soga, Tomoyoshi; Kranc, Kamil R; Pollard, Patrick J

    2014-01-01

    The hypoxia-inducible factor (HIF) prolyl hydroxylase domain enzymes (PHDs) regulate the stability of HIF protein by post-translational hydroxylation of two conserved prolyl residues in its α subunit in an oxygen-dependent manner. Trans-4-prolyl hydroxylation of HIFα under normal oxygen (O2) availability enables its association with the von Hippel-Lindau (VHL) tumor suppressor pVHL E3 ligase complex, leading to the degradation of HIFα via the ubiquitin-proteasome pathway. Due to the obligatory requirement of molecular O2 as a co-substrate, the activity of PHDs is inhibited under hypoxic conditions, resulting in stabilized HIFα, which dimerizes with HIFβ and, together with transcriptional co-activators CBP/p300, activates the transcription of its target genes. As a key molecular regulator of adaptive response to hypoxia, HIF plays important roles in multiple cellular processes and its overexpression has been detected in various cancers. The HIF1α isoform in particular has a strong impact on cellular metabolism, most notably by promoting anaerobic, whilst inhibiting O2-dependent, metabolism of glucose. The PHD enzymes also seem to have HIF-independent functions and are subject to regulation by factors other than O2, such as by metabolic status, oxidative stress, and abnormal levels of endogenous metabolites (oncometabolites) that have been observed in some types of cancers. In this review, we aim to summarize current understandings of the function and regulation of PHDs in cancer with an emphasis on their roles in metabolism. PMID:27774472

  11. Transmembrane prolyl 4-hydroxylase is a fourth prolyl 4-hydroxylase regulating EPO production and erythropoiesis.

    PubMed

    Laitala, Anu; Aro, Ellinoora; Walkinshaw, Gail; Mäki, Joni M; Rossi, Maarit; Heikkilä, Minna; Savolainen, Eeva-Riitta; Arend, Michael; Kivirikko, Kari I; Koivunen, Peppi; Myllyharju, Johanna

    2012-10-18

    An endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the α subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm(-/-) mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1α and Hif-2α protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm(-/-) mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497-treated Hif-p4h-2 hypomorphic (Hif-p4h-2(gt/gt)) and Hif-p4h-3(-/-) mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm(-/-) and wild-type mice, but caused higher increases in both values in the Hif-p4h-2(gt/gt) mice and in hematocrit value in the Hif-p4h-3(-/-) mice than in the wild-type. Hif-p4h-2(gt/gt)/P4h-tm(-/-) double gene-modified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2(gt/gt) or P4h-tm(-/-) mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis.

  12. Engineering of an industrial polyoxin producer for the rational production of hybrid peptidyl nucleoside antibiotics.

    PubMed

    Zhai, Lipeng; Lin, Shuangjun; Qu, Dongjing; Hong, Xuechuan; Bai, Linquan; Chen, Wenqing; Deng, Zixin

    2012-07-01

    Polyoxins and nikkomycins are potent antifungal peptidyl nucleoside antibiotics, which inhibit fungal cell wall biosynthesis. They consist of a nucleoside core and one or two independent peptidyl moieties attached to the core at different sites. Making mutations and introducing heterologous genes into an industrial Streptomyces aureochromogenes polyoxin producer, resulted in the production of four polyoxin-nikkomycin hybrid antibiotics designated as polyoxin N and nikkoxin B-D, whose structures were confirmed using high resolution MS and NMR. Two of the hybrid antibiotics, polyoxin N and nikkoxin D, were significantly more potent against some human or plant fungal pathogens than their parents. The data provides an example for rational generation of novel peptidyl nucleoside antibiotics in an industrial producer.

  13. Ambient Temperature Synthesis of High Enantiopurity N-Protected Peptidyl Ketones by Peptidyl Thiol Ester–Boronic Acid Cross-Coupling

    PubMed Central

    Yang, Hao; Li, Hao; Wittenberg, Rüdiger; Egi, Masahiro; Huang, Wenwei; Liebeskind, Lanny S.

    2009-01-01

    α-Amino acid thiol esters derived from N-protected mono-, di-, and tripeptides couple with aryl, π-electron-rich heteroaryl, or alkenyl boronic acids in the presence of stoichiometric Cu(I) thiophene-2-carboxylate (CuTC) and catalytic Pd2(dba)3/triethylphosphite to generate the corresponding N-protected peptidyl ketones in good to excellent yields and in high enantiopurity. Triethylphosphite plays a key role as a supporting ligand by mitigating an undesired palladium-catalyzed decarbonylation-β-elimination of the α-amino thiol esters. The peptidyl ketone synthesis proceeds at room temperature under non-basic conditions and demonstrates a high tolerance to functionality. PMID:17263394

  14. Cyclophilin-promoted folding of mouse dihydrofolate reductase does not include the slow conversion of the late-folding intermediate to the active enzyme.

    PubMed

    von Ahsen, O; Lim, J H; Caspers, P; Martin, F; Schönfeld, H J; Rassow, J; Pfanner, N

    2000-03-31

    Cyclophilins accelerate slow protein folding reactions in vitro by catalyzing the cis/trans isomerization of peptidyl-prolyl bonds. Cyclophilins were reported to be involved in a variety of cellular functions, including the promotion of protein folding by use of the substrate mouse dihydrofolate reductase (DHFR). The interaction of cyclophilin with DHFR has only been studied under limited conditions so far, not taking into account that native DHFR exists in equilibrium with a non-native late-folding intermediate. Here we report a systematic analysis of catalysis of DHFR folding by cyclophilins. The specific ligand methotrexate traps DHFR in its native state, permitting a specific analysis of the action of cyclophilin on both denatured DHFR with non-native prolyl bonds and denatured DHFR with all-native prolyl bonds. Cyclophilins from yeast and Neurospora crassa as well as the related prolyl isomerase b from Escherichia coli promote the folding of different forms of DHFR to the enzymatically active form, demonstrating the generality of cyclophilin-catalyzed folding of DHFR. The slow equilibrium between the late-folding intermediate and native DHFR suggests that prolyl isomerization may be required for this final phase of conversion to native DHFR. However, by reversible trapping of the intermediate, we analyze the slow interconversion between native and late-folding conformations in the backward and forward reactions and show a complete independence of cyclophilin. We conclude that cyclophilin catalyzes folding of DHFR, but surprisingly not in the last slow folding step. Copyright 2000 Academic Press.

  15. Peptidyl aldehydes as slow-binding inhibitors of dual-specificity phosphatases.

    PubMed

    Park, Junguk; Fu, Hua; Pei, Dehua

    2004-02-09

    Peptidyl aldehydes were tested for inhibition of dual-specificity phosphatases VH1 and VHR. The most potent compound, cinnamaldehyde-Gly-Glu-Glu (Cinn-GEE), acted as a slow-binding inhibitor with K(I)* values of 18 and 288 microM against VH1 and VHR, respectively.

  16. Peptidyl transferase centre of bacterial ribosomes: substrate specificity and binding sites.

    PubMed Central

    Krayevsky, A A; Kukhanova, M K; Gottikh, B P

    1975-01-01

    A detailed scheme of the Peptidyl Transferase Centre of bacterial ribosomes is proposed by summarizing the literature data on the substrate specificity of the acceptor and donor sites. According to the proposed scheme only the elements of the donor and acceptor having a stable structure bind with the ribosome. The present paper proposes such main elements for the donor and acceptor. PMID:802510

  17. Biological role of prolyl 3-hydroxylation in type IV collagen.

    PubMed

    Pokidysheva, Elena; Boudko, Sergei; Vranka, Janice; Zientek, Keith; Maddox, Kerry; Moser, Markus; Fässler, Reinhard; Ware, Jerry; Bächinger, Hans Peter

    2014-01-07

    Collagens constitute nearly 30% of all proteins in our body. Type IV collagen is a major and crucial component of basement membranes. Collagen chains undergo several posttranslational modifications that are indispensable for proper collagen function. One of these modifications, prolyl 3-hydroxylation, is accomplished by a family of prolyl 3-hydroxylases (P3H1, P3H2, and P3H3). The present study shows that P3H2-null mice are embryonic-lethal by embryonic day 8.5. The mechanism of the unexpectedly early lethality involves the interaction of non-3-hydroxylated embryonic type IV collagen with the maternal platelet-specific glycoprotein VI (GPVI). This interaction results in maternal platelet aggregation, thrombosis of the maternal blood, and death of the embryo. The phenotype is completely rescued by producing double KOs of P3H2 and GPVI. Double nulls are viable and fertile. Under normal conditions, subendothelial collagens bear the GPVI-binding sites that initiate platelet aggregation upon blood exposure during injuries. In type IV collagen, these sites are normally 3-hydroxylated. Thus, prolyl 3-hydroxylation of type IV collagen has an important function preventing maternal platelet aggregation in response to the early developing embryo. A unique link between blood coagulation and the ECM is established. The newly described mechanism may elucidate some unexplained fetal losses in humans, where thrombosis is often observed at the maternal/fetal interface. Moreover, epigenetic silencing of P3H2 in breast cancers implies that the interaction between GPVI and non-3-hydroxylated type IV collagen might also play a role in the progression of malignant tumors and metastasis.

  18. Isoform-specific Inhibition of Cyclophilins

    PubMed Central

    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

    2009-01-01

    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. PMID:19480458

  19. Characterization of two carnation petal prolyl 4 hydroxylases.

    PubMed

    Vlad, Florina; Tiainen, Päivi; Owen, Carolyn; Spano, Thodhoraq; Daher, Firas Bou; Oualid, Fatiha; Senol, Namik Ozer; Vlad, Daniela; Myllyharju, Johanna; Kalaitzis, Panagiotis

    2010-10-01

    Prolyl 4-hydroxylases (P4Hs) catalyze the proline hydroxylation, a major post-translational modification, of hydroxyproline-rich glycoproteins. Two carnation petal P4H cDNAs, (Dianthus caryophyllus prolyl 4-hydroxylase) DcP4H1 and DcP4H2, were identified and characterized at the gene expression and biochemical level in order to investigate their role in flower senescence. Both mRNAs showed similar patterns of expression with stable transcript abundance during senescence progression and differential tissue-specific expression with DcP4H1 and DcP4H2 strongly expressed in ovaries and stems, respectively. Recombinant DcP4H1 and DcP4H2 proteins were produced and their catalytic properties were determined. Pyridine 2,4-dicarboxylate (PDCA) was identified as a potent inhibitor of the in vitro enzyme activity of both P4Hs and used to determine whether inhibition of proline hydroxylation in petals is involved in senescence progression of cut carnation flowers. PDCA suppressed the climacteric ethylene production indicating a strong correlation between the inhibition of DcP4H1 and DcP4H2 activity in vitro by PDCA and the suppression of climacteric ethylene production in cut carnation flowers.

  20. Evolutionary relationships of the prolyl oligopeptidase family enzymes.

    PubMed

    Venäläinen, Jarkko I; Juvonen, Risto O; Männistö, Pekka T

    2004-07-01

    The prolyl oligopeptidase (POP) family of serine proteases includes prolyl oligopeptidase, dipeptidyl peptidase IV, acylaminoacyl peptidase and oligopeptidase B. The enzymes of this family specifically hydrolyze oligopeptides with less than 30 amino acids. Many of the POP family enzymes have evoked pharmaceutical interest as they have roles in the regulation of peptide hormones and are involved in a variety of diseases such as dementia, trypanosomiasis and type 2 diabetes. In this study we have clarified the evolutionary relationships of these four POP family enzymes and analyzed POP sequences from different sources. The phylogenetic trees indicate that the four enzymes were present in the last common ancestor of all life forms and that the beta-propeller domain has been part of the family for billions of years. There are striking differences in the mutation rates between the enzymes and POP was found to be the most conserved enzyme of this family. However, the localization of this enzyme has changed throughout evolution, as three archaeal POPs seem to be membrane bound and one third of the bacterial as well as two eukaryotic POPs were found to be secreted out of the cell. There are also considerable distinctions between the mutation rates of the different substrate binding subsites of POP. This information may help in the development of species-specific POP inhibitors.

  1. Chemistry of coordinated nitroxyl. Reagent-specific protonations of trans-Re(CO)(2)(NO)(PR(3))(2) (R = Ph, Cy) that give the neutral nitroxyl complexes cis,trans-ReCl(CO)(2)(NH=O)(PR(3))(2) or the cationic hydride complex [trans,trans-ReH(CO)(2)(NO)(PPh(3))(2)(+)][SO(3)CF(3)(-)].

    PubMed

    Southern, J S; Green, M T; Hillhouse, G L; Guzei, I A; Rheingold, A L

    2001-11-05

    The reactions of hydrochloric and triflic acids with the five-coordinate nitrosyl complexes trans-Re(CO)(2)(NO)(PR(3))(2) (2a, R = Ph; 2b, R = Cy) have been investigated. Reaction of anhydrous HCl with 2 results in a formal protonation of the nitrosyl ligand and addition of chloride to the metal, giving the neutral nitroxyl complex cis,trans-ReCl(CO)(2)(NH=O)(PR(3))(2) (3a, R = Ph; 3b, R = Cy). Reaction of Brønsted bases with 3a or 3b results in clean conversion of 3 to 2 when the base is appropriately strong (pK(b) approximately 7). Addition of HOSO(2)CF(3) to solutions of 2a results in protonation at the metal and formation of the cationic rhenium hydride [trans,trans-ReH(CO)(2)(NO)(PPh(3))(2)(+)][SO(3)CF(3)(-)] (4) in 74% yield; the deuteride [trans,trans-Re((2)H)(CO)(2)(NO)(PPh(3))(2)(+)][SO(3)CF(3)(-)] (4-d) was analogously prepared from (2)HOSO(2)CF(3). 4 crystallized from CH(2)Cl(2)/Et(2)O solution in the orthorhombic space group Pnma, with a = 17.2201(2) A, b = 23.6119(3) A, c = 9.2380(2) A, and Z = 4. The least-squares refinement converged to R(F) = 0.039 and R(wF(2)()) = 0.063 for the 4330 unique data with I > 2 sigma(I). The structure of 4 shows that the hydride (Re-H = 1.74 A) occupies the position trans to the linear nitrosyl ligand (Re-N-O = 178.1(4) degrees ) in the pseudooctahedral complex cation. Complex 4 does not react with chloride to give 3a. DFT calculations carried out on free nitroxyl and its model complexes [Re(CO)(5)(NH=O)(+)] (5), [mer,trans-Re(CO)(3)(NH=O)(PH(3))(2)(+)] (6), and cis,trans-ReCl(CO)(2)(NH=O)(PH(3))(2) (7) indicate that coordinated nitroxyl acts as both a sigma-donor and pi-acceptor ligand, consistent with the observed trend for nu(NO) in free HN=O (1563 cm(-1)), [mer,trans-Re(CO)(3)(NH=O)(PPh(3))(2)(+)] (1, 1391 cm(-1)), 3a (1376 cm(-1)), and 3b (1335 cm(-1)).

  2. EF4 disengages the peptidyl-tRNA CCA end and facilitates back-translocation on the 70S ribosome.

    PubMed

    Zhang, Dejiu; Yan, Kaige; Liu, Guangqiao; Song, Guangtao; Luo, Jiejian; Shi, Yi; Cheng, Erchao; Wu, Shan; Jiang, Taijiao; Lou, Jizhong; Gao, Ning; Qin, Yan

    2016-02-01

    EF4 catalyzes tRNA back-translocation through an unknown mechanism. We report cryo-EM structures of Escherichia coli EF4 in post- and pretranslocational ribosomes (Post- and Pre-EF4) at 3.7- and 3.2-Å resolution, respectively. In Post-EF4, peptidyl-tRNA occupies the peptidyl (P) site, but the interaction between its CCA end and the P loop is disrupted. In Pre-EF4, the peptidyl-tRNA assumes a unique position near the aminoacyl (A) site, denoted the A site/EF4 bound (A/4) site, with a large displacement at its acceptor arm. Mutagenesis analyses suggest that a specific region in the EF4 C-terminal domain (CTD) interferes with base-pairing between the peptidyl-tRNA 3'-CCA and the P loop, whereas the EF4 CTD enhances peptidyl-tRNA interaction at the A/4 site. Therefore, EF4 induces back-translocation by disengaging the tRNA's CCA end from the peptidyl transferase center of the translating ribosome.

  3. Erythromycin, carbomycin, and spiramycin inhibit protein synthesis by stimulating the dissociation of peptidyl-tRNA from ribosomes.

    PubMed

    Menninger, J R; Otto, D P

    1982-05-01

    In mutant Escherichia coli with temperature-sensitive peptidyl-tRNA hydrolase (aminoacyl-tRNA hydrolase; EC 3.1.1.29), peptidyl-tRNA accumulates at the nonpermissive temperature (40 degrees C), and the cells die. These consequences of high temperature were enhanced if the cells were first treated with erythromycin, carbomycin, or spiramycin at doses sufficient to inhibit protein synthesis in wild-type cells but not sufficient to kill either mutant or wild-type cells at the permissive temperature (30 degrees C). Since peptidyl-tRNA hydrolase in he mutant cells is inactivated rapidly and irreversibly at 40 degrees C, the enhanced accumulation of peptidyl-tRNA and killing were the result of enhanced dissociation, stimulated by the antibiotics, of peptidyl-tRNA from ribosomes. The implications of these findings for inhibition of cell growth and protein synthesis are discussed. Certain alternative interpretations are shown to be inconsistent with the relevant data. Previous conflicting observations on the effects of macrolide antibiotics are explained in terms of our observations. We conclude that erythromycin, carbomycin, and spiramycin (and probably all macrolides) have as a primary mechanism of action the stimulation of dissociation of peptidyl-tRNA from ribosomes, probably during translocation.

  4. Erythromycin, carbomycin, and spiramycin inhibit protein synthesis by stimulating the dissociation of peptidyl-tRNA from ribosomes.

    PubMed Central

    Menninger, J R; Otto, D P

    1982-01-01

    In mutant Escherichia coli with temperature-sensitive peptidyl-tRNA hydrolase (aminoacyl-tRNA hydrolase; EC 3.1.1.29), peptidyl-tRNA accumulates at the nonpermissive temperature (40 degrees C), and the cells die. These consequences of high temperature were enhanced if the cells were first treated with erythromycin, carbomycin, or spiramycin at doses sufficient to inhibit protein synthesis in wild-type cells but not sufficient to kill either mutant or wild-type cells at the permissive temperature (30 degrees C). Since peptidyl-tRNA hydrolase in he mutant cells is inactivated rapidly and irreversibly at 40 degrees C, the enhanced accumulation of peptidyl-tRNA and killing were the result of enhanced dissociation, stimulated by the antibiotics, of peptidyl-tRNA from ribosomes. The implications of these findings for inhibition of cell growth and protein synthesis are discussed. Certain alternative interpretations are shown to be inconsistent with the relevant data. Previous conflicting observations on the effects of macrolide antibiotics are explained in terms of our observations. We conclude that erythromycin, carbomycin, and spiramycin (and probably all macrolides) have as a primary mechanism of action the stimulation of dissociation of peptidyl-tRNA from ribosomes, probably during translocation. PMID:6179465

  5. Conformationally constrained analogues of L-prolyl-l-leucylglycinamide

    SciTech Connect

    Yu, K.L.

    1986-01-01

    The tripeptide, L-prolyl-L-leucylglycinamide (PLG), has been shown to modulate the pharmacological response of the neurotransmitter, dopamine, in the central nervous system. Many physical studies have suggested that PLG can exist in a type II ..beta..-bend conformation. In this study several types of conformationally constrained analogues capable of mimicking different types of conformations of PLG have been designed to answer two questions: (1) Is the type II ..beta..-bend the bioactive conformation of PLG. (2) Does the Leu-Gly amide bond of PLG need to be in a cis or trans configuration in order for it to bind to its receptor. The analogues of PLG that have been synthesized include the following: (1) Lactam analogues, (2) Cyclic peptides, (3) Olefinic analogue, and (4) Tetazole analogues. The analogues synthesized were tested in a (/sup 3/H)-ADTN binding assay to determine their ability to enhance the binding of this dopamine agonist to dopamine receptors.

  6. Prolyl carboxypeptidase mRNA expression in the mouse brain.

    PubMed

    Jeong, Jin Kwon; Diano, Sabrina

    2014-01-13

    Prolyl carboxypeptidase (PRCP), a serine protease, is widely expressed in the body including liver, lung, kidney and brain, with a variety of known substrates such as plasma prekallikrein, bradykinin, angiotensins II and III, and α-MSH, suggesting its role in the processing of tissue-specific substrates. In the brain, PRCP has been shown to inactivate hypothalamic α-MSH, thus modulating melanocortin signaling in the control of energy metabolism. While its expression pattern has been reported in the hypothalamus, little is known on the distribution of PRCP throughout the mouse brain. This study was undertaken to determine PRCP expression in the mouse brain. Radioactive in situ hybridization was performed to determine endogenous PRCP mRNA expression. In addition, using a gene-trap mouse model for PRCP deletion, X-gal staining was performed to further determine PRCP distribution. Results from both approaches showed that PRCP gene is broadly expressed in the brain.

  7. Bioavailable affinity label for collagen prolyl 4-hydroxylase

    PubMed Central

    Vasta, James D.; Higgin, Joshua J.; Kersteen, Elizabeth A.

    2013-01-01

    Collagen is the most abundant protein in animals. Its prevalent 4-hydroxyproline residues contribute greatly to its conformational stability. The hydroxyl groups arise from a post-translational modification catalyzed by the non-heme iron-dependent enzyme, collagen prolyl 4-hydroxylase (P4H). Here, we report that 4-oxo-5,6-epoxyhexanoate, a mimic of the α-ketoglutarate co-substrate, inactivates human P4H. The inactivation installs a ketone functionality in P4H, providing a handle for proteomic experiments. Caenorhabditis elegans exposed to the esterified epoxy ketone displays the phenotype of a worm lacking P4H. Thus, this affinity label can be used to mediate collagen stability in an animal, as is desirable in the treatment of a variety of fibrotic diseases. PMID:23702396

  8. Bone matrix hypermineralization in prolyl-3 hydroxylase 1 deficient mice.

    PubMed

    Fratzl-Zelman, Nadja; Bächinger, Hans-Peter; Vranka, Janice A; Roschger, Paul; Klaushofer, Klaus; Rauch, Frank

    2016-04-01

    Lack of prolyl 3-hydroxylase 1 (P3H1) due to mutations in P3H1 results in severe forms of recessive osteogenesis imperfecta. In the present study, we investigated the bone tissue characteristics of P3H1 null mice. Histomorphometric analyses of cancellous bone in the proximal tibia and lumbar vertebra in 1-month and 3-month old mice demonstrated that P3H1 deficient mice had low trabecular bone volume and low mineral apposition rate, but normal osteoid maturation time and normal osteoblast and osteoclast surfaces. Quantitative backscattered electron imaging revealed that the bone mineralization density distribution was shifted towards higher values, indicating hypermineralization of bone matrix. It thus appears that P3H1 deficiency leads to decreased deposition of extracellular matrix by osteoblasts and increased incorporation of mineral into the matrix. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Aliphatic peptidyl hydroperoxides as a source of secondary oxidation in hydroxyl radical protein footprinting.

    PubMed

    Saladino, Jessica; Liu, Mian; Live, David; Sharp, Joshua S

    2009-06-01

    Hydroxyl radical footprinting is a technique for studying protein structure and binding that entails oxidizing a protein system of interest with diffusing hydroxyl radicals, and then measuring the amount of oxidation of each amino acid. One important issue in hydroxyl radical footprinting is limiting amino acid oxidation by secondary oxidants to prevent uncontrolled oxidation, which can cause amino acids to appear more solvent accessible than they really are. Previous work suggested that hydrogen peroxide was the major secondary oxidant of concern in hydroxyl radical footprinting experiments; however, even after elimination of all hydrogen peroxide, some secondary oxidation was still detected. Evidence is presented for the formation of peptidyl hydroperoxides as the most abundant product upon oxidation of aliphatic amino acids. Both reverse phase liquid chromatography and catalase treatment were shown to be ineffective at eliminating peptidyl hydroperoxides. The ability of these peptidyl hydroperoxides to directly oxidize methionine is demonstrated, suggesting the value of methionine amide as an in situ protectant. Hydroxyl radical footprinting protocols require the use of an organic sulfide or similar peroxide scavenger in addition to removal of hydrogen peroxide to successfully eradicate all secondary oxidizing species and prevent uncontrolled oxidation of sulfur-containing residues.

  10. Oxygen isotopic substitution of peptidyl phosphates for modification-specific mass spectrometry.

    PubMed

    Shi, Yu; Yao, Xudong

    2007-11-15

    The first method of isotopic substitution of a nonbridging oxygen atom in pre-existing phosphates on peptides is reported, solving a long-standing, challenging issue in the sample preparation of phosphopeptides. Peptidyl phosphates, phosphate groups on phosphopeptides, are converted to phosphoramidates with carbodiimide assistance. Acid-catalyzed hydrolysis of the newly formed phosphoramidates incorporates one oxygen atom from H2(16)O or H2(18)O, producing peptidyl phosphates-16O1 or -18O1, respectively. The oxygen labels are stable under common separation and analysis conditions. This labeling method causes minimal structural alteration to peptidyl phosphates and allows the direct application of established phosphate-specific marker ions to the mass spectrometric analysis of differentially labeled phosphopeptide pairs. Using phosphotyrosinyl peptides as model analytes, the characteristic 16O1- and 18O1-labeled phosphotyrosine immonium ions at m/z 216.043 and 218.047 are used for developing a method of phosphopeptide quantitation that is independent of the amino acid sequence of the peptides. From analysis by tandem parallel fragmentation mass spectrometry, it is clear that the phosphate-specific marker ions authentically inherit the quantitative information from precursor phosphopeptides. The dynamic range for relative quantitation of differentially labeled phosphopeptides is at least 2 orders of magnitude for experiments run on a quadrupole time-of-flight mass spectrometer. The use of 16O1 and 18O1 labeling for counting the number of phosphate groups on peptides is also demonstrated.

  11. Structure of the prolyl-tRNA synthetase from the eukaryotic pathogen Giardia lamblia

    SciTech Connect

    Larson, Eric T.; Kim, Jessica E.; Napuli, Alberto J.; Verlinde, Christophe L. M. J.; Fan, Erkang; Zucker, Frank H.; Van Voorhis, Wesley C.; Buckner, Frederick S.; Hol, Wim G. J.; Merritt, Ethan A.

    2012-09-01

    The structure of Giardia prolyl-tRNA synthetase cocrystallized with proline and ATP shows evidence for half-of-the-sites activity, leading to a corresponding mixture of reaction substrates and product (prolyl-AMP) in the two active sites of the dimer. The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolution crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine.

  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. 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

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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

  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. 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.

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. Discovery of novel selenium derivatives as Pin1 inhibitors by high-throughput screening

    SciTech Connect

    Subedi, Amit; Shimizu, Takeshi; Ryo, Akihide; Sanada, Emiko; Watanabe, Nobumoto; Osada, Hiroyuki

    2016-06-03

    Peptidyl prolyl cis/trans isomerization by Pin1 regulates various oncogenic signals during cancer progression, and its inhibition through multiple approaches has established Pin1 as a therapeutic target. However, lack of simplified screening systems has limited the discovery of potent Pin1 inhibitors. We utilized phosphorylation-dependent binding of Pin1 to its specific substrate to develop a screening system for Pin1 inhibitors. Using this system, we screened a chemical library, and identified a novel selenium derivative as Pin1 inhibitor. Based on structure-activity guided chemical synthesis, we developed more potent Pin1 inhibitors that inhibited cancer cell proliferation. -- Highlights: •Novel screening for Pin1 inhibitors based on Pin1 binding is developed. •A novel selenium compound is discovered as Pin1 inhibitor. •Activity guided chemical synthesis of selenium derivatives resulted potent Pin1 inhibitors.

  10. 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.

  11. 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

  12. Targeting the molecular chaperone SlyD to inhibit bacterial growth with a small molecule

    PubMed Central

    Kumar, Amit; Balbach, Jochen

    2017-01-01

    Molecular chaperones are essential molecules for cell growth, whereby they maintain protein homeostasis. Because of their central cellular function, bacterial chaperones might be potential candidates for drug targets. Antimicrobial resistance is currently one of the greatest threats to human health, with gram-negative bacteria being of major concern. We found that a Cu2+ complex readily crosses the bacterial cell wall and inhibits SlyD, which is a molecular chaperone, cis/trans peptidyl prolyl isomerise (PPIase) and involved in various other metabolic pathways. The Cu2+ complex binds to the active sites of SlyD, which suppresses its PPIase and chaperone activities. Significant cell growth retardation could be observed for pathogenic bacteria (e.g., Staphylococcus aureus and Pseudomonas aeruginosa). We anticipate that rational development of drugs targeting molecular chaperones might help in future control of pathogenic bacterial growth, in an era of rapidly increasing antibiotic resistance. PMID:28176839

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. Cyclo(l-Leucyl-l-Prolyl) Produced by Achromobacter xylosoxidans Inhibits Aflatoxin Production by Aspergillus parasiticus

    PubMed Central

    Yan, Pei-Sheng; Song, Yuan; Sakuno, Emi; Nakajima, Hiromitsu; Nakagawa, Hiroyuki; Yabe, Kimiko

    2004-01-01

    Aflatoxins are potent carcinogenic and toxic substances that are produced primarily by Aspergillus flavus and Aspergillus parasiticus. We found that a bacterium remarkably inhibited production of norsolorinic acid, a precursor of aflatoxin, by A. parasiticus. This bacterium was identified as Achromobacter xylosoxidans based on its 16S ribosomal DNA sequence and was designated A. xylosoxidans NFRI-A1. A. xylosoxidans strains commonly showed similar inhibition. The inhibitory substance(s) was excreted into the medium and was stable after heat, acid, or alkaline treatment. Although the bacterium appeared to produce several inhibitory substances, we finally succeeded in purifying a major inhibitory substance from the culture medium using Diaion HP20 column chromatography, thin-layer chromatography, and high-performance liquid chromatography. The purified inhibitory substance was identified as cyclo(l-leucyl-l-prolyl) based on physicochemical methods. The 50% inhibitory concentration for aflatoxin production by A. parasiticus SYS-4 (= NRRL2999) was 0.20 mg ml−1, as determined by the tip culture method. High concentrations (more than 6.0 mg ml−1) of cyclo(l-leucyl-l-prolyl) further inhibited fungal growth. Similar inhibitory activities were observed with cyclo(d-leucyl-d-prolyl) and cyclo(l-valyl-l-prolyl), whereas cyclo(d-prolyl-l-leucyl) and cyclo(l-prolyl-d-leucyl) showed weaker activities. Reverse transcription-PCR analyses showed that cyclo(l-leucyl-l-prolyl) repressed transcription of the aflatoxin-related genes aflR, hexB, pksL1, and dmtA. This is the first report of a cyclodipeptide that affects aflatoxin production. PMID:15574949

  2. Unveiling Prolyl Oligopeptidase Ligand Migration by Comprehensive Computational Techniques

    PubMed Central

    Kotev, Martin; Lecina, Daniel; Tarragó, Teresa; Giralt, Ernest; Guallar, Víctor

    2015-01-01

    Prolyl oligopeptidase (POP) is a large 80 kDa protease, which cleaves oligopeptides at the C-terminal side of proline residues and constitutes an important pharmaceutical target. Despite the existence of several crystallographic structures, there is an open debate about migration (entrance and exit) pathways for ligands, and their coupling with protein dynamics. Recent studies have shown the capabilities of molecular dynamics and classical force fields in describing spontaneous binding events and nonbiased ligand migration pathways. Due to POP’s size and to the buried nature of its active site, an exhaustive sampling by means of conventional long enough molecular dynamics trajectories is still a nearly impossible task. Such a level of sampling, however, is possible with the breakthrough protein energy landscape exploration technique. Here, we present an exhaustive sampling of POP with a known inhibitor, Z-pro-prolinal. In >3000 trajectories Z-pro-prolinal explores all the accessible surface area, showing multiple entrance events into the large internal cavity through the pore in the β-propeller domain. Moreover, we modeled a natural substrate binding and product release by predicting the entrance of an undecapeptide substrate, followed by manual active site cleavage and nonbiased exit of one of the products (a dipeptide). The product exit shows preference from a flexible 18-amino acid residues loop, pointing to an overall mechanism where entrance and exit occur in different sites. PMID:25564858

  3. Prolyl oligopeptidase is inhibited in relapsing-remitting multiple sclerosis

    PubMed Central

    2010-01-01

    Background Multiple sclerosis (MS) is a complex, inflammatory and neurodegenerative disease of the central nervous system leading to long-term disability. Recent studies indicate a close association between inflammation and neurodegeneration in all lesions and disease stages of MS. Prolyl oligopeptidase (POP) is a proline-specific serine protease that cleaves several neuroactive peptides. This peptidase has been implicated in neurodegeneration, as well as in the modulation of the inflammatory response. Methods We examined plasma POP and the levels of an endogenous POP inhibitor from relapsing remitting MS patients and compared these with healthy controls, by monitoring the fluorescent changes due to standard fluorescently labelled substrate cleavage. We analysed the data in relationship to patient age and disease disability status. Results We observed a significant decrease in POP activity in plasma of relapsing remitting MS patients relative to healthy controls, coupled with an increase of POP endogenous inhibitor. The POP activity was also correlated with patient age and disability status. The lowered POP activity from plasma of MS patients could be rescued by reductants Conclusions The decrease in circulating POP activity measured in MS is reverted by reductants. This suggests that POP inactivation in MS might be a result of the oxidative conditions prevailing in the plasma of the diseased patients. Plasma levels of POP activity as well as those of their endogenous inhibitor are suggested as biomarkers of inflammation and oxidative stress in MS. PMID:20370893

  4. The role of HIF prolyl hydroxylases in tumour growth

    PubMed Central

    Jokilehto, Terhi; Jaakkola, Panu M

    2010-01-01

    Abstract Tumour hypoxia is a well-known microenvironmental factor that causes cancer progression and resistance to cancer treatment. This involves multiple mechanisms of which the best-understood ones are mediated through transcriptional gene activation by the hypoxia-inducible factors (HIFs). HIFs in turn are regulated in response to oxygen availability by a family of iron- and 2-oxoglutarate-dependent dioxygenases, the HIF prolyl hydroxylases (PHDs). PHDs inactivate HIFs in normoxia by activating degradation of the HIF-α subunit but release HIF activation in poorly oxygenated conditions. The function of HIF in tumours is fairly well characterized but our understanding on the outcome of PHDs in tumours is much more limited. Here we review the function of PHDs on the HIF system, the expression of PHDs in human tumours as well as their putative function in cancer. The PHDs may have either tumour promoting or suppressing activity. Their outcome in cancer depends on the cell and cancer type-specific expression and on the availability of diverse natural PHD inhibitors in tumours. Moreover, besides the action of PHDs on HIF, recent data suggest PHD function in non-HIF signalling. Together the data illustrate a complex operation of the oxygen sensors in cancer. PMID:20178464

  5. A digestive prolyl carboxypeptidase in Tenebrio molitor larvae.

    PubMed

    Goptar, Irina A; Shagin, Dmitry A; Shagina, Irina A; Mudrik, Elena S; Smirnova, Yulia A; Zhuzhikov, Dmitry P; Belozersky, Mikhail A; Dunaevsky, Yakov E; Oppert, Brenda; Filippova, Irina Yu; Elpidina, Elena N

    2013-06-01

    Prolyl carboxypeptidase (PRCP) is a lysosomal proline specific serine peptidase that also plays a vital role in the regulation of physiological processes in mammals. In this report, we isolate and characterize the first PRCP in an insect. PRCP was purified from the anterior midgut of larvae of a stored product pest, Tenebrio molitor, using a three-step chromatography strategy, and it was determined that the purified enzyme was a dimer. The cDNA of PRCP was cloned and sequenced, and the predicted protein was identical to the proteomic sequences of the purified enzyme. The substrate specificity and kinetic parameters of the enzyme were determined. The T. molitor PRCP participates in the hydrolysis of the insect's major dietary proteins, gliadins, and is the first PRCP to be ascribed a digestive function. Our collective data suggest that the evolutionary enrichment of the digestive peptidase complex in insects with an area of acidic to neutral pH in the midgut is a result of the incorporation of lysosomal peptidases, including PRCP. Published by Elsevier Ltd.

  6. Purification and Characterization of a Prolyl Aminopeptidase from Debaryomyces hansenii

    PubMed Central

    Bolumar, Tomás; Sanz, Yolanda; Aristoy, M.-Concepción; Toldrá, Fidel

    2003-01-01

    A prolyl aminopeptidase (PAP) (EC 3.4.11.5) was isolated from the cell extract of Debaryomyces hansenii CECT12487. The enzyme was purified by selective fractionation with protamine and ammonium sulfate, followed by two chromatography steps, which included gel filtration and anion-exchange chromatography. The PAP was purified 248-fold, with a recovery yield of 1.4%. The enzyme was active in a broad pH range (from 5 to 9.5), with pH and temperature optima at 7.5 and 45°C. The molecular mass was estimated to be around 370 kDa. The presence of inhibitors of serine and aspartic proteases, bestatin, puromycin, reducing agents, chelating agents, and different cations did not have any effect on the enzyme activity. Only iodoacetate, p-chloromercuribenzoic acid, and Hg2+, which are inhibitors of cysteine proteases, markedly reduced the enzyme activity. The Km for proline-7-amido-4-methylcoumarin was 40 μM. The enzyme exclusively hydrolyzed N-terminal-proline-containing substrates. This is the first report on the identification and purification of this type of aminopeptidase in yeast, which may contribute to the scarce knowledge about D. hansenii proteases and their possible roles in meat fermentation. PMID:12513999

  7. Protein engineering of improved prolyl endopeptidases for celiac sprue therapy

    PubMed Central

    Ehren, Jennifer; Govindarajan, Sridhar; Morón, Belén; Minshull, Jeremy; Khosla, Chaitan

    2008-01-01

    Due to their unique ability to cleave immunotoxic gluten peptides endoproteolytically, prolyl endopeptidases (PEPs) are attractive oral therapeutic candidates for protecting celiac sprue patients from the toxic effects of dietary gluten. Enhancing the activity and stability of PEPs under gastric conditions (low pH, high pepsin concentration) is a challenge for protein engineers. Using a combination of sequence- and structure-based approaches together with machine learning algorithms, we have identified improved variants of the Sphingomonas capsulata PEP, a target of clinical relevance. Through two rounds of iterative mutagenesis and analysis, variants with as much as 20% enhanced specific activity at pH 4.5 and 200-fold greater resistance to pepsin were identified. Our results vividly reinforce the concept that conservative changes in proteins, especially in hydrophobic residues within tightly packed regions, can profoundly influence protein structure and function in ways that are difficult to predict entirely from first principles and must therefore be optimized through iterative design and analytical cycles. Incubation with whole wheat bread under simulated gastric conditions also suggests that some variants have pharmacologically significant improvements in gluten detoxification activity. PMID:18836204

  8. Versatility of prolyl oligopeptidase B in peptide macrocyclization.

    PubMed

    Sgambelluri, Robert Michael; Smith, Miranda O; Walton, Jonathan D

    2017-09-02

    Cyclic peptides are promising compounds for new chemical biological tools and therapeutics due to their structural diversity, resistance to proteases, and membrane permeability. Amatoxins, the toxic principles of poisonous mushrooms, are biosynthesized on ribosomes as 35-mer precursor peptides which are ultimately converted to hydroxylated bicyclic octapeptides. The initial cyclization steps, catalyzed by a dedicated prolyl oligopeptidase (POPB), involves removal of the 10-amino acid leader sequence from the precursor peptide and transpeptidation to produce a monocyclic octapeptide intermediate. The utility of POPB as a general catalyst for peptide cyclization was systematically characterized using a range of precursor peptide substrates produced either in E. coli or chemically. Substrates produced in E. coli were expressed either individually or in mixtures produced by codon mutagenesis. A total of 127 novel peptide substrates were tested, of which POPB could cyclize 100. Peptides of 7 to 16 residues were cyclized at least partially. Synthetic 25mer precursor peptide substrates containing modified amino acids including D-Ala, β-Ala, N-methyl-Ala, and 4-hydroxy-Pro were also successfully cyclized. Although a phalloidin heptapeptide with all L amino acids was not cyclized, partial cyclization was seen when L-Thr at position #5 was replaced with the naturally occurring D amino acid. POPB should have broad applicability as a general catalyst for macrocyclization of peptides containing 7 to at least 16 amino acids, with an optimum of 8-9 residues.

  9. Prolyl hydroxylase 3 inhibited the tumorigenecity of gastric cancer cells.

    PubMed

    Cui, Lei; Qu, Jianguo; Dang, Shengchun; Mao, Zhengfa; Wang, Xuqing; Fan, Xin; Sun, Kang; Zhang, Jianxin

    2014-09-01

    Gastric cancer is one of the most common malignancies and the second leading cause of cancer-related death in the world, and it is very urgent to develop novel therapeutic strategies. Although HIF-1α is the most highly characterized target of prolyl hydroxylase 3 (PHD3), PHD3 has been shown to regulate several signal pathways independent of HIF-1α. Here, we found that the expression of PHD3 was decreased in the clinical gastric cancer samples and reversely correlated with tumor size and tumor stage. Over-expression of PHD3 in the gastric cancer cells significantly inhibited cell growth in vitro and in vivo, while knockdown the expression of PHD3 promoted the tumorigenecity of gastric cancer cells. Mechanistically, it showed that PHD3 downregulated the expression of beta-catenin and inhibited beta-catenin/T-cell factor (TCF) signaling. Taken together, our findings demonstrate that PHD3 inhibits gastric cancer by suppressing the beta-catenin/TCF signaling and PHD3 might be an important therapeutic target in gastric cancer.

  10. Neutron diffraction analysis of Pseudomonas aeruginosa peptidyl-tRNA hydrolase 1.

    PubMed

    McFeeters, Hana; Vandavasi, Venu Gopal; Weiss, Kevin L; Coates, Leighton; McFeeters, Robert L

    2016-03-01

    Perdeuterated peptidyl-tRNA hydrolase 1 from Pseudomonas aeruginosa was crystallized for structural analysis using neutron diffraction. Crystals of perdeuterated protein were grown to 0.15 mm(3) in size using batch crystallization in 22.5% polyethylene glycol 4000, 100 mM Tris pH 7.5, 10%(v/v) isopropyl alcohol with a 20-molar excess of trilysine as an additive. Neutron diffraction data were collected from a crystal at room temperature using the MaNDi single-crystal diffractometer at Oak Ridge National Laboratory.

  11. Photolysis of a peptide with N-peptidyl-7-nitroindoline units using two-photon absorption

    PubMed Central

    Hatch, Kevin A.; Ornelas, Alfredo; Williams, Kaitlyn N.; Boland, Thomas; Michael, Katja; Li, Chunqiang

    2016-01-01

    N-acyl-7-nitroindolines have been used as caged compounds to photorelease active molecules by a one- or two-photon excitation mechanism in biological systems. Here, we report the photolysis of a polypeptide that contains 7-nitroindoline units as linker moieties in its peptide backbone for potential materials engineering applications. Upon two-photon excitation with femtosecond laser light at 710 nm the photoreactive amide bond in N-peptidyl-7-nitroindolines is cleaved rendering short peptide fragments. Thus, this photochemical process changes the molecular composition at the laser focal volume. Gel modifications of this peptide can potentially be used for three-dimensional microstructure fabrication. PMID:27896004

  12. 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

  13. 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.

  14. Ribosome release factor RF4 and termination factor RF3 are involved in dissociation of peptidyl-tRNA from the ribosome.

    PubMed Central

    Heurgué-Hamard, V; Karimi, R; Mora, L; MacDougall, J; Leboeuf, C; Grentzmann, G; Ehrenberg, M; Buckingham, R H

    1998-01-01

    Peptidyl-tRNA dissociation from ribosomes is an energetically costly but apparently inevitable process that accompanies normal protein synthesis. The drop-off products of these events are hydrolysed by peptidyl-tRNA hydrolase. Mutant selections have been made to identify genes involved in the drop-off of peptidyl-tRNA, using a thermosensitive peptidyl-tRNA hydrolase mutant in Escherichia coli. Transposon insertions upstream of the frr gene, which encodes RF4 (ribosome release or recycling factor), restored growth to this mutant. The insertions impaired expression of the frr gene. Mutations inactivating prfC, encoding RF3 (release factor 3), displayed a similar phenotype. Conversely, production of RF4 from a plasmid increased the thermosensitivity of the peptidyl-tRNA hydrolase mutant. In vitro measurements of peptidyl-tRNA release from ribosomes paused at stop signals or sense codons confirmed that RF3 and RF4 were able to stimulate peptidyl-tRNA release from ribosomes, and showed that this action of RF4 required the presence of translocation factor EF2, known to be needed for the function of RF4 in ribosome recycling. When present together, the three factors were able to stimulate release up to 12-fold. It is suggested that RF4 may displace peptidyl-tRNA from the ribosome in a manner related to its proposed function in removing deacylated tRNA during ribosome recycling. PMID:9451005

  15. cDNA cloning of porcine brain prolyl endopeptidase and identification of the active-site seryl residue

    SciTech Connect

    Rennex, D.; Hemmings, B.A.; Hofsteenge, J.; Stone, S.R. )

    1991-02-26

    Prolyl endopeptidase is a cytoplasmic serine protease. The enzyme was purified from porcine kidney, and oligonucleotides based on peptide sequences from this protein were used to isolate a cDNA clone from a porcine brain library. This clone contained the complete coding sequence of prolyl endopeptidase and encoded a polypeptide with a molecular mass of 80751 Da. The deduced amino acid sequence of prolyl endopeptidase showed no sequence homology with other known serine proteases. ({sup 3}H)Diisopropyl fluorophosphate was used to identify the active-site serine of prolyl endopeptidase. One labeled peptide was isolated and sequenced. The sequence surrounding the active-site serine was Asn-Gly-Gly-Ser-Asn-Gly-Gly. This sequence is different from the active-site sequences of other known serine proteases. This difference and the lack of overall homology with the known families of serine proteases suggest that prolyl endopeptidase represents a new type of serine protease.

  16. Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases

    NASA Astrophysics Data System (ADS)

    Chowdhury, Rasheduzzaman; Leung, Ivanhoe K. H.; Tian, Ya-Min; Abboud, Martine I.; Ge, Wei; Domene, Carmen; Cantrelle, François-Xavier; Landrieu, Isabelle; Hardy, Adam P.; Pugh, Christopher W.; Ratcliffe, Peter J.; Claridge, Timothy D. W.; Schofield, Christopher J.

    2016-08-01

    The response to hypoxia in animals involves the expression of multiple genes regulated by the αβ-hypoxia-inducible transcription factors (HIFs). The hypoxia-sensing mechanism involves oxygen limited hydroxylation of prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of HIFα isoforms, as catalysed by prolyl hydroxylases (PHD 1-3). Prolyl hydroxylation promotes binding of HIFα to the von Hippel-Lindau protein (VHL)-elongin B/C complex, thus signalling for proteosomal degradation of HIFα. We reveal that certain PHD2 variants linked to familial erythrocytosis and cancer are highly selective for CODD or NODD. Crystalline and solution state studies coupled to kinetic and cellular analyses reveal how wild-type and variant PHDs achieve ODD selectivity via different dynamic interactions involving loop and C-terminal regions. The results inform on how HIF target gene selectivity is achieved and will be of use in developing selective PHD inhibitors.

  17. Potent Inhibition of Feline Coronaviruses with Peptidyl Compounds Targeting Coronavirus 3C-like Protease

    PubMed Central

    Kim, Yunjeong; Mandadapu, Sivakoteswara Rao; Groutas, William C.; Chang, Kyeong-Ok

    2012-01-01

    Feline coronavirus infection is common among domestic and exotic felid species and usually associated with mild or asymptomatic enteritis; however, feline infectious peritonitis (FIP) is a fatal disease of cats that is caused by systemic infection with a feline infectious peritonitis virus (FIPV), a variant of feline enteric coronavirus (FECV). Currently, there is no specific treatment approved for FIP despite the importance of FIP as the leading infectious cause of death in young cats. During the replication process, coronavirus produces viral polyproteins that are processed into mature proteins by viral proteases, the main protease (3C-like [3CL] protease) and the papain-like protease. Since the cleavages of viral polyproteins are an essential step for virus replication, blockage of viral protease is an attractive target for therapeutic intervention. Previously, we reported the generation of broad-spectrum peptidyl inhibitors against viruses that possess a 3C or 3CL protease. In this study, we further evaluated the antiviral effects of the peptidyl inhibitors against feline coronaviruses, and investigated the interaction between our protease inhibitor and a cathepsin B inhibitor, an entry blocker, against feline coronaviruses in cell culture. Herein we report that our compounds behave as reversible, competitive inhibitors of 3CL protease, potently inhibited the replication of feline coronaviruses (EC50 in a nanomolar range) and, furthermore, the combination of cathepsin B and 3CL protease inhibitors led to a strong synergistic interaction against feline coronaviruses in cell culture systems. PMID:23219425

  18. Potent inhibition of feline coronaviruses with peptidyl compounds targeting coronavirus 3C-like protease.

    PubMed

    Kim, Yunjeong; Mandadapu, Sivakoteswara Rao; Groutas, William C; Chang, Kyeong-Ok

    2013-02-01

    Feline coronavirus infection is common among domestic and exotic felid species and usually associated with mild or asymptomatic enteritis; however, feline infectious peritonitis (FIP) is a fatal disease of cats that is caused by systemic infection with a feline infectious peritonitis virus (FIPV), a variant of feline enteric coronavirus (FECV). Currently, there is no specific treatment approved for FIP despite the importance of FIP as the leading infectious cause of death in young cats. During the replication process, coronavirus produces viral polyproteins that are processed into mature proteins by viral proteases, the main protease (3C-like [3CL] protease) and the papain-like protease. Since the cleavages of viral polyproteins are an essential step for virus replication, blockage of viral protease is an attractive target for therapeutic intervention. Previously, we reported the generation of broad-spectrum peptidyl inhibitors against viruses that possess a 3C or 3CL protease. In this study, we further evaluated the antiviral effects of the peptidyl inhibitors against feline coronaviruses, and investigated the interaction between our protease inhibitor and a cathepsin B inhibitor, an entry blocker, against a feline coronavirus in cell culture. Herein we report that our compounds behave as reversible, competitive inhibitors of 3CL protease, potently inhibited the replication of feline coronaviruses (EC(50) in a nanomolar range) and, furthermore, combination of cathepsin B and 3CL protease inhibitors led to a strong synergistic interaction against feline coronaviruses in a cell culture system.

  19. Madumycin II inhibits peptide bond formation by forcing the peptidyl transferase center into an inactive state

    PubMed Central

    Osterman, Ilya A.; Khabibullina, Nelli F.; Komarova, Ekaterina S.; Kasatsky, Pavel; Kartsev, Victor G.; Bogdanov, Alexey A.; Dontsova, Olga A.

    2017-01-01

    Abstract The emergence of multi-drug resistant bacteria is limiting the effectiveness of commonly used antibiotics, which spurs a renewed interest in revisiting older and poorly studied drugs. Streptogramins A is a class of protein synthesis inhibitors that target the peptidyl transferase center (PTC) on the large subunit of the ribosome. In this work, we have revealed the mode of action of the PTC inhibitor madumycin II, an alanine-containing streptogramin A antibiotic, in the context of a functional 70S ribosome containing tRNA substrates. Madumycin II inhibits the ribosome prior to the first cycle of peptide bond formation. It allows binding of the tRNAs to the ribosomal A and P sites, but prevents correct positioning of their CCA-ends into the PTC thus making peptide bond formation impossible. We also revealed a previously unseen drug-induced rearrangement of nucleotides U2506 and U2585 of the 23S rRNA resulting in the formation of the U2506•G2583 wobble pair that was attributed to a catalytically inactive state of the PTC. The structural and biochemical data reported here expand our knowledge on the fundamental mechanisms by which peptidyl transferase inhibitors modulate the catalytic activity of the ribosome. PMID:28505372

  20. A new peptidyl fluorescent chemosensors for the selective detection of mercury ions based on tetrapeptide.

    PubMed

    Thirupathi, Ponnaboina; Lee, Keun-Hyeung

    2013-12-15

    A novel peptidyl chemosensor (PySO2-His-Gly-Gly-Lys(PySO2)-NH2, 1) was synthesized by incorporation of two pyrene (Py) fluorophores into the tetrapeptide using sulfonamide group. Compound 1 exhibited selective fluorescence response towards Hg(II) over the other metal ions in aqueous buffered solutions. Furthermore, 1 with the potent binding affinity (Kd=120 nM) for Hg(II) detected Hg(II) without interference of other metal ions such as Ag(I), Cu(II), Cd(II), and Pb(II). The binding mode of 1 with Hg(II) was investigated by UV absorbance spectroscopy, (1)H NMR titration experiment, and pH titration experiment. The addition of Hg(II) induced a significant decrease in both excimer and monomer emissions of the pyrene fluorescence. Hg(II) interacted with the sulfonamide groups and the imidazole group of His in the peptidyl chemosensor and then two pyrene fluorophores were close to each other in the peptide. The decrease of both excimer and monomer emission was mainly due to the excimer/monomer emission change by dimerization of two pyrene fluorophores and a quenching effect of Hg(II).

  1. Affinity labeling of a reactive sulfhydryl residue at the peptidyl transferase P site in Drosophila ribosomes.

    PubMed

    Fabijanski, S; Pellegrini, M

    1979-12-11

    An affinity label has been prepared that is specific for the P site of a eucaryotic peptidyl transferase, that of Drosophila melanogaster. It has the sequence C-A-C-C-A-(Ac[3H]Leu) with a mercury atom added at the C-5 position of all three cytosine residues (referred to as the mercurated fragment). This label is an analogue of the 3' terminus of N-acetylleucyl-tRNA. The mercurated fragment binds specifically to the P site of peptidyl transferase. It participates fully in peptide bond formation as judged by its ability to transfer N-acetylleucine to puromycin with at least the same efficiency as a nonmercurated fragment. Once bound to the P site, the mercurated fragment reacts covalently with a ribosomal protein(s). This affinity-labeling process can be effectively competed by nonmercurated fragment, which indicates a site-specific reaction. The covalent attachment of the affinity label to a ribosomal protein(s) occurs through the formation of a mercury-sulfur bond, as judged by its lability in the presence of thiol reducing agents. The major ribosomal protein labeled at the P site of D. melanogaster was found to be a small, basic protein. The electrophoretic behavior of this protein parallels that of major P site proteins found in Escherichia coli ribosomes and in other eucaryotes. These results suggest conservation of some of the overall properties of the P site proteins from these organisms.

  2. 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.

  3. Cyclosporin A Impairs the Secretion and Activity of ADAMTS13 (A Disintegrin and Metalloprotease with Thrombospondin Type 1 Repeat)*

    PubMed Central

    Hershko, Klilah; Simhadri, Vijaya L.; Blaisdell, Adam; Hunt, Ryan C.; Newell, Jordan; Tseng, Sandra C.; Hershko, Alon Y.; Choi, Jae Won; Sauna, Zuben E.; Wu, Andrew; Bram, Richard J.; Komar, Anton A.; Kimchi-Sarfaty, Chava

    2012-01-01

    The protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat) cleaves multimers of von Willebrand factor, thus regulating platelet aggregation. ADAMTS13 deficiency leads to the fatal disorder thrombotic thrombocytopenic purpura (TTP). It has been observed that cyclosporin A (CsA) treatment, particularly in transplant patients, may sometimes be linked to the development of TTP. Until now, the reason for such a link was unclear. Here we provide evidence demonstrating that cyclophilin B (CypB) activity plays an important role in the secretion of active ADAMTS13. We found that CsA, an inhibitor of CypB, reduces the secretion of ADAMTS13 and leads to conformational changes in the protein resulting in diminished ADAMTS13 proteolytic activity. A direct, functional interaction between CypB (which possesses peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone functions) and ADAMTS13 is demonstrated using immunoprecipitation and siRNA knockdown of CypB. Finally, CypB knock-out mice were found to have reduced ADAMTS13 levels. Taken together, our findings indicate that cyclophilin-mediated activity is an important factor affecting secretion and activity of ADAMTS13. The large number of proline residues in ADAMTS13 is consistent with the important role of cis-trans isomerization in the proper folding of this protein. These results altogether provide a novel mechanistic explanation for CsA-induced TTP in transplant patients. PMID:23144461

  4. Cyclosporin A impairs the secretion and activity of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat).

    PubMed

    Hershko, Klilah; Simhadri, Vijaya L; Blaisdell, Adam; Hunt, Ryan C; Newell, Jordan; Tseng, Sandra C; Hershko, Alon Y; Choi, Jae Won; Sauna, Zuben E; Wu, Andrew; Bram, Richard J; Komar, Anton A; Kimchi-Sarfaty, Chava

    2012-12-28

    The protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat) cleaves multimers of von Willebrand factor, thus regulating platelet aggregation. ADAMTS13 deficiency leads to the fatal disorder thrombotic thrombocytopenic purpura (TTP). It has been observed that cyclosporin A (CsA) treatment, particularly in transplant patients, may sometimes be linked to the development of TTP. Until now, the reason for such a link was unclear. Here we provide evidence demonstrating that cyclophilin B (CypB) activity plays an important role in the secretion of active ADAMTS13. We found that CsA, an inhibitor of CypB, reduces the secretion of ADAMTS13 and leads to conformational changes in the protein resulting in diminished ADAMTS13 proteolytic activity. A direct, functional interaction between CypB (which possesses peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone functions) and ADAMTS13 is demonstrated using immunoprecipitation and siRNA knockdown of CypB. Finally, CypB knock-out mice were found to have reduced ADAMTS13 levels. Taken together, our findings indicate that cyclophilin-mediated activity is an important factor affecting secretion and activity of ADAMTS13. The large number of proline residues in ADAMTS13 is consistent with the important role of cis-trans isomerization in the proper folding of this protein. These results altogether provide a novel mechanistic explanation for CsA-induced TTP in transplant patients.

  5. Proline isomerization in the C-terminal region of HSP27.

    PubMed

    Alderson, T Reid; Benesch, Justin L P; Baldwin, Andrew J

    2017-07-01

    In mammals, small heat-shock proteins (sHSPs) typically assemble into interconverting, polydisperse oligomers. The dynamic exchange of sHSP oligomers is regulated, at least in part, by molecular interactions between the α-crystallin domain and the C-terminal region (CTR). Here we report solution-state nuclear magnetic resonance (NMR) spectroscopy investigations of the conformation and dynamics of the disordered and flexible CTR of human HSP27, a systemically expressed sHSP. We observed multiple NMR signals for residues in the vicinity of proline 194, and we determined that, while all observed forms are highly disordered, the extra resonances arise from cis-trans peptidyl-prolyl isomerization about the G193-P194 peptide bond. The cis-P194 state is populated to near 15% at physiological temperatures, and, although both cis- and trans-P194 forms of the CTR are flexible and dynamic, both states show a residual but differing tendency to adopt β-strand conformations. In NMR spectra of an isolated CTR peptide, we observed similar evidence for isomerization involving proline 182, found within the IPI/V motif. Collectively, these data indicate a potential role for cis-trans proline isomerization in regulating the oligomerization of sHSPs.

  6. Highly efficient gluten degradation with a newly identified prolyl endoprotease: implications for celiac disease.

    PubMed

    Stepniak, Dariusz; Spaenij-Dekking, Liesbeth; Mitea, Cristina; Moester, Martine; de Ru, Arnoud; Baak-Pablo, Renee; van Veelen, Peter; Edens, Luppo; Koning, Frits

    2006-10-01

    Celiac disease is a T cell-driven intolerance to wheat gluten. The gluten-derived T cell epitopes are proline-rich and thereby highly resistant to proteolytic degradation within the gastrointestinal tract. Oral supplementation with prolyl oligopeptidases has therefore been proposed as a potential therapeutic approach. The enzymes studied, however, have limitations as they are irreversibly inactivated by pepsin and acidic pH, both present in the stomach. As a consequence, these enzymes will fail to degrade gluten before it reaches the small intestine, the site where gluten induces inflammatory T cell responses that lead to celiac disease. We have now determined the usefulness of a newly identified prolyl endoprotease from Aspergillus niger for this purpose. Gluten and its peptic/tryptic digest were treated with prolyl endoprotease, and the destruction of the T cell epitopes was tested using mass spectrometry, T cell proliferation assays, ELISA, reverse-phase HPLC, SDS-PAGE, and Western blotting. We observed that the A. niger prolyl endoprotease works optimally at 4-5 pH, remains stable at 2 pH, and is completely resistant to digestion with pepsin. Moreover, the A. niger-derived enzyme efficiently degraded all tested T cell stimulatory peptides as well as intact gluten molecules. On average, the endoprotease from A. niger degraded gluten peptides 60 times faster than a prolyl oligopeptidase. Together these results indicate that the enzyme from A. niger efficiently degrades gluten proteins. Future studies are required to determine if the prolyl endoprotease can be used as an oral supplement to reduce gluten intake in patients.

  7. Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation

    PubMed Central

    Scotti, John S.; Leung, Ivanhoe K. H.; Ge, Wei; Bentley, Michael A.; Paps, Jordi; Kramer, Holger B.; Lee, Joongoo; Aik, WeiShen; Choi, Hwanho; Paulsen, Steinar M.; Bowman, Lesley A. H.; Loik, Nikita D.; Horita, Shoichiro; Ho, Chia-hua; Kershaw, Nadia J.; Tang, Christoph M.; Claridge, Timothy D. W.; Preston, Gail M.; McDonough, Michael A.; Schofield, Christopher J.

    2014-01-01

    The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolyl-hydroxylation in human hypoxia sensing has ancient origins. PMID:25197067

  8. Binding kinetics and duration of in vivo action of novel prolyl oligopeptidase inhibitors.

    PubMed

    Venäläinen, Jarkko I; Garcia-Horsman, J Arturo; Forsberg, Markus M; Jalkanen, Aaro; Wallén, Erik A A; Jarho, Elina M; Christiaans, Johannes A M; Gynther, Jukka; Männistö, Pekka T

    2006-02-28

    Prolyl oligopeptidase (POP) is a serine protease that specifically hydrolyses small peptides at the carboxyl end of the proline residue. POP has gained pharmaceutical interest, since its inhibitors have been shown to have antiamnesic properties in rat. We examined the effect of the 2(S)-substituents CN and COCH(2)OH at the P1 site of the parent inhibitors isophthalic acid 2(S)-(cyclopentanecarbonyl)pyrrolidine-l-prolyl-pyrrolidine amide and 4-phenylbutanoyl-l-prolyl-pyrrolidine and bulky 5-t-butyl group at the P2 site l-prolyl residue of the parent inhibitor 4-phenylbutanoyl-l-prolyl-pyrrolidine on the binding kinetics to the enzyme. In addition, we studied the duration of POP inhibition in the rat tissues in vivo after i.p. administration. CN and COCH(2)OH substituents at the P1 site pyrrolidine group were found to greatly increase the affinity of the inhibitor and the enzyme-inhibitor complex half-life. In addition, 5-t-butyl group at the P2 site l-prolyl residue increased the dissociation half-life of the enzyme-inhibitor complex, without much affecting the inhibitory potency. The duration of the inhibition in the rat tissues followed the inhibition kinetic properties in that the compounds with fast dissociation produced shorter inhibition in the rat tissues than the compounds with slow dissociation. The duration of POP inhibition of compounds was evidently not governed by their serum clearance. The fact that the in vivo pharmacodynamic behaviour of POP inhibitors can be predicted by their in vitro-properties may be of importance when designing therapeutically useful POP inhibitors.

  9. Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation.

    PubMed

    Scotti, John S; Leung, Ivanhoe K H; Ge, Wei; Bentley, Michael A; Paps, Jordi; Kramer, Holger B; Lee, Joongoo; Aik, WeiShen; Choi, Hwanho; Paulsen, Steinar M; Bowman, Lesley A H; Loik, Nikita D; Horita, Shoichiro; Ho, Chia-hua; Kershaw, Nadia J; Tang, Christoph M; Claridge, Timothy D W; Preston, Gail M; McDonough, Michael A; Schofield, Christopher J

    2014-09-16

    The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolyl-hydroxylation in human hypoxia sensing has ancient origins.

  10. Isolation of translating ribosomes containing peptidyl-tRNAs for functional and structural analyses.

    PubMed

    Shirole, Nitin; Balasubramanian, Sreeram; Yanofsky, Charles; Cruz-Vera, Luis

    2011-02-25

    Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination. These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism. Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism. Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA. This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled m

  11. Secretory activity of mast cell during stress: effect of prolyl-glycyl-proline and Semax.

    PubMed

    Umarova, B A; Kopylova, G N; Smirnova, E A; Guseva, A A; Zhuikova, S E

    2003-10-01

    Stress increased secretory activity of mast cells in the mesentery and subcutaneous fat of rats. Intraperitoneal injection of Semax and prolyl-glycyl-proline in doses of 0.05 and 1 mg/kg, respectively, 1 h before stress abolished this effect. The test preparations did not modulate secretory activity of mast cells in unstressed animals. Semax and prolyl-glycyl-proline in vitro prevented activation of mast cells with synacten and acetylcholine. The stabilizing effect of peptides on mast cells probably determines their antiulcer activity.

  12. A steady-state kinetic analysis of the prolyl-4-hydroxylase mechanism.

    PubMed

    Soskel, N T; Kuby, S A

    1981-01-01

    Published kinetic data by Kivirikko, et al. on the prolyl-4-hydroxylase reaction have been re-evaluated using the overall steady-state velocity equation in the forward and reverse directions for an ordered ter ter kinetic mechanism. Qualitatively, the published data for prolyl-4-hydroxylase appear to fit the predicted patterns for this kinetic mechanism. More kinetic data are needed to confirm these results and to quantitate the kinetic parameters but, tentatively, the order of substrate addition would appear to be alpha-ketoglutarate, oxygen, and peptide; and the order of product release would be hydroxylated peptide (or collagen), carbon dioxide, and succinate.

  13. Uncovering the enzymatic pKa of the ribosomal peptidyl transferase reaction utilizing a fluorinated puromycin derivative.

    PubMed

    Okuda, Kensuke; Seila, Amy C; Strobel, Scott A

    2005-05-03

    The ribosome-catalyzed peptidyl transferase reaction displays a complex pH profile resulting from two functional groups whose deprotonation is important for the reaction, one within the A-site substrate and a second unidentified group thought to reside in the rRNA peptidyl transferase center. Here we report the synthesis and activity of the beta,beta-difluorophenylalanyl derivative of puromycin, an A-site substrate. The fluorine atoms reduce the pK(a) of the nucleophilic alpha-amino group (<5.0) such that it is deprotonated at all pHs amenable to ribosomal analysis (pH 5.2-9.5). In the 50S modified fragment assay, this substrate reacts substantially faster than puromycin at neutral or acidic pH. The reaction follows a simplified pH profile that is dependent only upon deprotonation of a titratable group within the ribosomal active site. This feature will simplify characterization of the peptidyl transferase reaction mechanism. On the basis of the reaction efficiency of the doubly fluorinated substrate compared to the unfluorinated derivative, the Bronsted coefficient for the nucleophile is estimated to be substantially smaller than that reported for uncatalyzed aminolysis reactions, which has important mechanistic implications for the peptidyl transferase reaction.

  14. The pleuromutilin drugs tiamulin and valnemulin bind to the RNA at the peptidyl transferase centre on the ribosome.

    PubMed

    Poulsen, S M; Karlsson, M; Johansson, L B; Vester, B

    2001-09-01

    The pleuromutilin antibiotic derivatives, tiamulin and valnemulin, inhibit protein synthesis by binding to the 50S ribosomal subunit of bacteria. The action and binding site of tiamulin and valnemulin was further characterized on Escherichia coli ribosomes. It was revealed that these drugs are strong inhibitors of peptidyl transferase and interact with domain V of 23S RNA, giving clear chemical footprints at nucleotides A2058-9, U2506 and U2584-5. Most of these nucleotides are highly conserved phylogenetically and functionally important, and all of them are at or near the peptidyl transferase centre and have been associated with binding of several antibiotics. Competitive footprinting shows that tiamulin and valnemulin can bind concurrently with the macrolide erythromycin but compete with the macrolide carbomycin, which is a peptidyl transferase inhibitor. We infer from these and previous results that tiamulin and valnemulin interact with the rRNA in the peptidyl transferase slot on the ribosomes in which they prevent the correct positioning of the CCA-ends of tRNAs for peptide transfer.

  15. Dissociation rates of peptidyl-tRNA from the P-site of E.coli ribosomes.

    PubMed

    Karimi, R; Ehrenberg, M

    1996-03-01

    We studied the dissociation rates of peptidyl-tRNA from the P-site of poly(U)-programmed wild-type Escherichia coli ribosomes, hyperaccurate variants altered in S12 (SmD, SmP) and error-prone variants (Ram) altered in S4 or S5. The experiments were carried out in the presence and absence of streptomycin, and the effects of neomycin were tested in the wild-type ribosomes. Binding of peptidyl-tRNA to the P-site of wild-type ribosomes is much stronger than to their A-site. Addition of streptomycin dramatically reduces its affinity for the P-site. The S12 alternations make the P-site binding of peptidyl-tRNA much tighter, and the S4, S5 alterations make it weaker than in the case of the wild-type. We find that when binding of peptidyl-tRNA to the A-site is weak, then the affinity for the P-site is stronger, and vice versa. From these results, we formulate a hypothesis for the actions of streptomycin and neomycin based on deformations of the 16S rRNA tertiary structure. The results are also used to interpret some in vivo experiments on translational processivity.

  16. Design, synthesis and biological activity of novel non-peptidyl endothelin converting enzyme inhibitors, 1-phenyl-tetrazole-formazan analogues.

    PubMed

    Yamazaki, Kazuto; Hasegawa, Hirohiko; Umekawa, Kayo; Ueki, Yasuyuki; Ohashi, Naohito; Kanaoka, Masaharu

    2002-05-06

    A novel non-peptidyl endothelin converting enzyme inhibitor was obtained through a pharmacophore analysis of known inhibitors and three-dimensional structure database search. Analogues of the new inhibitor were designed using the structure-activity relationship of known inhibitors and synthesized. In anesthetized rats, intraperitoneal administration of the analogues suppressed the pressor responses induced by big endothelin-1.

  17. Peptidylation for the determination of low-molecular-weight compounds by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

    PubMed

    Tang, Feng; Cen, Si-Ying; He, Huan; Liu, Yi; Yuan, Bi-Feng; Feng, Yu-Qi

    2016-05-23

    Determination of low-molecular-weight compounds by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been a great challenge in the analytical research field. Here we developed a universal peptide-based derivatization (peptidylation) strategy for the sensitive analysis of low-molecular-weight compounds by MALDI-TOF-MS. Upon peptidylation, the molecular weights of target analytes increase, thus avoiding serious matrix ion interference in the low-molecular-weight region in MALDI-TOF-MS. Since peptides typically exhibit good signal response during MALDI-TOF-MS analysis, peptidylation endows high detection sensitivities of low-molecular-weight analytes. As a proof-of-concept, we analyzed low-molecular-weight compounds of aldehydes and thiols by the developed peptidylation strategy. Our results showed that aldehydes and thiols can be readily determined upon peptidylation, thus realizing the sensitive and efficient determination of low-molecular-weight compounds by MALDI-TOF-MS. Moreover, target analytes also can be unambiguously detected in biological samples using the peptidylation strategy. The established peptidylation strategy is a universal strategy and can be extended to the sensitive analysis of various low-molecular-weight compounds by MALDI-TOF-MS, which may be potentially used in areas such as metabolomics.

  18. Inhibition of the ribosomal peptidyl transferase reaction by the mycarose moiety of the antibiotics carbomycin, spiramycin and tylosin.

    PubMed

    Poulsen, S M; Kofoed, C; Vester, B

    2000-12-01

    Many antibiotics, including the macrolides, inhibit protein synthesis by binding to ribosomes. Only some of the macrolides affect the peptidyl transferase reaction. The 16-member ring macrolide antibiotics carbomycin, spiramycin, and tylosin inhibit peptidyl transferase. All these have a disaccharide at position 5 in the lactone ring with a mycarose moiety. We have investigated the functional role of this mycarose moiety. The 14-member ring macrolide erythromycin and the 16-member ring macrolides desmycosin and chalcomycin do not inhibit the peptidyl transferase reaction. These drugs have a monosaccharide at position 5 in the lactone ring. The presence of mycarose was correlated with inhibition of peptidyl transferase, footprints on 23 S rRNA and whether the macrolide can compete with binding of hygromycin A to the ribosome. The binding sites of the macrolides to Escherichia coli ribosomes were investigated by chemical probing of domains II and V of 23 S rRNA. The common binding site is around position A2058, while effects on U2506 depend on the presence of the mycarose sugar. Also, protection at position A752 indicates that a mycinose moiety at position 14 in 16-member ring macrolides interact with hairpin 35 in domain II. Competitive footprinting of ribosomal binding of hygromycin A and macrolides showed that tylosin and spiramycin reduce the hygromycin A protections of nucleotides in 23 S rRNA and that carbomycin abolishes its binding. In contrast, the macrolides that do not inhibit the peptidyl transferase reaction bind to the ribosomes concurrently with hygromycin A. Data are presented to argue that a disaccharide at position 5 in the lactone ring of macrolides is essential for inhibition of peptide bond formation and that the mycarose moiety is placed near the conserved U2506 in the central loop region of domain V 23 S rRNA.

  19. Purification, characterization and the use of recombinant prolyl oligopeptidase from Myxococcus xanthus for gluten hydrolysis.

    PubMed

    Kocadag Kocazorbaz, Ebru; Zihnioglu, Figen

    2017-01-01

    Prolyl oligopeptidase (POP, EC 3.4.21.26) is a cytosolic serine protease that hydrolyses proline containing small peptides. The members of prolyl oligopeptidase family play important roles in many physiological processes such as neurodegenerative diseases, maturation and degradation of peptide hormones. Thus the enzyme has been purified and characterized from various sources to elucidate the potential use as therapeutics. In this study recombinant Myxococcus xanthus prolyl oligopeptidase expressed in E. coli was purified 60.3 fold, using metal-chelate affinity and gel permeation chromatography. The recombinant enzyme had a monomeric molecular weight of 70 kDa. Isoelectric point of the enzyme was found to be approximately 6.3 by two-dimensional polyacrylamide gel electrophoresis. The optimum pH and temperature was estimated as 7.5 and 37 °C, respectively. The purified enzyme was stable in a pH range of 6.0-8.5 and thermally stable up to 37 °C. The Km and Vmax values were 0.2 mM and 3.42 μmol/min/mg. The proteolytic activity was inhibited by active-site inhibitors of serine protease, Z-Pro-Prolinal, PMSF, and metal ions, Cd(2+), and Hg(2+). Furthermore, the hydrolysis efficiency of the recombinant prolyl oligopeptidase was investigated with wheat gluten. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Collagen prolyl3-hydroxylation: a major role for a minor post-translational modification?

    PubMed Central

    Hudson, David M.; Eyre, David R.

    2014-01-01

    Prolyl 3-hydroxylation is a rare but conserved post-translational modification in many collagen types and, when defective, may be linked to a number of human diseases with musculoskeletal and potentially ocular and renal pathologies. Prolyl 3-hydroxylase-1 (P3H1), the enzyme responsible for converting proline to 3-hydroxyproline (3Hyp) in type I collagen, requires the coenzyme CRTAP for activity. Mass spectrometric analysis showed that the Crtap−/− mouse was missing 3-hydroxyproline in type I collagen α-chains. This finding led to the discovery mutations in genes encoding the P3H1 complex as a cause of recessively inherited osteogenesis imperfecta (brittle bone disease). Since then, many additional 3Hyp sites have been identified in various collagen types and classified based on observed substrate and tissue specificity. P3H1 is part of a family of gene products that also includes isoenzymes P3H2 and P3H3 as well as CRTAP and Sc65. It is believed these isoenzymes and coenzymes have evolved different collagen substrate site and tissue specificities in their activities. The post-translational fingerprinting of collagens will be essential in understanding the basic role and extent of regulated variations of prolyl 3-hydroxylation in collagen. We believe that prolyl 3-hydroxylation is a functionally significant collagen post-translational modification and can be a cause of disease when absent. PMID:23772978

  1. A gene responsible for prolyl-hydroxylation of moss-produced recombinant human erythropoietin

    PubMed Central

    Parsons, Juliana; Altmann, Friedrich; Graf, Manuela; Stadlmann, Johannes; Reski, Ralf; Decker, Eva L.

    2013-01-01

    Recombinant production of pharmaceutical proteins is crucial, not only for personalized medicine. While most biopharmaceuticals are currently produced in mammalian cell culture, plant-made pharmaceuticals gain momentum. Post-translational modifications in plants are similar to those in humans, however, existing differences may affect quality, safety and efficacy of the products. A frequent modification in higher eukaryotes is prolyl-4-hydroxylase (P4H)-catalysed prolyl-hydroxylation. P4H sequence recognition sites on target proteins differ between humans and plants leading to non-human posttranslational modifications of recombinant human proteins produced in plants. The resulting hydroxyprolines display the anchor for plant-specific O-glycosylation, which bears immunogenic potential for patients. Here we describe the identification of a plant gene responsible for non-human prolyl-hydroxylation of human erythropoietin (hEPO) recombinantly produced in plant (moss) bioreactors. Targeted ablation of this gene abolished undesired prolyl-hydroxylation of hEPO and thus paves the way for plant-made pharmaceuticals humanized via glyco-engineering in moss bioreactors. PMID:24145658

  2. A gene responsible for prolyl-hydroxylation of moss-produced recombinant human erythropoietin.

    PubMed

    Parsons, Juliana; Altmann, Friedrich; Graf, Manuela; Stadlmann, Johannes; Reski, Ralf; Decker, Eva L

    2013-10-22

    Recombinant production of pharmaceutical proteins is crucial, not only for personalized medicine. While most biopharmaceuticals are currently produced in mammalian cell culture, plant-made pharmaceuticals gain momentum. Post-translational modifications in plants are similar to those in humans, however, existing differences may affect quality, safety and efficacy of the products. A frequent modification in higher eukaryotes is prolyl-4-hydroxylase (P4H)-catalysed prolyl-hydroxylation. P4H sequence recognition sites on target proteins differ between humans and plants leading to non-human posttranslational modifications of recombinant human proteins produced in plants. The resulting hydroxyprolines display the anchor for plant-specific O-glycosylation, which bears immunogenic potential for patients. Here we describe the identification of a plant gene responsible for non-human prolyl-hydroxylation of human erythropoietin (hEPO) recombinantly produced in plant (moss) bioreactors. Targeted ablation of this gene abolished undesired prolyl-hydroxylation of hEPO and thus paves the way for plant-made pharmaceuticals humanized via glyco-engineering in moss bioreactors.

  3. 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

  4. Structural basis for the interaction of antibiotics with peptidyl transferase center in eubacteria

    SciTech Connect

    Schlunzen, Frank; Zarivach, Raz; Harms, Jörg; Bashan, Anat; Tocilj, Ante; Albrecht, Renate; Yonath, Ada; Franceschi, Francois

    2009-10-07

    Ribosomes, the site of protein synthesis, are a major target for natural and synthetic antibiotics. Detailed knowledge of antibiotic binding sites is central to understanding the mechanisms of drug action. Conversely, drugs are excellent tools for studying the ribosome function. To elucidate the structural basis of ribosome-antibiotic interactions, we determined the high-resolution X-ray structures of the 50S ribosomal subunit of the eubacterium Deinococcus radiodurans, complexed with the clinically relevant antibiotics chloramphenicol, clindamycin and the three macrolides erythromycin, clarithromycin and roxithromycin. We found that antibiotic binding sites are composed exclusively of segments of 23S ribosomal RNA at the peptidyl transferase cavity and do not involve any interaction of the drugs with ribosomal proteins. Here we report the details of antibiotic interactions with the components of their binding sites. Our results also show the importance of putative Mg{sup +2} ions for the binding of some drugs. This structural analysis should facilitate rational drug design.

  5. Inhibition of prolyl hydroxylation during collagen biosynthesis in human skin fibroblast cultures by ethyl 3,4-dihydroxybenzoate.

    PubMed

    Majamaa, K; Sasaki, T; Uitto, J

    1987-10-01

    The enzymatically catalyzed formation of 4-hydroxyproline plays a key role in the intracellular biosynthesis of collagen, since a critical number of 4-hydroxyprolyl residues is required for synthesis and secretion of triple-helical procollagen molecules under physiologic conditions. The enzyme catalyzing the conversion of prolyl residues to 4-hydroxyproline, prolyl 4-hydroxylase, requires ferrous ion, alpha-ketoglutarate, and ascorbate for its activity. 3,4-Dihydroxybenzoic acid has been known to act as potent competitive inhibitor of purified prolyl 4-hydroxylase with respect to one or several of the cofactors or cosubstrates of the enzyme. 3,4-Dihydroxybenzoic acid, however, is a poor inhibitor of prolyl hydroxylation in intact cells, probably due to its polarity not allowing it to enter the cells. In this study, several hydrophobic modifications of 3,4-dihydroxybenzoic acid were tested in human skin fibroblast cultures for their efficacy to inhibit the synthesis of 4-hydroxyproline. The results indicated that the ethyl ester of 3,4-dihydroxybenzoic acid was an efficient inhibitor of prolyl hydroxylation in fibroblast cultures, with Ki of approximately 0.4 mM. Ethyl 3,4-dihydroxybenzoate had little, if any, effect on the hydroxylation of lysyl residues, and it did not affect total protein synthesis or DNA replication in these cells. To test the hypothesis that ethyl 3,4-dihydroxybenzoate might serve as a potential antifibrotic agent, its efficacy in inhibiting prolyl hydroxylation in scleroderma fibroblasts was also tested. The results indicated that the synthesis of 4-hydroxyproline in scleroderma cell cultures was similarly reduced by ethyl 3,4-dihydroxybenzoate. Thus, structural analogs of the cofactors or cosubstrates of prolyl 4-hydroxylase, such as ethyl 3,4-dihydroxybenzoate tested here or its further modifications, may serve as inhibitors of posttranslational hydroxylation of prolyl residues also in vivo. These compounds could potentially provide a novel

  6. Small Molecule Binding, Docking, and Characterization of the Interaction between Pth1 and Peptidyl-tRNA

    SciTech Connect

    Hames, Mary C; McFeeters, Hana; Holloway, W Blake; Stanley, Christopher B; Urban, Volker S; McFeeters, Robert L

    2013-01-01

    Bacterial Pth1 is essential for viability. Pth1 cleaves the ester bond between the peptide and nucleotide of peptidyl-tRNA generated from aborted translation, expression of mini-genes, and short ORFs. We have determined the shape of the Pth1:peptidyl-tRNA complex using small angle neutron scattering. Binding of piperonylpiperazine, a small molecule constituent of a combinatorial synthetic library common to most compounds with inhibitory activity, was mapped to Pth1 via NMR spectroscopy. We also report computational docking results, modeling piperonylpiperazine binding based on chemical shift perturbation mapping. Overall these studies promote Pth1 as a novel antibiotic target, contributing to understanding how Pth1 interacts with its substrate, advancing the current model for cleavage, and demonstrating feasibility of small molecule inhibition.

  7. Ribosomal protein L5 helps anchor peptidyl-tRNA to the P-site in Saccharomyces cerevisiae.

    PubMed Central

    Meskauskas, A; Dinman, J D

    2001-01-01

    Our previous demonstration that mutants of 5S rRNA called mof9 can specifically alter efficiencies of programmed ribosomal frameshifting (PRF) suggested a role for this ubiquitous molecule in the maintenance of translational reading frame, though the repetitive nature of the 5S rDNA gene (>100 copies/cell) inhibited more detailed analyses. However, given the known interactions between 5S rRNA and ribosomal protein L5 (previously called L1 or YL3) encoded by an essential, single-copy gene, we monitored the effects of a series of well-defined rpl5 mutants on PRF and virus propagation. Consistent with the mof9 results, we find that the rpl5 mutants promoted increased frameshifting efficiencies in both the -1 and +1 directions, and conferred defects in the ability of cells to propagate two endogenous viruses. Biochemical analyses demonstrated that mutant ribosomes had decreased affinities for peptidyl-tRNA. Pharmacological studies showed that sparsomycin, a peptidyltransferase inhibitor that specifically increases the binding of peptidyl-tRNA with ribosomes, was antagonistic to the frameshifting defects of the most severe mutant, and the extent of sparsomycin resistance correlated with the severity of the frameshifting defects in all of the mutants. These results provide biochemical and physiological evidence that one function of L5 is to anchor peptidyl-tRNA to the P-site. A model is presented describing how decreased affinity of ribosomes for peptidyl-tRNA can affect both -1 and +1 frameshifting, and for the effects of sparsomycin. PMID:11497428

  8. In vitro and ex vivo inhibition of hepatitis A virus 3C proteinase by a peptidyl monofluoromethyl ketone.

    PubMed

    Morris, T S; Frormann, S; Shechosky, S; Lowe, C; Lall, M S; Gauss-Müller, V; Purcell, R H; Emerson, S U; Vederas, J C; Malcolm, B A

    1997-05-01

    Hepatitis A virus (HAV) 3C proteinase is the enzyme responsible for the processing of the viral polyprotein. Although a cysteine proteinase, it displays an active site configuration like those of the mammalian serine proteinases (Malcolm, B. A. Protein Science 1995, 4, 1439). A peptidyl monofluoromethyl ketone (peptidyl-FMK) based on the preferred peptide substrates for HAV 3C proteinase was generated by first coupling the precursor, N,N-dimethylglutamine fluoromethylalcohol, to the tripeptide, Ac-Leu-Ala-Ala-OH, and then oxidizing the product to the corresponding peptidyl-FMK (Ac-LAAQ'-FMK). This molecule was found to be an irreversible inactivator of HAV 3C with a second-order rate constant of 3.3 x 10(2) M-1 s-1. 19F NMR spectroscopy indicates the displacement of fluoride on inactivation of the enzyme by the fluoromethyl ketone. NMR spectroscopy of the complex between the 13C-labeled inhibitor and the HAV 3C proteinase indicates that an (alkylthio)methyl ketone is formed. Studies of polyprotein processing, using various substrates generated by in vitro transcription/translation, demonstrated efficient blocking of even the most rapid proteolytic events such as cleavage of the 2A-2B and 2C-3A junctions. Subsequent ex vivo studies, to test for antiviral activity, show a 25-fold reduction in progeny virus production as the result of treatment with 5 microM inhibitor 24 h post-infection.

  9. Reliable semi-synthesis of hydrolysis-resistant 3'-peptidyl-tRNA conjugates containing genuine tRNA modifications.

    PubMed

    Graber, Dagmar; Moroder, Holger; Steger, Jessica; Trappl, Krista; Polacek, Norbert; Micura, Ronald

    2010-10-01

    The 3'-peptidyl-tRNA conjugates that possess a hydrolysis-resistant ribose-3'-amide linkage instead of the natural ester linkage would represent valuable substrates for ribosomal studies. Up to date, access to these derivatives is severely limited. Here, we present a novel approach for the reliable synthesis of non-hydrolyzable 3'-peptidyl-tRNAs that contain all the respective genuine nucleoside modifications. In short, the approach is based on tRNAs from natural sources that are site-specifically cleaved within the TΨC loop by using DNA enzymes to obtain defined tRNA 5'-fragments carrying the modifications. After dephosphorylation of the 2',3'-cyclophosphate moieties from these fragments, they are ligated to the respective 3'-peptidylamino-tRNA termini that were prepared following the lines of a recently reported solid-phase synthesis. By this novel concept, non-hydrolyzable 3'-peptidyl-tRNA conjugates possessing all natural nucleoside modifications are accessible in highly efficient manner.

  10. Geometry matters: inverse cytotoxic relationship for cis/trans-Ru(ii) polypyridyl complexes from cis/trans-[PtCl2(NH3)2].

    PubMed

    Wachter, Erin; Zamora, Ana; Heidary, David K; Ruiz, José; Glazer, Edith C

    2016-08-09

    Two thermally activated ruthenium(ii) polypyridyl complexes, cis-Ru(bpy)2Cl2 and trans-Ru(qpy)Cl2 were investigated to determine the impact of the geometric arrangement of the exchangable ligands on the potential of the compounds to act as chemotherapeutics. In contrast to the geometry requirements for cisplatin, trans-Ru(qpy)Cl2 was 7.1-9.5× more cytotoxic than cis-Ru(bpy)2Cl2. This discovery could open up a new area of metal-based chemotherapeutic research.

  11. Mechanistic control of product selectivity. Reactions between cis-/trans cis-/trans-[OsVI(tpy)(Cl)2(N)]+ and triphenylphosphine sulfide.

    PubMed

    Huynh, M H; White, P S; Meyer, T J

    2000-06-26

    Reactions between the Os(VI)-nitrido complexes cis- and trans-[Os(VI)(tpy)(Cl)2(N)]+ (tpy is 2,2':6',2"-terpyridine) and triphenylphosphine sulfide, SPPh3, give the corresponding Os(IV)-phosphoraniminato, [Os(IV)(tpy)(Cl)2(NPPh3)]+, and Os(II)-thionitrosyl, [Os(II)(tpy)(Cl)2(NS)]+, complexes as products. The Os-N bond length and Os-N-P angle in cis-[Os(IV)(tpy)(Cl)2(NPPh3)](PF6) are 2.077(6) A and 138.4(4) degrees. The rate law for formation of cis- and trans-[Os(IV)(tpy)(Cl)2(NPPh3)]+ is first order in both [Os(VI)(tpy)(Cl)2(N)]+ and SPPh3 with ktrans(25 degrees C, CH3CN) = 24.6 +/- 0.6 M(-1) s(-1) and kcis(25 degrees C, CH3CN) = 0.84 +/- 0.09 M(-1) s(-1). As found earlier for [Os(II)(tpm)(Cl)2(NS)]+, both cis- and trans-[Os(II)(tpy)(Cl)2(NS)]+ react with PPh3 to give [Os(IV)(tpy)(Cl)2(NPPh3)]+ and SPPh3. For both complexes, the reaction is first order in each reagent with ktrans(25 degrees C, CH3CN) = (6.79 +/- 0.08) x 10(2) M(-1) s(-1) and kcis(25 degrees C, CH3CN) = (2.30 +/- 0.07) x 10(2) M(-1) s(-1). The fact that both reactions occur rules out mechanisms involving S atom transfer. These results can be explained by invoking a common intermediate, [Os(IV)(tpy)(Cl)2(NSPPh3)]+, which undergoes further reaction with PPh3 to give [Os(IV)(tpy)(Cl)2(NPPh3)]+ and SPPh3 or with [Os(VI)(tpy)(Cl)2(N)]+ to give [Os(IV)(tpy)(Cl)2(NPPh3)]+ and [Os(II)(tpy)(Cl)2(NS)]+.

  12. Prolyl hydroxylation regulates protein degradation, synthesis, and splicing in human induced pluripotent stem cell-derived cardiomyocytes.

    PubMed

    Stoehr, Andrea; Yang, Yanqin; Patel, Sajni; Evangelista, Alicia M; Aponte, Angel; Wang, Guanghui; Liu, Poching; Boylston, Jennifer; Kloner, Philip H; Lin, Yongshun; Gucek, Marjan; Zhu, Jun; Murphy, Elizabeth

    2016-06-01

    Protein hydroxylases are oxygen- and α-ketoglutarate-dependent enzymes that catalyse hydroxylation of amino acids such as proline, thus linking oxygen and metabolism to enzymatic activity. Prolyl hydroxylation is a dynamic post-translational modification that regulates protein stability and protein-protein interactions; however, the extent of this modification is largely uncharacterized. The goals of this study are to investigate the biological consequences of prolyl hydroxylation and to identify new targets that undergo prolyl hydroxylation in human cardiomyocytes. We used human induced pluripotent stem cell-derived cardiomyocytes in combination with pulse-chase amino acid labelling and proteomics to analyse the effects of prolyl hydroxylation on protein degradation and synthesis. We identified 167 proteins that exhibit differences in degradation with inhibition of prolyl hydroxylation by dimethyloxalylglycine (DMOG); 164 were stabilized. Proteins involved in RNA splicing such as serine/arginine-rich splicing factor 2 (SRSF2) and splicing factor and proline- and glutamine-rich (SFPQ) were stabilized with DMOG. DMOG also decreased protein translation of cytoskeletal and sarcomeric proteins such as α-cardiac actin. We searched the mass spectrometry data for proline hydroxylation and identified 134 high confidence peptides mapping to 78 unique proteins. We identified SRSF2, SFPQ, α-cardiac actin, and cardiac titin as prolyl hydroxylated. We identified 29 prolyl hydroxylated proteins that showed a significant difference in either protein degradation or synthesis. Additionally, we performed next-generation RNA sequencing and showed that the observed decrease in protein synthesis was not due to changes in mRNA levels. Because RNA splicing factors were prolyl hydroxylated, we investigated splicing ± inhibition of prolyl hydroxylation and detected 369 alternative splicing events, with a preponderance of exon skipping. This study provides the first extensive

  13. Altered dipeptidyl peptidase IV and prolyl endopeptidase activities in chronic tonsillitis, tonsillar hyperplasia and adenoid hyperplasia.

    PubMed

    Larrinaga, Gorka; Pérez, Itxaro; Sanz, Begoña; Zarrazquin, Idoia; Casis, Luis; Anta, Jose Antonio; Martínez, Agustin; Santaolalla, Francisco

    2011-03-01

    To analyse peptidase activities in the removed tonsils and adenoids from patients with chronic tonsillitis, tonsillar hyperplasia and adenoid hyperplasia. We have analyzed 48 tissue samples from patients undergoing tonsillectomy and adenoidectomy for chronic tonsillitis, tonsillar hyperplasia or adenoid hyperplasia. Tonsillectomy and adenoidectomy samples were collected and frozen for later enzyme analysis. The catalytic activity of a pool of peptidases (dipeptidyl peptidase IV, prolyl endopeptidase, aminopeptidase A, aminopeptidase N, aspartyl aminopeptidase, aminopeptidase B, neutral endopeptidase, pyroglutamyl peptidase I, puromycin-sensitive aminopeptidase and cystinyl aminopeptidase) was measured fluorometrically. The activity of prolyl endopeptidase was higher in tonsillar hyperplasia and adenoid hyperplasia than in chronic tonsillitis. On the contrary, dipeptidyl peptidase IV activity was higher in chronic tonsillitis than in hypertrophic tissues. When data were stratified by age and gender, dipeptidyl peptidase IV was also found to be more active in adult and male chronic tonsillitis tissues. Inversely, dipeptidyl peptidase IV activity was higher in tissues of females with tonsillar hyperplasia. These data indicate the involvement of dipeptidyl peptidase IV and prolyl endopeptidase in the mechanisms underlying chronic tonsillitis, tonsillar hyperplasia and adenoid hyperplasia. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  14. Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases

    PubMed Central

    Chowdhury, Rasheduzzaman; Leung, Ivanhoe K. H.; Tian, Ya-Min; Abboud, Martine I.; Ge, Wei; Domene, Carmen; Cantrelle, François-Xavier; Landrieu, Isabelle; Hardy, Adam P.; Pugh, Christopher W.; Ratcliffe, Peter J.; Claridge, Timothy D. W.; Schofield, Christopher J.

    2016-01-01

    The response to hypoxia in animals involves the expression of multiple genes regulated by the αβ-hypoxia-inducible transcription factors (HIFs). The hypoxia-sensing mechanism involves oxygen limited hydroxylation of prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of HIFα isoforms, as catalysed by prolyl hydroxylases (PHD 1–3). Prolyl hydroxylation promotes binding of HIFα to the von Hippel–Lindau protein (VHL)–elongin B/C complex, thus signalling for proteosomal degradation of HIFα. We reveal that certain PHD2 variants linked to familial erythrocytosis and cancer are highly selective for CODD or NODD. Crystalline and solution state studies coupled to kinetic and cellular analyses reveal how wild-type and variant PHDs achieve ODD selectivity via different dynamic interactions involving loop and C-terminal regions. The results inform on how HIF target gene selectivity is achieved and will be of use in developing selective PHD inhibitors. PMID:27561929

  15. High-performance liquid chromatographic method for the determination of prolyl peptides in urine.

    PubMed

    Codini, M; Palmerini, C A; Fini, C; Lucarelli, C; Floridi, A

    1991-01-04

    A rapid and accurate method is described for the determination of prolyl peptides in urine, with specific reference to the dipeptide prolylhydroxyproline, and free hydroxyproline and proline. Free amino acids and peptides were isolated from urine on cation-exchange minicolumns, and free imino acids and prolyl-N-terminal peptides were selectively derivatized with 4-chloro-7-nitrobenzofurazan, after reaction of amino acids and N-terminal aminoacyl peptides with o-phthalaldehyde. The highly fluorescent adducts of imino acids and prolyl peptides were separated on a Spherisorb ODS 2 column by isocratic elution for 12 min using as mobile phase 17.5 mM aqueous trifluoracetic acid solution containing 12.5% acetonitrile (eluent A), followed by gradient elution from eluent A to 40% of 17.5 mM aqueous trifluoroacetic acid solution containing 80% acetonitrile in 20 min. Analytes of interest, in particular the dipeptide prolylhydroxyproline, can be easily quantified by fluorimetric detection (epsilon ex = 470 nm, epsilon em = 530 nm) without interference from primary amino-containing compounds.

  16. Cellular Oxygen Sensing: Crystal Structure of Hypoxia-Inducible Factor Prolyl Hydroxylase (PHD2)

    SciTech Connect

    McDonough,M.; Li, V.; Flashman, E.; Chowdhury, R.; Mohr, C.; Lienard, B.; Zondlo, J.; Oldham, N.; Clifton, I.; et al.

    2006-01-01

    Cellular and physiological responses to changes in dioxygen levels in metazoans are mediated via the posttranslational oxidation of hypoxia-inducible transcription factor (HIF). Hydroxylation of conserved prolyl residues in the HIF-{alpha} subunit, catalyzed by HIF prolyl-hydroxylases (PHDs), signals for its proteasomal degradation. The requirement of the PHDs for dioxygen links changes in dioxygen levels with the transcriptional regulation of the gene array that enables the cellular response to chronic hypoxia; the PHDs thus act as an oxygen-sensing component of the HIF system, and their inhibition mimics the hypoxic response. We describe crystal structures of the catalytic domain of human PHD2, an important prolyl-4-hydroxylase in the human hypoxic response in normal cells, in complex with Fe(II) and an inhibitor to 1.7 Angstroms resolution. PHD2 crystallizes as a homotrimer and contains a double-stranded {beta}-helix core fold common to the Fe(II) and 2-oxoglutarate-dependant dioxygenase family, the residues of which are well conserved in the three human PHD enzymes (PHD 1-3). The structure provides insights into the hypoxic response, helps to rationalize a clinically observed mutation leading to familial erythrocytosis, and will aid in the design of PHD selective inhibitors for the treatment of anemia and ischemic disease.

  17. Gallate, the component of HIF-inducing catechins, inhibits HIF prolyl hydroxylase

    SciTech Connect

    Tsukiyama, Fuyo; Nakai, Yumi; Yoshida, Masataka; Tokuhara, Takahiro; Hirota, Kiichi; Sakai, Akiko; Hayashi, Hideyuki . E-mail: hayashi@art.osaka-med.ac.jp; Katsumata, Takahiro

    2006-12-08

    Catechins have recently been reported to increase the cellular content of the hypoxia-inducible factor (HIF)-1{alpha} within mammalian cells. These catechins have a gallate moiety as a common structure. We now report that n-propyl gallate (nPG) also increases the HIF-1{alpha} protein in the rat heart-derived H9c2 cells. The increase was dose-dependent and reached a maximum at 2-4 h after the addition of nPG to the cells. nPG did not change the HIF-1{alpha} mRNA level, showing that the increase is a posttranscriptional event. Although nPG did not inhibit the HIF prolyl hydroxylase, gallate, the hydrolysis product of nPG, inhibited the enzyme completely at submillimolar concentrations. Model building studies on the human HIF prolyl hydroxylase 2 showed that the two phenolate oxygen atoms of gallate form a chelate with the active site Fe{sup 2+}, while the carboxyl group of gallate forms a strong ionic/hydrogen bonding interaction with Arg383, explaining why nPG, which has an esterified carboxyl group, is unable to inhibit the hydroxylase. Together with the observation that gallate was detected in the H9c2 cells treated with nPG, these results suggest that nPG incorporated into the cells is hydrolyzed and the released gallate inhibits the HIF prolyl hydroxylase, thereby reducing the HIF degradation rate and increasing the HIF-1{alpha} content.

  18. Deimination level and peptidyl arginine deiminase 2 expression are elevated in astrocytes with increased incubation temperature.

    PubMed

    Enriquez-Algeciras, Mabel; Bhattacharya, Sanjoy K; Serra, Horacio M

    2015-09-01

    Astrocytes respond to environmental cues, including changes in temperatures. Increased deimination, observed in many progressive neurological diseases, is thought to be contributed by astrocytes. We determined the level of deimination and expression of peptidyl arginine deiminase 2 (PAD2) in isolated primary astrocytes in response to changes on either side (31°C and 41°C) of the optimal temperature (37°C). We investigated changes in the astrocytes by using a number of established markers and accounted for cell death with the CellTiter-Blue assay. We found increased expression of glial fibrillary acidic protein, ALDH1L1, and J1-31, resulting from increased incubation temperature and increased expression of TSP1, S100β, and AQP4, resulting from decreased incubation temperature vs. optimal temperature, suggesting activation of different biochemical pathways in astrocytes associated with different incubation temperatures. Mass spectrometric analyses support such trends. The PAD2 level was increased only as a result of increased incubation temperature with a commensurate increased level of deimination. Actin cytoskeleton and iso[4]LGE, a lipid peroxidase modification, also showed an increase with higher incubation temperature. Altogether, these results suggest that temperature, as an environmental cue, activates astrocytes in a different manner on either side of the optimal temperature and that increase in deimination is associated only with the higher temperature side of the spectrum.

  19. Enzymatic Modification of Soluble Cyanophycin Using the Type II Peptidyl Arginine Deiminase from Oryctolagus cuniculus.

    PubMed

    Wiefel, Lars; Steinbüchel, Alexander

    2016-07-01

    An increased structural variety expands the number of putative applications for cyanophycin (multi-l-arginyl-poly-[l-aspartic acid], CGP). Therefore, structural modifications of CGP are of major interest; these are commonly obtained by modification and optimization of the bacterial producing strain or by chemical modification. In this study, an enzymatic modification of arginine side chains from lysine-rich CGP is demonstrated using the peptidyl arginine deiminase from Oryctolagus cuniculus, purified from Escherichia coli after heterologous expression. About 10% of the arginine side chains are converted to citrulline which corresponds to 4% of the polymer's total side chains. An inhibition of the reaction in the presence of small amounts of l-citrulline is observed, thereby explaining the low conversion rate. CGP dipeptides can be modified with about 7.5 mol% of the Asp-Arg dipeptides being converted to Asp-Cit. These results show that the enzymatic modification of CGP is feasible, opening up a whole new area of possible CGP modifications for further research.

  20. Generating Functional Recombinant NRPS Enzymes in the Laboratory Setting via Peptidyl Carrier Protein Engineering.

    PubMed

    Owen, Jeremy G; Calcott, Mark J; Robins, Katherine J; Ackerley, David F

    2016-11-17

    Non-ribosomal peptide synthetases (NRPSs) are modular enzymatic assembly lines where substrates and intermediates undergo rounds of transformation catalyzed by adenylation (A), condensation (C), and thioesterase (TE) domains. Central to the NRPS biosynthesis are peptidyl carrier protein (PCP) domains, small, catalytically inactive domains that shuttle substrates and intermediates between the catalytic modules and govern product release from TE domains. There is strong interest in recombination of NRPS systems to generate new chemical entities. However, the intrinsic complexity of these systems has been a major challenge. Here, we employ domain substitution and random mutagenesis to recapitulate NRPS evolution, focusing on PCP domains. Using NRPS model systems that produce two different pigmented molecules, pyoverdine and indigoidine, we found that only evolutionarily specialized recombinant PCP domains could interact effectively with the native TE domain for product release. Overall, we highlight that substituted PCP domains require very minor changes to result in functional NRPSs, and infer that positive selection pressure may improve recombinant NRPS outcomes.

  1. Homochiral preference in peptide synthesis in ribosome: role of amino terminal, peptidyl terminal, and U2620.

    PubMed

    Thirumoorthy, Krishnan; Nandi, Nilashis

    2007-08-23

    Experimental studies have shown that peptide synthesis in ribosome exhibits a homochiral preference. We present, for the first time, an analysis of the origin of the phenomenon using hybrid quantum chemical studies based on a model of peptidyl transferase center from the crystal structure of the ribosomal part of Haloarcula marismortui. The study quantitatively shows that the observed homochiral preference is due to the difference in the nonbonded interaction between amino acids at the A- and P-terminals as well as due to the difference in interaction with the U2620 residue. A major part of the discrimination comes from the variation of nonbonded interaction of rotating A-terminal during the approach of the former toward the P-terminal. The difference indicates that, during the rotatory motion between A- and P-terminals for the proximal positioning of the reactant for reaction to occur, the interaction for a L-L pair is far less repulsive compared to the same process for a D-L pair. The activation barriers for L-L and D-L pairs of the neutral state of phenylalanine leading to corresponding dipeptides are also compared. The corresponding difference in rate constants is 40-fold. The study provides an understanding of how preferred addition of L-L pairs of amino acids rather than D-L pairs leads to retention of homochirality in peptides.

  2. Peptidyl alpha-keto amide inhibitor of calpain blocks excitotoxic damage without affecting signal transduction events.

    PubMed

    Caba, Ebru; Brown, Queenie B; Kawasaki, Brian; Bahr, Ben A

    2002-03-15

    The cysteine protease calpain is activated by calcium and has a wide range of substrates. Calpain-mediated cellular damage is associated with many neuropathologies, and calpain also plays a role in signal transduction events that are essential for cell maintenance, including the activation of important kinases and transcription factors. In the present study, the hippocampal slice culture was used as a model of excitotoxicity to test whether the neuroprotection elicited by selective calpain inhibition is associated with changes in cell signaling. Peptidyl alpha-keto amide and alpha-keto acid inhibitors reduced both calpain-mediated cytoskeletal damage and the concomitant synaptic deterioration resulting from an N-methyl-D-aspartate exposure. The alpha-keto amide CX295 was protective when infused into slice cultures before or after the excitotoxic episode. The slices protected with CX295 exhibited normal activation levels of mitogen-activated protein kinase and the transcription factor nuclear factor-kappaB. Thus, selective inhibition of calpain provides neuroprotection without influencing critical signaling pathways.

  3. Highly sensitive ratiometric detection of heparin and its oversulfated chondroitin sulfate contaminant by fluorescent peptidyl probe.

    PubMed

    Mehta, Pramod Kumar; Lee, Hyeri; Lee, Keun-Hyeung

    2017-05-15

    The selective and sensitive detection of heparin, an anticoagulant in clinics as well as its contaminant oversulfated chondroitin sulfate (OSCS) is of great importance. We first reported a ratiometric sensing method for heparin as well as OSCS contaminants in heparin using a fluorescent peptidyl probe (Pep1, pyrene-GSRKR) and heparin-digestive enzyme. Pep1 exhibited a highly sensitive ratiometric response to nanomolar concentration of heparin in aqueous solution over a wide pH range (2~11) and showed highly selective ratiometric response to heparin among biological competitors such as hyaluronic acid and chondroitin sulfate. Pep1 showed a linear ratiometric response to nanomolar concentrations of heparin in aqueous solutions and in human serum samples. The detection limit for heparin was calculated to be 2.46nM (R(2)=0.99) in aqueous solutions, 2.98nM (R(2)=0.98) in 1% serum samples, and 3.43nM (R(2)=0.99) in 5% serum samples. Pep1 was applied to detect the contaminated OSCS in heparin with heparinase I, II, and III, respectively. The ratiometric sensing method using Pep1 and heparinase II was highly sensitive, fast, and efficient for the detection of OSCS contaminant in heparin. Pep1 with heparinase II could detect as low as 0.0001% (w/w) of OSCS in heparin by a ratiometric response.

  4. Exploration of novel piperazine or piperidine constructed non-covalent peptidyl derivatives as proteasome inhibitors.

    PubMed

    Zhuang, Rangxiao; Gao, Lixin; Lv, Xiaoqing; Xi, Jianjun; Sheng, Li; Zhao, Yanmei; He, Ruoyu; Hu, Xiaobei; Shao, Yidan; Pan, Xuwang; Liu, Shourong; Huang, Weiwei; Zhou, Yubo; Li, Jia; Zhang, Jiankang

    2017-01-27

    A series of novel piperazine or piperidine-containing non-covalent peptidyl derivatives possessing a neopentyl-asparagine residue were designed, synthesized and evaluated as proteasome inhibitors. All target compounds were screened for their 20S proteasome chymotrypsin-like inhibitory activities, and 15 ones displayed more potent activities than carfilzomib with IC50 values lower than 10 nM. Subsequently, the most potent 10 analogues were tested for their cytotoxic activities against two multiple myeloma (MM) cell lines RPMI-8226 and MM-1S. Based on these experiments, selected derivatives were further evaluated for their ex vivo and in vivo blood cell proteasome inhibitory activities. The most potential compound 35 (proteasome inhibition IC50: 1.2 ± 0.1 nM) with potent anti-proliferation (IC50: RPMI-8226 8.4 ± 0.8 nM; MM-1S: 6.3 ± 0.8 nM), ex vivo and in vivo activities also had a prolonged half life in plasma, which demonstrated that the enzymatic stabilities of this series of compounds have been improved by constructing a six-membered ring into the peptide skeleton. All the experiments confirmed the correctness of design concept, which made this series of compounds potential leads for exploring new anti-MM drugs. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  5. The arginine attenuator peptide interferes with the ribosome peptidyl transferase center.

    PubMed

    Wei, Jiajie; Wu, Cheng; Sachs, Matthew S

    2012-07-01

    The fungal arginine attenuator peptide (AAP) is encoded by a regulatory upstream open reading frame (uORF). The AAP acts as a nascent peptide within the ribosome tunnel to stall translation in response to arginine (Arg). The effect of AAP and Arg on ribosome peptidyl transferase center (PTC) function was analyzed in Neurospora crassa and wheat germ translation extracts using the transfer of nascent AAP to puromycin as an assay. In the presence of a high concentration of Arg, the wild-type AAP inhibited PTC function, but a mutated AAP that lacked stalling activity did not. While AAP of wild-type length was most efficient at stalling ribosomes, based on primer extension inhibition (toeprint) assays and reporter synthesis assays, a window of inhibitory function spanning four residues was observed at the AAP's C terminus. The data indicate that inhibition of PTC function by the AAP in response to Arg is the basis for the AAP's function of stalling ribosomes at the uORF termination codon. Arg could interfere with PTC function by inhibiting peptidyltransferase activity and/or by restricting PTC A-site accessibility. The mode of PTC inhibition appears unusual because neither specific amino acids nor a specific nascent peptide chain length was required for AAP to inhibit PTC function.

  6. The Arginine Attenuator Peptide Interferes with the Ribosome Peptidyl Transferase Center

    PubMed Central

    Wei, Jiajie; Wu, Cheng

    2012-01-01

    The fungal arginine attenuator peptide (AAP) is encoded by a regulatory upstream open reading frame (uORF). The AAP acts as a nascent peptide within the ribosome tunnel to stall translation in response to arginine (Arg). The effect of AAP and Arg on ribosome peptidyl transferase center (PTC) function was analyzed in Neurospora crassa and wheat germ translation extracts using the transfer of nascent AAP to puromycin as an assay. In the presence of a high concentration of Arg, the wild-type AAP inhibited PTC function, but a mutated AAP that lacked stalling activity did not. While AAP of wild-type length was most efficient at stalling ribosomes, based on primer extension inhibition (toeprint) assays and reporter synthesis assays, a window of inhibitory function spanning four residues was observed at the AAP's C terminus. The data indicate that inhibition of PTC function by the AAP in response to Arg is the basis for the AAP's function of stalling ribosomes at the uORF termination codon. Arg could interfere with PTC function by inhibiting peptidyltransferase activity and/or by restricting PTC A-site accessibility. The mode of PTC inhibition appears unusual because neither specific amino acids nor a specific nascent peptide chain length was required for AAP to inhibit PTC function. PMID:22508989

  7. Changes produced by bound tryptophan in the ribosome peptidyl transferase center in response to TnaC, a nascent leader peptide

    PubMed Central

    Cruz-Vera, Luis Rogelio; Gong, Ming; Yanofsky, Charles

    2006-01-01

    Studies in vitro have established that free tryptophan induces tna operon expression by binding to the ribosome that has just completed synthesis of TnaC-tRNAPro, the peptidyl-tRNA precursor of the leader peptide of this operon. Tryptophan acts by inhibiting Release Factor 2-mediated cleavage of this peptidyl-tRNA at the tnaC stop codon. Here we analyze the ribosomal location of free tryptophan, the changes it produces in the ribosome, and the role of the nascent TnaC-tRNAPro peptide in facilitating tryptophan binding and induction. The positional changes of 23S rRNA nucleotides that occur during induction were detected by using methylation protection and binding/competition assays. The ribosome-TnaC-tRNAPro complexes analyzed were formed in vitro; they contained either wild-type TnaC-tRNAPro or its nonfunctional substitute, TnaC(W12R)-tRNAPro. Upon comparing these two peptidyl-tRNA-ribosome complexes, free tryptophan was found to block methylation of nucleotide A2572 of wild-type ribosome-TnaC-tRNAPro complexes but not of ribosome-TnaC(W12R)-tRNAPro complexes. Nucleotide A2572 is in the ribosomal peptidyl transferase center. Tryptophanol, a noninducing competitor of tryptophan, was ineffective in blocking A2572 methylation; however, it did reverse the protective effect of tryptophan. Free tryptophan inhibited puromycin cleavage of TnaC-tRNAPro; it also inhibited binding of the antibiotic sparsomycin. These effects were not observed with TnaC(W12R)-tRNAPro mutant complexes. These findings establish that Trp-12 of TnaC-tRNAPro is required for introducing specific changes in the peptidyl transferase center of the ribosome that activate free tryptophan binding, resulting in peptidyl transferase inhibition. Free tryptophan appears to act at or near the binding sites of several antibiotics in the peptidyl transferase center. PMID:16505360

  8. Hypoxia-inducible Factor Prolyl 4-Hydroxylase Inhibition A TARGET FOR NEUROPROTECTION IN THE CENTRAL NERVOUS SYSTEM*

    PubMed Central

    Siddiq, Ambreena; Ayoub, Issam A.; Chavez, Juan C.; Aminova, Leila; Shah, Sapan; LaManna, Joseph C.; Patton, Stephanie M.; Connor, James R.; Cherny, Robert A.; Volitakis, Irene; Bush, Ashley I.; Langsetmo, Ingrid; Seeley, Todd; Gunzler, Volkmar; Ratan, Rajiv R.

    2008-01-01

    Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases are a family of iron- and 2-oxoglutarate-dependent dioxygenases that negatively regulate the stability of several proteins that have established roles in adaptation to hypoxic or oxidative stress. These proteins include the transcriptional activators HIF-1α and HIF-2α. The ability of the inhibitors of HIF prolyl 4-hydroxylases to stabilize proteins involved in adaptation in neurons and to prevent neuronal injury remains unclear. We reported that structurally diverse low molecular weight or peptide inhibitors of the HIF prolyl 4-hydroxylases stabilize HIF-1α and up-regulate HIF-dependent target genes (e.g. enolase, p21waf1/cip1, vascular endothelial growth factor, or erythropoietin) in embryonic cortical neurons in vitro or in adult rat brains in vivo. We also showed that structurally diverse HIF prolyl 4-hydroxylase inhibitors prevent oxidative death in vitro and ischemic injury in vivo. Taken together these findings identified low molecular weight and peptide HIF prolyl 4-hydroxylase inhibitors as novel neurological therapeutics for stroke as well as other diseases associated with oxidative stress. PMID:16227210

  9. Peptidyl arginine deiminase-4 activation exacerbates kidney ischemia-reperfusion injury.

    PubMed

    Ham, Ahrom; Rabadi, May; Kim, Mihwa; Brown, Kevin M; Ma, Zhe; D'Agati, Vivette; Lee, H Thomas

    2014-11-01

    Peptidyl arginine deiminase (PAD)4 is a nuclear enzyme that catalyzes the posttranslational conversion of arginine residues to citrulline. Posttranslational protein citrullination has been implicated in several inflammatory autoimmune diseases, including rheumatoid arthritis, colitis, and multiple sclerosis. Here, we tested the hypothesis that PAD4 contributes to ischemic acute kidney injury (AKI) by exacerbating the inflammatory response after renal ischemia-reperfusion (I/R). Renal I/R injury in mice increased PAD4 activity as well as PAD4 expression in the mouse kidney. After 30 min of renal I/R, vehicle-treated mice developed severe AKI with large increases in plasma creatinine. In contrast, mice pretreated with PAD4 inhibitors (2-chloroamidine or streptonigrin) had significantly reduced renal I/R injury. Further supporting a critical role for PAD4 in generating ischemic AKI, mice pretreated with recombinant human PAD4 (rPAD4) protein and subjected to mild (20 min) renal I/R developed exacerbated ischemic AKI. Consistent with the hypothesis that PAD4 regulates renal tubular inflammation after I/R, mice treated with a PAD4 inhibitor had significantly reduced renal neutrophil chemotactic cytokine (macrophage inflammatory protein-2 and keratinocyte-derived cytokine) expression and had decreased neutrophil infiltration. Furthermore, mice treated with rPAD4 had significantly increased renal tubular macrophage inflammatory protein-2 and keratinocyte-derived cytokine expression as well as increased neutrophil infiltration and necrosis. Finally, cultured mouse kidney proximal tubules treated with rPAD4 had significantly increased proinflammatory chemokine expression compared with vehicle-treated cells. Taken together, our results suggest that PAD4 plays a critical role in renal I/R injury by increasing renal tubular inflammatory responses and neutrophil infiltration after renal I/R.

  10. Crystal structure of delta-chymotrypsin bound to a peptidyl chloromethyl ketone inhibitor.

    PubMed

    Mac Sweeney, A; Birrane, G; Walsh, M A; O'Connell, T; Malthouse, J P; Higgins, T M

    2000-03-01

    Chymotrypsin is a member of the trypsin family of serine proteases and is one of the first proteins successfully studied by X-ray crystallography. It is secreted into the intestine as the inactive precursor chymotrypsinogen; four sequential cleavages of the peptide bonds following residues 13, 15, 146 and 148 occur to generate the active pi, delta, kappa and alpha forms of chymotrypsin. (13)C NMR has shown [O'Connell & Malthouse (1995). Biochem. J. 307, 353-359] that when the delta form of chymotrypsin is inhibited by 2-(13)C-enriched benzyloxycarbonylglycylglycylphenylalanyl chloromethane, a tetrahedral adduct is formed which is thought to be analogous to the tetrahedral intermediate formed during catalysis. This inhibitor complex has been crystallized as a dimer in space group P4(1)2(1)2. The structure has been refined at 2.14 A resolution to an R value of 21.2% (free R = 25.2%). Conformational differences between delta-chymotrypsin and chymotrypsinogen in the region of the flexible autolysis loop (residues 145-150) were observed. This is the first crystal structure of delta-chymotrypsin and includes two residues which are disordered in previous crystal structures of active chymotrypsin. A difference of 11.3 A(2) between the average B values of the monomers within the asymmetric unit is caused by lattice-disordering effects approximating to rotation of the molecules about a crystallographic screw axis. The substrate-binding mode of the inhibitor was similar to other chymotrypsin peptidyl inhibitor complexes, but this is the first published chymotrypsin structure in which the tetrahedral chloromethyl ketone transition-state analogue is observed. This structure is compared with that of a similar tetrahedral transition-state analogue which does not alkylate the active-site histidine residue.

  11. Evaluation of diverse peptidyl motifs for cellular delivery of semiconductor quantum dots.

    PubMed

    Gemmill, Kelly Boeneman; Muttenthaler, Markus; Delehanty, James B; Stewart, Michael H; Susumu, Kimihiro; Dawson, Philip E; Medintz, Igor L

    2013-07-01

    Cell-penetrating peptides (CPPs) have rapidly become a mainstay technology for facilitating the delivery of a wide variety of nanomaterials to cells and tissues. Currently, the library of CPPs to choose from is still limited, with the HIV TAT-derived motif still being the most used. Among the many materials routinely delivered by CPPs, nanoparticles are of particular interest for a plethora of labeling, imaging, sensing, diagnostic, and therapeutic applications. The development of nanoparticle-based technologies for many of these uses will require access to a much larger number of functional peptide motifs that can both facilitate cellular delivery of different types of nanoparticles to cells and be used interchangeably in the presence of other peptides and proteins on the same surface. Here, we evaluate the utility of four peptidyl motifs for their ability to facilitate delivery of luminescent semiconductor quantum dots (QDs) in a model cell culture system. We find that an LAH4 motif, derived from a membrane-inserting antimicrobial peptide, and a chimeric sequence that combines a sweet arrow peptide with a portion originating from the superoxide dismutase enzyme provide effective cellular delivery of QDs. Interestingly, a derivative of the latter sequence lacking just a methyl group was found to be quite inefficient, suggesting that even small changes can have significant functional outcomes. Delivery was effected using 1 h incubation with cells, and fluorescent counterstaining strongly suggests an endosomal uptake process that requires a critical minimum number or ratio of peptides to be displayed on the QD surface. Concomitant cytoviability testing showed that the QD-peptide conjugates are minimally cytotoxic in the model COS-1 cell line tested. Potential applications of these peptides in the context of cellular delivery of nanoparticles and a variety of other (bio)molecules are discussed.

  12. Peptidyl arginine deiminase-4 activation exacerbates kidney ischemia-reperfusion injury

    PubMed Central

    Ham, Ahrom; Rabadi, May; Kim, Mihwa; Brown, Kevin M.; Ma, Zhe; D'Agati, Vivette

    2014-01-01

    Peptidyl arginine deiminase (PAD)4 is a nuclear enzyme that catalyzes the posttranslational conversion of arginine residues to citrulline. Posttranslational protein citrullination has been implicated in several inflammatory autoimmune diseases, including rheumatoid arthritis, colitis, and multiple sclerosis. Here, we tested the hypothesis that PAD4 contributes to ischemic acute kidney injury (AKI) by exacerbating the inflammatory response after renal ischemia-reperfusion (I/R). Renal I/R injury in mice increased PAD4 activity as well as PAD4 expression in the mouse kidney. After 30 min of renal I/R, vehicle-treated mice developed severe AKI with large increases in plasma creatinine. In contrast, mice pretreated with PAD4 inhibitors (2-chloroamidine or streptonigrin) had significantly reduced renal I/R injury. Further supporting a critical role for PAD4 in generating ischemic AKI, mice pretreated with recombinant human PAD4 (rPAD4) protein and subjected to mild (20 min) renal I/R developed exacerbated ischemic AKI. Consistent with the hypothesis that PAD4 regulates renal tubular inflammation after I/R, mice treated with a PAD4 inhibitor had significantly reduced renal neutrophil chemotactic cytokine (macrophage inflammatory protein-2 and keratinocyte-derived cytokine) expression and had decreased neutrophil infiltration. Furthermore, mice treated with rPAD4 had significantly increased renal tubular macrophage inflammatory protein-2 and keratinocyte-derived cytokine expression as well as increased neutrophil infiltration and necrosis. Finally, cultured mouse kidney proximal tubules treated with rPAD4 had significantly increased proinflammatory chemokine expression compared with vehicle-treated cells. Taken together, our results suggest that PAD4 plays a critical role in renal I/R injury by increasing renal tubular inflammatory responses and neutrophil infiltration after renal I/R. PMID:25164081

  13. 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.

  14. Understanding the role of PIN1 in hepatocellular carcinoma

    PubMed Central

    Cheng, Chi-Wai; Leong, Ka-Wai; Tse, Eric

    2016-01-01

    PIN1 is a peptidyl-prolyl cis/trans isomerase that binds and catalyses isomerization of the specific motif comprising a phosphorylated serine or threonine residue preceding a proline (pSer/Thr-Pro) in proteins. PIN1 can therefore induce conformational and functional changes of its interacting proteins that are regulated by proline-directed serine/threonine phosphorylation. Through this phosphorylation-dependent prolyl isomerization, PIN1 fine-tunes the functions of key phosphoproteins (e.g., cyclin D1, survivin, β-catenin and x-protein of hepatitis B virus) that are involved in the regulation of cell cycle progression, apoptosis, proliferation and oncogenic transformation. PIN1 has been found to be over-expressed in many cancers, including human hepatocellular carcinoma (HCC). It has been shown previously that overexpression of PIN1 contributes to the development of HCC in-vitro and in xenograft mouse model. In this review, we first discussed the aberrant transcription factor expression, miRNAs dysregulation, PIN1 gene promoter polymorphisms and phosphorylation of PIN1 as potential mechanisms underlying PIN1 overexpression in cancers. Furthermore, we also examined the role of PIN1 in HCC tumourigenesis by reviewing the interactions between PIN1 and various cellular and viral proteins that are involved in β-catenin, NOTCH, and PI3K/Akt/mTOR pathways, apoptosis, angiogenesis and epithelial-mesenchymal transition. Finally, the potential of PIN1 inhibitors as an anti-cancer therapy was explored and discussed. PMID:28018099

  15. 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

  16. Alterations at the peptidyl transferase centre of the ribosome induced by the synergistic action of the streptogramins dalfopristin and quinupristin

    PubMed Central

    Harms, Jörg M; Schlünzen, Frank; Fucini, Paola; Bartels, Heike; Yonath, Ada

    2004-01-01

    Background The bacterial ribosome is a primary target of several classes of antibiotics. Investigation of the structure of the ribosomal subunits in complex with different antibiotics can reveal the mode of inhibition of ribosomal protein synthesis. Analysis of the interactions between antibiotics and the ribosome permits investigation of the specific effect of modifications leading to antimicrobial resistances. Streptogramins are unique among the ribosome-targeting antibiotics because they consist of two components, streptogramins A and B, which act synergistically. Each compound alone exhibits a weak bacteriostatic activity, whereas the combination can act bactericidal. The streptogramins A display a prolonged activity that even persists after removal of the drug. However, the mode of activity of the streptogramins has not yet been fully elucidated, despite a plethora of biochemical and structural data. Results The investigation of the crystal structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with the clinically relevant streptogramins quinupristin and dalfopristin reveals their unique inhibitory mechanism. Quinupristin, a streptogramin B compound, binds in the ribosomal exit tunnel in a similar manner and position as the macrolides, suggesting a similar inhibitory mechanism, namely blockage of the ribosomal tunnel. Dalfopristin, the corresponding streptogramin A compound, binds close to quinupristin directly within the peptidyl transferase centre affecting both A- and P-site occupation by tRNA molecules. Conclusions The crystal structure indicates that the synergistic effect derives from direct interaction between both compounds and shared contacts with a single nucleotide, A2062. Upon binding of the streptogramins, the peptidyl transferase centre undergoes a significant conformational transition, which leads to a stable, non-productive orientation of the universally conserved U2585. Mutations of this rRNA base are known to yield

  17. Assay of prolyl 4-hydroxylase by the chromatographic determination of [14C]succinic acid on ion-exchange minicolumns.

    PubMed Central

    Cunliffe, C J; Franklin, T J; Gaskell, R M

    1986-01-01

    An assay for prolyl 4-hydroxylase (EC 1.14.11.2) is described which measures succinic acid produced during the decarboxylation of 2-oxoglutaric acid in the presence of poly(L-Pro-Gly-L-Pro). [1-14C]Succinic acid was separated from its precursor 2-oxo[5-14C]glutaric acid by using ion-exchange minicolumns. The contamination of succinic acid by 2-oxoglutaric acid was approx. 1%, and the recovery of succinic acid was 100%. Kinetic parameters of prolyl 4-hydroxylase measured by the assay showed good agreement with published values. Our experience indicates that the measurement of prolyl 4-hydroxylase by the production of succinic acid is especially suited to investigations involving large numbers of assays. PMID:3028379

  18. Intracellular Delivery of Peptidyl Ligands by Reversible Cyclization: Discovery of a PDZ Domain Inhibitor that Rescues CFTR Activity**

    PubMed Central

    Qian, Ziqing; Xu, Xiaohua; Amacher, Jeanine F.; Madden, Dean R.; Cormet-Boyaka, Estelle

    2015-01-01

    We report a general strategy for intracellular delivery of linear peptidyl ligands by fusing them with a cell-penetrating peptide and cyclizing the fusion peptides through a disulfide bond. The resulting cyclic peptides are cell permeable and have improved proteolytic stability. Once inside the cell, the disulfide bond is reduced to produce linear, biologically active peptides. This strategy was applied to generate a cell-permeable peptide substrate for real-time detection of intracellular caspase activities during apoptosis and a CAL-PDZ domain inhibitor for potential treatment of cystic fibrosis. PMID:25785567

  19. Substrate-dependent, non-hyperbolic kinetics of pig brain prolyl oligopeptidase and its tight binding inhibition by JTP-4819.

    PubMed

    Venäläinen, Jarkko I; Juvonen, Risto O; Forsberg, Markus M; Garcia-Horsman, Arturo; Poso, Antti; Wallen, Erik A A; Gynther, Jukka; Männistö, Pekka T

    2002-08-01

    Prolyl oligopeptidase (POP) is a cytosolic serine protease that hydrolyses small peptides at the carboxyl end of the proline residue. It has raised pharmaceutical interest, since its inhibitors have been shown to have antiamnesic properties. We studied prolyl oligopeptidase kinetics with two 7-amino-4-methylcoumarin derivatives: Z-Gly-Pro-AMC and Suc-Gly-Pro-AMC. Z-Gly-Pro-AMC was found to obey standard Henri-Michaelis-Menten kinetics with a K(m) of 30+/-3 microM, whereas Suc-Gly-Pro-AMC exhibited substrate inhibition kinetics with K(m) and K(is) of 510+/-150 and 270+/-90 microM, respectively. Autodock simulations revealed that either the succinyl or the AMC-end of Suc-Gly-Pro-AMC may bind to the S'1 subsite of the active site. We believe that non-specifically bound Suc-Gly-Pro-AMC allows the simultaneous binding of second substrate molecule to the active site and this leads in substrate inhibition. In addition, we demonstrated that the inhibition type of a well characterized prolyl oligopeptidase inhibitor, JTP-4819, is competitive tight binding with a K(ic) of 0.045+/-0.008 nM. We suggest that due to the high concentration of prolyl oligopeptidase in the brain (0.12 nmol/g pig brain), the tight binding nature of the inhibition should be considered when using brain homogenate as the enzyme source in prolyl oligopeptidase inhibition measurements. This is of importance in studying structure-activity relationships of potent prolyl oligopeptidase inhibitors.

  20. Comparative biochemical analysis of three bacterial prolyl endopeptidases: implications for coeliac sprue.

    PubMed

    Shan, Lu; Marti, Thomas; Sollid, Ludvig M; Gray, Gary M; Khosla, Chaitan

    2004-10-15

    Prolyl endopeptidases have potential for treating coeliac sprue, a disease of the intestine caused by proteolytically resistant peptides from proline-rich prolamins of wheat, barley and rye. We compared the properties of three similar bacterial prolyl endopeptidases, including the known enzymes from Flavobacterium meningosepticum (FM) and Sphingomonas capsulate (SC) and a novel enzyme from Myxococcus xanthus (MX). These enzymes were interrogated with reference chromogenic substrates, as well as two related gluten peptides (PQPQLPYPQPQLP and LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF), believed to play a key role in coeliac sprue pathogenesis. In vitro and in vivo studies were conducted to evaluate the activity, specificity and acid/protease stability of the enzymes. All peptidases were relatively resistant to acid, pancreatic proteases and membrane peptidases of the small intestinal mucosa. Although their activities against reference substrates were similar, the enzymes exhibited substantial differences with respect to chain length and subsite specificity. SC hydrolysed PQPQLPYPQPQLP well, but had negligible activity against LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF. In contrast, the FM and MX peptidases cleaved both substrates, although the FM enzyme acted more rapidly on LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF than MX. Whereas the FM enzyme showed a preference for Pro-Gln bonds, SC cleaved both Pro-Gln and Pro-Tyr bonds with comparable efficiency, and MX had a modest preference for Pro-(Tyr/Phe) sites over Pro-Gln sites. While a more comprehensive understanding of sequence and chain-length specificity may be needed to assess the relative utility of alternative prolyl endopeptidases for treating coeliac sprue, our present work has illustrated the diverse nature of this class of enzymes from the standpoint of proteolysing complex substrates such as gluten.

  1. The structural basis of cysteine aminoacylation of tRNAPro by prolyl-tRNA synthetases

    PubMed Central

    Kamtekar, Satwik; Kennedy, W. Dexter; Wang, Jimin; Stathopoulos, Constantinos; Söll, Dieter; Steitz, Thomas A.

    2003-01-01

    Cysteinyl-tRNA synthetase is an essential enzyme required for protein synthesis. Genes encoding this protein have not been identified in Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, or Methanopyrus kandleri. It has previously been proposed that the prolyl-tRNA synthetase (ProRS) enzymes in these organisms recognize either proline or cysteine and can aminoacylate their cognate tRNAs through a dual-specificity mechanism. We report five crystal structures at resolutions between 2.6 and 3.2 Å: apo M. jannaschii ProRS, and M. thermautotrophicus ProRS in apo form and in complex with cysteinyl-sulfamoyl-, prolyl-sulfamoyl-, and alanyl-sulfamoyl-adenylates. These aminoacyl-adenylate analogues bind to a single active-site pocket and induce an identical set of conformational changes in loops around the active site when compared with the ligand-free conformation of ProRS. The cysteinyl- and prolyl-adenylate analogues have similar, nanomolar affinities for M. thermautotrophicus ProRS. Homology modeling of tRNA onto these adenylate complexes places the 3′-OH of A76 in an appropriate position for the transfer of any of the three amino acids to tRNA. Thus, these structures explain recent biochemical experiments showing that M. jannaschii ProRS misacylates tRNAPro with cysteine, and argue against the proposal that these archaeal ProRS enzymes possess the dual capacity to aminoacylate both tRNAPro and tRNACys with their cognate amino acids. PMID:12578991

  2. The site of hydrolysis by rabbit reticulocyte peptidyl-tRNA hydrolase is the 3'-AMP terminus of susceptible tRNA substrates.

    PubMed

    Gross, M; Crow, P; White, J

    1992-01-25

    The preceding paper (Gross, M., Starn, T.K., Rundquist, C., Crow, P., White, J., Olin, A., and Wagner, T. (1992) J. Biol. Chem. 267, 2073-2079) reported the purification and partial characterization of rabbit reticulocyte peptidyl-tRNA hydrolase. In this article we demonstrate that, unlike bacterial and yeast peptidyl-tRNA hydrolase which act by deacylation, the reticulocyte enzyme hydrolyzes N-acylaminoacyl-tRNA to N-acylaminoacyl-AMP. Reticulocyte lysate has a separate enzyme, that we have isolated and termed aminoacyl-AMP deacylase, which hydrolyzes N-acylaminoacyl-AMP and aminoacyl-AMP, recycling the amino acid and nucleotide components. The action of this enzyme is relatively specific for the N-acylaminoacyl-AMP generated by peptidyl-tRNA hydrolase, since it is much less active with N-acylaminoacyl-adenosine and inactive with N-acylaminoacyl-ACCAC, N-acylaminoacyl-tRNA, or aminoacyl-tRNA. The tRNA product of peptidyl-tRNA hydrolase action is tRNA missing only its 3'-AMP terminus (tRNA(c-c)), since reaminoacylation requires tRNA nucleotidyltransferase but not CTP. The 3' exonucleolytic action of reticulocyte peptidyl-tRNA hydrolase is specific to susceptible tRNA substrates, since it does not hydrolyze CACCA, CACCA-N-acylamino acid, polyuridylic acid, or the 3' polyadenylate tail of globin mRNA, and, since its ability to hydrolyze Escherichia coli f[3H]Met-tRNA(fMet) is not reduced by excess 5 S or 28 S ribosomal RNA and is reduced only slightly by excess tRNA(c-c). Reticulocyte peptidyl-tRNA hydrolase also hydrolyzes th 3'-AMP terminus of deacylated tRNA. This property may explain why the 3'-terminal AMP of tRNA undergoes turnover in reticulocytes and reticulocyte lysate, since we find that such turnover in gel-filtered reticulocyte lysate is increased under conditions where aminoacylation is reduced.

  3. Anticoagulant Effects of Heparin Complexes with Prolyl-Glycine Peptide and Glycine and Proline Amino Acids.

    PubMed

    Grigorieva, M E; Obergan, T Yu; Maystrenko, E S; Kalugina, M D

    2016-05-01

    The study demonstrates the formation of heparin complexes with prolyl-glycine peptide and proline and glycine amino acids. The method was developed for in vitro production of these complexes at 1:1 dipeptide to heparin molar ratio and 2:1 amino acid to heparin molar ratio. These complexes, unlike the constituents, proline and glycine, exhibited significant anticoagulant, antiplatelet, and fibrin-depolymerization activities of varying degree in vitro and in vivo. The heparin-dipeptide complex produced maximum effect. The dipeptide by itself also showed anticoagulant properties, but less pronounced than in the complex with heparin.

  4. The adaptation of diastereomeric S-prolyl dipeptide derivatives to the quantitative estimation of R- and S-leucine enantiomers.

    NASA Technical Reports Server (NTRS)

    Bonner, W. A.

    1972-01-01

    Description of methods developed for the preparation of N-TFA-S(-)prolyl chloride and for synthesizing from it N-TFA-S-prolyl(and S)-leucine methyl ester diastereomers without detectible racemization. These diastereomers prepared by these methods were subjected to GC analyses under conditions permitting baseline separation of two diastereomeric peaks, while peak areas were measured with an electronic digital integrator. Under these conditions, it was found that the known enantiomeric compositions could be duplicated experimentally to an absolute error of only 0.0 to 0.6% over the entire composition range.

  5. The adaptation of diastereomeric S-prolyl dipeptide derivatives to the quantitative estimation of R- and S-leucine enantiomers.

    NASA Technical Reports Server (NTRS)

    Bonner, W. A.

    1972-01-01

    Description of methods developed for the preparation of N-TFA-S(-)prolyl chloride and for synthesizing from it N-TFA-S-prolyl(and S)-leucine methyl ester diastereomers without detectible racemization. These diastereomers prepared by these methods were subjected to GC analyses under conditions permitting baseline separation of two diastereomeric peaks, while peak areas were measured with an electronic digital integrator. Under these conditions, it was found that the known enantiomeric compositions could be duplicated experimentally to an absolute error of only 0.0 to 0.6% over the entire composition range.

  6. An indigenous posttranscriptional modification in the ribosomal peptidyl transferase center confers resistance to an array of protein synthesis inhibitors

    PubMed Central

    Toh, Seok-Ming; Mankin, Alexander S.

    2017-01-01

    A number of nucleotide residues in ribosomal RNA undergo specific posttranscriptional modification. The roles of most modifications are unclear, but their clustering in the functionally-important regions of rRNA suggest that they might either directly affect the activity or assembly of the ribosome or modulate its interactions with ligands. Of the 25 modified nucleotides in E. coli 23S rRNA, 14 are located in the peptidyl transferase center, the main antibiotic target in the large ribosomal subunit. Since nucleotide modifications have been closely associated with both antibiotic sensitivity and antibiotic resistance, the loss of some of these posttranscriptional modifications may affect the susceptibility of bacteria to antibiotics. We investigated the antibiotic sensitivity of E. coli cells in which the genes of eight rRNA modifying enzymes targeting the PTC were individually inactivated. The lack of pseudouridine at position 2504 of 23S rRNA was found to significantly increase the susceptibility of bacteria to peptidyl transferase inhibitors. Therefore, this indigenous posttranscriptional modification may have evolved as an intrinsic resistance mechanism protecting bacteria against natural antibiotics. PMID:18554609

  7. Inhibition of the CaaX proteases Rce1p and Ste24p by peptidyl (acyloxy)methyl ketones

    PubMed Central

    Porter, Stephen B.; Hildebrandt, Emily R.; Breevoort, Sarah R.; Mokry, David Z.; Dore, Timothy M.; Schmidt, Walter K.

    2007-01-01

    The CaaX proteases Rce1p and Ste24p can independently promote a proteolytic step required for the maturation of certain isoprenylated proteins. Although functionally related, Rce1p and Ste24p are unrelated in primary sequence. They have distinct enzymatic properties, which are reflected in part by their distinct inhibitor profiles. Moreover, Rce1p has an undefined catalytic mechanism, whereas Ste24p is an established zinc-dependent metalloprotease. This study demonstrates that both enzymes are inhibited by peptidyl (acyloxy)methyl ketones (AOMKs), making these compounds the first documented dual specificity inhibitors of the CaaX proteases. Further investigation of AOMK-mediated inhibition reveals that varying the peptidyl moiety can significantly alter the inhibitory properties of AOMKs toward Rce1p and Ste24p and that these enzymes display subtle differences in sensitivity to AOMKs. This observation suggests that this compound class could potentially be engineered to be selective for either of the CaaX proteases. We also demonstrate that the reported sensitivity of Rce1p to TPCK is substrate-dependent, which significantly alters the interpretation of certain reports having used TPCK sensitivity for mechanistic classification of Rce1p. Finally, we show that an AOMK inhibits the isoprenylcysteine carboxyl methyltransferase Ste14p. In sum, our observations raise important considerations regarding the specificity of agents targeting enzymes involved in the maturation of isoprenylated proteins, some of which are being developed as anti-cancer therapeutic agents. PMID:17467817

  8. Complexity generation in fungal peptidyl alkaloid biosynthesis: a two-enzyme pathway to the hexacyclic MDR export pump inhibitor ardeemin

    PubMed Central

    Haynes, Stuart W.; Gao, Xue; Tang, Yi; Walsh, Christopher T.

    2013-01-01

    Ardeemins are hexacyclic peptidyl alkaloids isolated from Aspergillus fischeri as agents that block efflux of anticancer drugs by (MultiDrug Resistance) MDR export pumps. To evaluate the biosynthetic logic and enzymatic machinery for ardeemin framework assembly, we sequenced the A. fischeri genome and identified the ardABC gene cluster. Through both genetic deletions and biochemical characterizations of purified ArdA and ArdB we show this ArdAB enzyme pair is sufficient to convert anthranilate (Ant), l-Ala and l-Trp to ardeemin. ArdA is a 430 kDa trimodular nonribosomal peptide synthase (NRPS) that converts the three building blocks into a fumiquinazoline (FQ) regioisomer termed ardeemin FQ. ArdB is a prenyltransferase that takes tricyclic ardeemin FQ and dimethylallyl diphosphate to the hexacyclic ardeemin scaffold via prenylation at C2 of the Trp-derived indole moiety with intramolecular capture by an amide NH of the fumiquinazoline ring. The two-enzyme ArdAB pathway reveals remarkable efficiency in construction of the hexacyclic peptidyl alkaloid scaffold. PMID:23330675

  9. Sites of interaction of the CCA end of peptidyl-tRNA with 23S rRNA.

    PubMed

    Moazed, D; Noller, H F

    1991-05-01

    Oligonucleotide fragments derived from the 3' CCA terminus of acylated tRNA, such as CACCA-(AcPhe), UACCA-(AcLeu), and CAACCA-(fMet), bind specifically to ribosomes in the presence of sparsomycin and methanol [Monro, R. E., Celma, M. L. & Vazquez, D. (1969) Nature (London) 222, 356-358]. All three oligonucleotides protect a characteristic set of bases in 23S rRNA from chemical probes: G2252, G2253, A2439, A2451, U2506, and U2585. A2602 shows enhanced reactivity. These account for most of the same bases that are protected when peptidyl-tRNA analogues such as AcPhe-tRNA are bound to the ribosomal P site, and correspond precisely to those bases whose protection is abolished by removal of the 3'-CA end of tRNA. We conclude that most of the observed interactions between tRNA and 23S rRNA in the 50S ribosomal P site involve the conserved CCA terminus of tRNA. Sparsomycin may inhibit protein synthesis by stabilizing interaction between the peptidyl-CCA and the 23S P site, preventing formation of the intermediate A/P hybrid state.

  10. Sites of interaction of the CCA end of peptidyl-tRNA with 23S rRNA.

    PubMed Central

    Moazed, D; Noller, H F

    1991-01-01

    Oligonucleotide fragments derived from the 3' CCA terminus of acylated tRNA, such as CACCA-(AcPhe), UACCA-(AcLeu), and CAACCA-(fMet), bind specifically to ribosomes in the presence of sparsomycin and methanol [Monro, R. E., Celma, M. L. & Vazquez, D. (1969) Nature (London) 222, 356-358]. All three oligonucleotides protect a characteristic set of bases in 23S rRNA from chemical probes: G2252, G2253, A2439, A2451, U2506, and U2585. A2602 shows enhanced reactivity. These account for most of the same bases that are protected when peptidyl-tRNA analogues such as AcPhe-tRNA are bound to the ribosomal P site, and correspond precisely to those bases whose protection is abolished by removal of the 3'-CA end of tRNA. We conclude that most of the observed interactions between tRNA and 23S rRNA in the 50S ribosomal P site involve the conserved CCA terminus of tRNA. Sparsomycin may inhibit protein synthesis by stabilizing interaction between the peptidyl-CCA and the 23S P site, preventing formation of the intermediate A/P hybrid state. Images PMID:2023922

  11. Alkaloids from Peumus boldus and their acetylcholinesterase, butyrylcholinesterase and prolyl oligopeptidase inhibition activity.

    PubMed

    Hošt'álková, Anna; Opletal, Lubomír; Kuneš, Jiří; Novák, Zdeněk; Hrabinová, Martina; Chlebek, Jakub; Čegan, Lukáš; Cahlíková, Lucie

    2015-04-01

    Eleven isoquinoline alkaloids (1-11) were isolated from dried leaves of Peumus boldus Mol. by standard chromatographic methods. The chemical structures were elucidated by MS, and 1D and 2D NMR spectroscopic analysis, and by comparison with literature data. Compounds isolated in sufficient amount were evaluated for their acetylcholinesterase, and butyrylcholinesterase inhibition activity using Ellman's method. In the prolyl oligopeptidase assay, Z-Gly-Pro-p-nitroanilide was used as substrate. Promising butyrylcholinesterase inhibition activities were demonstrated by two benzylisoquinoline alkaloids, reticuline (8) and N-methylcoclaurine (9), with IC50 values of 33.6 ± 3.0 µM and 15.0 ± 1.4 µM, respectively. Important prolyl oligopeptidase inhibition activities were shown by N-methyllaurotetanine (6) and sinoacutine (4) with IC50 values of 135.4 ± 23.2 µM and 143.1 ± 25.4 µM, respectively. Other tested compounds were considered inactive.

  12. Elicitor-induced prolyl hydroxylase from French bean (Phaseolus vulgaris). Localization, purification and properties.

    PubMed

    Bolwell, G P; Robbins, M P; Dixon, R A

    1985-08-01

    The enzyme prolyl hydroxylase (proline: 2-oxoglutarate dioxygenase, EC 1.14.11.12), induced in suspension-cultured cells of Phaseolus vulgaris L. (French bean) by treatment with an elicitor preparation from the phytopathogenic fungus Colletotrichum lindemuthianum, has been investigated. The enzyme, which catalyses the hydroxylation of poly-L-proline with the stoichiometric decarboxylation of 2-oxoglutarate, has been shown to be localized mainly in smooth endoplasmic reticulum. After solubilization from microsomal membranes, the hydroxylase was purified by ion-exchange chromatography and affinity chromatography on poly-L-proline-Sepharose 4B. The subunit Mr, as assessed by sodium dodecyl sulphate/poly-acrylamide-gel electrophoresis, was 65 000, the subunit apparently being recovered as a doublet: the subunits associate under non-denaturing conditions to give at least a tetramer. The bean hydroxylase has kinetic properties and cofactor requirements similar to those previously reported for the enzyme from other plants. Elicitor treatment of suspension-cultured bean cells leads to a rapid induction of prolyl hydroxylase activity concomitant with induction of a protein: arabinosyl-transferase and increased levels of an arabinosylated hydroxyproline-rich protein.

  13. Prolyl hydroxylase 2 inactivation enhances glycogen storage and promotes excessive neutrophilic responses.

    PubMed

    Sadiku, Pranvera; Willson, Joseph A; Dickinson, Rebecca S; Murphy, Fiona; Harris, Alison J; Lewis, Amy; Sammut, David; Mirchandani, Ananda S; Ryan, Eilise; Watts, Emily R; Thompson, A A Roger; Marriott, Helen M; Dockrell, David H; Taylor, Cormac T; Schneider, Martin; Maxwell, Patrick H; Chilvers, Edwin R; Mazzone, Massimilliano; Moral, Veronica; Pugh, Chris W; Ratcliffe, Peter J; Schofield, Christopher J; Ghesquiere, Bart; Carmeliet, Peter; Whyte, Moira Kb; Walmsley, Sarah R

    2017-09-01

    Fully activated innate immune cells are required for effective responses to infection, but their prompt deactivation and removal are essential for limiting tissue damage. Here, we have identified a critical role for the prolyl hydroxylase enzyme Phd2 in maintaining the balance between appropriate, predominantly neutrophil-mediated pathogen clearance and resolution of the innate immune response. We demonstrate that myeloid-specific loss of Phd2 resulted in an exaggerated inflammatory response to Streptococcus pneumonia, with increases in neutrophil motility, functional capacity, and survival. These enhanced neutrophil responses were dependent upon increases in glycolytic flux and glycogen stores. Systemic administration of a HIF-prolyl hydroxylase inhibitor replicated the Phd2-deficient phenotype of delayed inflammation resolution. Together, these data identify Phd2 as the dominant HIF-hydroxylase in neutrophils under normoxic conditions and link intrinsic regulation of glycolysis and glycogen stores to the resolution of neutrophil-mediated inflammatory responses. These results demonstrate the therapeutic potential of targeting metabolic pathways in the treatment of inflammatory disease.

  14. A family with erythrocytosis establishes a role for prolyl hydroxylase domain protein 2 in oxygen homeostasis.

    PubMed

    Percy, Melanie J; Zhao, Quan; Flores, Adrian; Harrison, Claire; Lappin, Terence R J; Maxwell, Patrick H; McMullin, Mary Frances; Lee, Frank S

    2006-01-17

    The number of red blood cells is normally tightly regulated by a classic homeostatic mechanism based on oxygen sensing in the kidney. Decreased oxygen delivery resulting from anemia induces the production of erythropoietin, which increases red cell production and hence oxygen delivery. Investigations of erythropoietin regulation identified the transcription factor hypoxia-inducible factor (HIF). HIF is now recognized as being a key regulator of genes that function in a comprehensive range of processes besides erythropoiesis, including energy metabolism and angiogenesis. HIF itself is regulated through the alpha-subunit, which is hydroxylated in the presence of oxygen by a family of three prolyl hydroxylase domain proteins (PHDs)/HIF prolyl hydroxylases/egg-laying-defective nine enzymes. Hydroxylation allows capture by the von Hippel-Lindau tumor suppressor gene product, ubiquitination, and destruction by the proteasome. Here we describe an inherited mutation in a mammalian PHD enzyme. We show that this mutation in PHD2 results in a marked decrease in enzyme activity and is associated with familial erythrocytosis, identifying a previously unrecognized cause of this condition. Our findings indicate that PHD2 is critical for normal regulation of HIF in humans.

  15. Contribution of epithelial innate immunity to systemic protection afforded by prolyl hydroxylase inhibition in murine colitis

    PubMed Central

    Keely, Simon; Campbell, Eric L.; Baird, Alan W.; Hansbro, Philip M.; Shalwitz, Robert A.; Kotsakis, Anna; McNamee, Eoin N.; Eltzschig, Holger K.; Kominsky, Douglas J.; Colgan, Sean P.

    2013-01-01

    Pharmacological stabilization of hypoxia-inducible factor (HIF) through prolyl hydroxylase (PHD) inhibition limits mucosal damage associated with models of murine colitis. However, little is known about how PHD inhibitors (PHDi) influence systemic immune function during mucosal inflammation or the relative importance of immunological changes to mucosal protection. We hypothesized that PHDi enhances systemic innate immune responses to colitis-associated bacteremia. Mice with colitis induced by TNBS were treated with AKB-4924, a new HIF-1 isoform-predominant PHDi and clinical, immunological and biochemical endpoints were assessed. Administration of AKB-4924 led to significantly reduced weight loss and disease activity compared to vehicle controls. Treated groups were pyrexic, but did not become subsequently hypothermic. PHDi treatment augmented epithelial barrier function and led to an approximately 50-fold reduction in serum endotoxin during colitis. AKB-4924 also decreased cytokines involved in pyrogenesis and hypothermia, significantly reducing serum levels of IL-1β, IL-6 and TNF-α, while increasing IL-10. Treatment offered no protection against colitis in epithelial-specific HIF-1α deficient mice, strongly implicating epithelial HIF-1α as the tissue target for AKB-4924-mediated protection. Taken together, these results indicate that inhibition of prolyl hydroxylase with AKB-4924 enhances innate immunity and identifies the epithelium is a central site of inflammatory protection afforded by PHDi in murine colitis. PMID:23695513

  16. 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

  17. Cloning and characterization of the rat HIF-1 alpha prolyl-4-hydroxylase-1 gene.

    PubMed

    Cobb, Ronald R; McClary, John; Manzana, Warren; Finster, Silke; Larsen, Brent; Blasko, Eric; Pearson, Jennifer; Biancalana, Sara; Kauser, Katalin; Bringmann, Peter; Light, David R; Schirm, Sabine

    2005-08-01

    Prolyl-4-hydroxylase domain-containing enzymes (PHDs) mediate the oxygen-dependent regulation of the heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1). Under normoxic conditions, one of the subunits of HIF-1, HIF-1alpha, is hydroxylated on specific proline residues to target HIF-1alpha for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, the hydroxylation by the PHDs is attenuated by lack of the oxygen substrate, allowing HIF-1 to accumulate, translocate to the nucleus, and mediate HIF-mediated gene transcription. In several mammalian species including humans, three PHDs have been identified. We report here the cloning of a full-length rat cDNA that is highly homologous to the human and murine PHD-1 enzymes and encodes a protein that is 416 amino acids long. Both cDNA and protein are widely expressed in rat tissues and cell types. We demonstrate that purified and crude baculovirus-expressed rat PHD-1 exhibits HIF-1alpha specific prolyl hydroxylase activity with similar substrate affinities and is comparable to human PHD-1 protein.

  18. Impact of estradiol supplementation on dual peptidyl drive of GH secretion in postmenopausal women.

    PubMed

    Veldhuis, J D; Evans, W S; Bowers, C Y

    2002-02-01

    As an indirect probe of estrogen-regulated hypothalamic somatostatin restraint, the present study monitors the ability of short-term oral E2 supplementation to modulate GH secretion during combined continuous stimulation by recombinant human GHRH [GHRH-(1-44)-amide] and the potent and selective synthetic GH-releasing peptide, GHRP-2. According to a simplified tripeptidyl model of GH neuroregulation, the effects of estrogen in this dual secretagogue paradigm should mirror alterations in endogenous somatostatinergic signaling. To this end, seven healthy postmenopausal women underwent frequent (10-min) blood sampling for 24 h during simultaneous i.v. infusion of GHRH and GHRP-2 each at a rate of 1 microg/kg x h on d 10 of randomly ordered placebo or 17beta-estradiol (E2) (1 mg orally twice daily) replacement. Serum GH concentrations (n = 280/subject) were assayed by chemiluminescence. The resultant GH time series was evaluated by deconvolution analysis, the approximate entropy statistic, and cosine regression to quantitate pulsatile, entropic (feedback-sensitive), and 24-h rhythmic GH release, respectively. Statistical comparisons revealed that E2 repletion increased the mean (+/- SEM) serum E2 concentration to 222 +/- 26 pg/ml from 16 +/- 1.7 pg/ml during placebo (P < 0.001) and suppressed the serum LH by 48% (P = 0.0033), serum FSH by 64% (P < 0.001), and serum IGF-I by 44% (P = 0.021). Double peptidyl secretagogue stimulation elevated mean 24-h serum GH concentrations to 8.1 +/- 1.0 microg/liter (placebo) and 7.7 +/- 0.89 microg/liter (E2; P = NS) and evoked prominently pulsatile patterns of GH secretion. No primary measure of pulsatile or basal GH release was altered by the disparate sex steroid milieu, i.e. GH secretory burst amplitudes of 0.62 +/- 0.93 (placebo) and 0.72 +/- 0.16 (E2) microg/liter x min, GH pulse frequencies of 27 +/- 1.8 (placebo) and 23 +/- 1.9 (E2) events/24 h, GH half-lives of 12 +/- 0.74 (placebo) and 15 +/- 4.5 (E2) min, and basal

  19. Antiamnesic effect of acyl-prolyl-containing dipeptide (GVS-111) in compression-induced damage to frontal cortex.

    PubMed

    Romanova, G A; Mirzoev, T K; Barskov, I V; Victorov, I V; Gudasheva, T A; Ostrovskaya, R U

    2000-09-01

    Antiamnestic effect of acyl-prolyl-containing dipeptide GVS-111 was demonstrated in rats with bilateral compression-induced damage to the frontal cortex. Both intraperitoneal and oral administration of the dipeptide improved retrieval of passive avoidance responses in rats with compression-induced cerebral ischemia compared to untreated controls.

  20. Cis-trans isomerizations of beta-carotene and lycopene: a theoretical study.

    PubMed

    Guo, Wen-Hsin; Tu, Cheng-Yi; Hu, Ching-Han

    2008-09-25

    The all-trans to mono-cis isomerizations of polyenes and two C40H56 carotenes, beta-carotene and lycopene, at the ground singlet (S0) and triplet (T1) states are studied by means of quantum chemistry computations. At the S0 state of polyenes containing n acetylene units (Pn), we find that the energy barrier of the central C=C rotation decreases with n. In contrast, however, at the T 1 state, the rotational barrier increases with n. For the C40H56 carotenes, the rotational barriers of lycopene are lower than those of their beta-carotene counterparts. This difference renders the rotational rates of lycopene to be 1-2 orders of magnitude higher than those of beta-carotene at room temperature. For both these carotenes, the barrier is lowest for the rotation toward the 13-cis isomer. The relative abundances are in the following order: all-trans > 9-cis > 13-cis > 15-cis. Although the 5-cis isomer of lycopene has the lowest energy among the cis isomers, its formation from the all-trans form is restricted, owing to a very large rotational barrier. The possible physiological implications of this study are discussed.

  1. Impact of water on the cis-trans photoisomerization of hydroxychalcones.

    PubMed

    Leydet, Yoann; Batat, Pinar; Jonusauskas, Gediminas; Denisov, Sergey; Lima, João Carlos; Parola, A Jorge; McClenaghan, Nathan D; Pina, Fernando

    2013-05-23

    The photochromism of a 2-hydroxychalcone has been studied in CH3CN and H2O/CH3OH (1/1, v/v), as well as in analogous deuterated solvents using steady-state (UV-vis absorption, (1)H and (13)C NMR) and time-resolved (ultrafast transient absorption and nanosecond flow flash photolysis) spectroscopies. Whereas the irradiation of trans-chalcone (Ct) under neutral pH conditions leads to the formation of the same final chromene derivative (B) in both media, two distinct photochemical mechanisms are proposed in agreement with thermodynamic and kinetic properties of the chemical reaction network at the ground state. Following light excitation, the first steps are identical in acetonitrile and aqueous solution: the Franck-Condon excited state rapidly populates the trans-chalcone singlet excited state (1)Ct* (LE), which evolves into a twisted state (1)P*. This excited state is directly responsible for the photochemistry in acetonitrile in the nanosecond time scale (16 ns) leading to the formation of cis-chalcone (Cc) through a simple isomerization process. The resulting cis-chalcone evolves into the chromene B through a tautomerization process in the ground state (τ = 10 ms). Unlike in acetonitrile, in H2O/CH3OH (1/1, v/v), the P* state becomes unstable and evolves into a new state attributed to the tautomer (1)Q*. This state directly evolves into B in one photochemical step through a consecutive ultrafast tautomerization process followed by electrocyclization. This last case represents a new hypothesis in the photochromism of 2-hydroxychalcone derivatives.

  2. Modelling of the cis-trans partitioning in the photoisomerizations of cyanines and stilbene derivatives

    NASA Astrophysics Data System (ADS)

    Caselli, M.; Momicchioli, F.; Ponterini, G.

    1993-12-01

    In the course of photoisomerization, polymethine cyanines as well as stilbene and its derivates decay from the S 1 potential energy minimum, corresponding to the perpendicular geometry, to yield either cis or trans ground-state molecules. The fraction of cis isomers obtained, α, spans a larger range of values for symmetric cyanines than for stilbene derivatives. It is argued that such different behaviour for the two classes of compounds should be traceable to the electronically different nature of their S 1 perp species. Making use of radiationless transition theory results, it is shown the relative location of the S 1 minimum and S 0 maximum along the internal rotation coordinate is crucial to the evaluation of α: even small differences between these critical twisting angles, which are more reasonably expected for polymethine cyanines than for stilbene-like compounds, may cause strong deviations from equipartitioning (α=0.5).

  3. IRIS Toxicological Review of cis- & trans-1,2-Dichloroethylene (Interagency Science Consultation Draft)

    EPA Science Inventory

    On September 24, 2009, the Toxicological Review of cis- and trans-1,2-dichloroethylene and the charge to external peer reviewers were released for external peer review and public comment. The Toxicological Review and charge were reviewed internally by EPA and by other federal ag...

  4. IRIS Toxicological Review of cis- & trans-1,2-Dichloroethylene (Interagency Science Discussion Draft)

    EPA Science Inventory

    EPA is releasing the draft report, Toxicological Review of cis-1,2-Dichloroethylene and trans-1,2-Dichloroethylene, that was distributed to Federal agencies and White House Offices for comment during the Science Discussion step of the IRIS Toxicological Review of Cis-& Trans-1,2-Dichloroethylene (External Review Draft)

    EPA Science Inventory

    EPA is conducting a peer review of the scientific basis supporting the human health hazard and dose-response assessment of cis- and trans-1,2-dichloroethylene that will appear in the Integrated Risk Information System (IRIS) database.

  5. Proline cis-trans isomerization is influenced by local lysine acetylation-deacetylation

    PubMed Central

    Howe, Françoise S.; Mellor, Jane

    2014-01-01

    Acetylation of lysine residues has several characterised functions in chromatin. These include neutralization of the lysine’s positive charge to directly influence histone tail-DNA/internucleosomal interactions or indirect effects via bromodomain-containing effector proteins. Recently, we described a novel function of lysine acetylation to influence proline isomerization and thus local protein conformation. We found that acetylation of lysine 14 in the histone H3 N-terminal tail (H3K14ac), an intrinsically disordered domain, increased the proportion of neighbouring proline 16 (H3P16) in the trans conformation. This conformation of the tail was associated with reduced tri-methylation on histone H3 lysine 4 (H3K4me3) due to both decreased methylation by the Set1 methyltransferase (with the me3-specific subunit Spp1) and increased demethylation by the demethylase Jhd2. Interestingly, H3K4me3 on individual genes was differentially affected by substitution of H3K14 or H3P16, with ribosomal protein genes losing the least H3K4me3 and environmental stress-induced genes losing the most. PMID:28357218

  6. Separate cis-trans Pathways Post-transcriptionally Regulate Murine CD154 (CD40 Ligand) Expression

    PubMed Central

    Hamilton, B. JoNell; Wang, Xiao-Wei; Collins, Jane; Bloch, Donald; Bergeron, Alan; Henry, Brian; Terry, Benjamin M.; Zan, Moe; Mouland, Andrew J.; Rigby, William F. C.

    2008-01-01

    We report a role for CA repeats in the 3′-untranslated region (3′-UTR) in regulating CD154 expression. Human CD154 is encoded by an unstable mRNA; this instability is conferred in cis by a portion of its 3′-UTR that includes a polypyrimidine-rich region and CA dinucleotide repeat. We demonstrate similar instability activity with the murine CD154 3′-UTR. This instability element mapped solely to a conserved 100-base CU-rich region alone, which we call a CU-rich response element. Surprisingly, the CA dinucleotide-rich region also regulated reporter expression but at the level of translation. This activity was associated with poly(A) tail shortening and regulated by heterogeneous nuclear ribonucleoprotein L levels. We conclude that the CD154 3′-UTR contains dual cis-acting elements, one of which defines a novel function for exonic CA dinucleotide repeats. These findings suggest a mechanism for the association of 3′-UTR CA-rich response element polymorphisms with CD154 overexpression and the subsequent risk of autoimmune disease. PMID:18640985

  7. Fluorescence of carotenoids. Effect of oxygenation and cis/trans isomerization

    NASA Astrophysics Data System (ADS)

    Jørgensen, Kevin; Stapelfeldt, Henrik; Skibsted, Leif H.

    1992-03-01

    C 40 carotenoids fall, with respect to fluorescence in homogeneous solution, into two distinct groups depending on the presence of a CO group in the molecule. Excitation spectra agree with absorption spectra for the carbonyl derivatives astaxanthin and canthaxanthin. In contrast, zeaxanthin and isomers of β-carotene have a twentyfold increase in fluorescence quantum yield for excitation around 350 nm compared to excitation near the absorption maximum (at approximatively 430 nm). These differences are interpreted in terms of the role of non-emitting 1(n, π*) states related to the CO group in facilitating non-radiative deactivation of higher 1(π, π*) states.

  8. 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

  9. Communication: One-photon phase control of cis-trans isomerization in retinal

    SciTech Connect

    Arango, Carlos A.; Brumer, Paul

    2013-02-21

    We computationally demonstrate the one-photon phase control of retinal isomerization under conditions of low laser intensity. The calculations, utilizing the multiconfigurational time dependent Hartree method, include coupling between the two modes that are active in isomerization and the background molecular vibrational environment. Noting previously unsuccessful computations highlights the significance of this result.

  10. PBOND: web server for the prediction of proline and non-proline cis/trans isomerization.

    PubMed

    Exarchos, Konstantinos P; Exarchos, Themis P; Papaloukas, Costas; Troganis, Anastassios N; Fotiadis, Dimitrios I

    2009-09-01

    PBOND is a web server that predicts the conformation of the peptide bond between any two amino acids. PBOND classifies the peptide bonds into one out of four classes, namely cis imide (cis-Pro), cis amide (cis-nonPro), trans imide (trans-Pro) and trans amide (trans-nonPro). Moreover, for every prediction a reliability index is computed. The underlying structure of the server consists of three stages: (1) feature extraction, (2) feature selection and (3) peptide bond classification. PBOND can handle both single sequences as well as multiple sequences for batch processing. The predictions can either be directly downloaded from the web site or returned via e-mail. The PBOND web server is freely available at http://195.251.198.21/pbond.html.

  11. The role of intersection topography in bond selectivity of cis-trans photoisomerization

    PubMed Central

    Ben-Nun, M.; Molnar, F.; Schulten, K.; Martínez, Todd J.

    2002-01-01

    Ab initio methods are used to characterize the ground and first excited state of the chromophore in the rhodopsin family of proteins: retinal protonated Schiff base. Retinal protonated Schiff base has five double bonds capable of undergoing isomerization. Upon absorption of light, the chromophore isomerizes and the character of the photoproducts (e.g., 13-cis and 11-cis) depends on the environment, protein vs. solution. Our ab initio calculations show that, in the absence of any specific interactions with the environment (e.g., discrete ordered charges in a protein), energetic considerations cannot explain the observed bond selectivity. We instead attribute the origin of bond selectivity to the shape (topography) of the potential energy surfaces in the vicinity of points of true degeneracy (conical intersections) between the ground and first excited electronic states. This provides a molecular example where a competition between two distinct but nearly isoenergetic photochemical reaction pathways is resolved by a topographical difference between two conical intersections. PMID:11854479

  12. IRIS Toxicological Review of cis- & trans-1,2-Dichloroethylene (Interagency Science Discussion Draft)

    EPA Science Inventory

    EPA is releasing the draft report, Toxicological Review of cis-1,2-Dichloroethylene and trans-1,2-Dichloroethylene, that was distributed to Federal agencies and White House Offices for comment during the Science Discussion step of the IRIS Toxicological Review of cis- & trans-1,2-Dichloroethylene (Interagency Science Consultation Draft)

    EPA Science Inventory

    On September 24, 2009, the Toxicological Review of cis- and trans-1,2-dichloroethylene and the charge to external peer reviewers were released for external peer review and public comment. The Toxicological Review and charge were reviewed internally by EPA and by other federal ag...

  13. IRIS Toxicological Review of Cis-& Trans-1,2-Dichloroethylene (External Review Draft)

    EPA Science Inventory

    EPA is conducting a peer review of the scientific basis supporting the human health hazard and dose-response assessment of cis- and trans-1,2-dichloroethylene that will appear in the Integrated Risk Information System (IRIS) database.

  14. Comparing long-range corrected functionals in the cis-trans isomerisation of the retinal chromophore

    NASA Astrophysics Data System (ADS)

    Rostov, Ivan V.; Kobayashi, Rika; Amos, Roger D.

    2012-10-01

    Earlier results for the 11-cis to all-trans isomerisation of the retinal chromophore after photoexcitation, studied using time-dependent density functional theory with the hybrid CAM-B3LYP functional, are compared with new results using other long-range corrected DFT functionals. The TDDFT S0 and S1 minimum energy paths have been compared with the approximate coupled-cluster method RI-CC2. All calculations were consistent in producing an additional avoided crossing minimum on the S 1 minimum energy path lying approximately halfway between the 11-cis and all-trans S1 minima. In this minimum on the S1 potential energy surface, the retinal chromophore has inverted bond order in its carbon chain and lower energy than it has in both the 11-cis and all-trans S1 minima.

  15. Proline cis-trans isomerization in staphylococcal nuclease: multi-substrate free energy perturbation calculations.

    PubMed Central

    Hodel, A.; Rice, L. M.; Simonson, T.; Fox, R. O.; Brünger, A. T.

    1995-01-01

    Staphylococcal nuclease A exists in two folded forms that differ in the isomerization state of the Lys 116-Pro 117 peptide bond. The dominant form (90% occupancy) adopts a cis peptide bond, which is observed in the crystal structure. NMR studies show that the relatively small difference in free energy between the cis and trans forms (delta Gcis-->trans approximately 1.2 kcal/mol) results from large and nearly compensating differences in enthalpy and entropy (delta Hcis-->trans approximately delta TScis-->trans approximately 10 kcal/mol). There is evidence from X-ray crystal structures that the structural differences between the cis and the trans forms of nuclease are confined to the conformation of residues 112-117, a solvated protein loop. Here, we obtain a thermodynamic and structural description of the conformational equilibrium of this protein loop through an exhaustive conformational search that identified several substates followed by free energy simulations between the substrates. By partitioning the search into conformational substates, we overcame the multiple minima problem in this particular case and obtained precise and reproducible free energy values. The protein and water environment was implicitly modeled by appropriately chosen nonbonded terms between the explicitly treated loop and the rest of the protein. These simulations correctly predicted a small free energy difference between the cis and trans forms composed of larger, compensating differences in enthalpy and entropy. The structural predictions of these simulations were qualitatively consistent with known X-ray structures of nuclease variants and yield a model of the unknown minor trans conformation. PMID:7613463

  16. Assembly and disassembly of the Golgi complex: two processes arranged in a cis-trans direction

    PubMed Central

    1992-01-01

    We have studied the disassembly and assembly of two morphologically and functionally distinct parts of the Golgi complex, the cis/middle and trans cisterna/trans network compartments. For this purpose we have followed the redistribution of three cis/middle- (GMPc-1, GMPc-2, MG 160) and two trans- (GMPt-1 and GMPt-2) Golgi membrane proteins during and after treatment of normal rat kidney (NRK) cells with brefeldin A (BFA). BFA induced complete disassembly of the cis/middle- and trans- Golgi complex and translocation of GMPc and GMPt to the ER. Cells treated for short times (3 min) with BFA showed extensive disorganization of both cis/middle- and trans-Golgi complexes. However, complete disorganization of the trans part required much longer incubations with the drug. Upon removal of BFA the Golgi complex was reassembled by a process consisting of three steps: (a) exist of cis/middle proteins from the ER and their accumulation into vesicular structures scattered throughout the cytoplasm; (b) gradual relocation and accumulation of the trans proteins in the vesicles containing the cis/middle proteins; and (c) assembly of the cisternae, and reconstruction of the Golgi complex within an area located in the vicinity of the centrosome from which the ER was excluded. Reconstruction of the cis/middle-Golgi complex occurred under temperature conditions inhibitory of the reorganization of the trans- Golgi complex, and was dependent on microtubules. Reconstruction of the trans-Golgi complex, disrupted with nocodazole after selective fusion of the cis/middle-Golgi complex with the ER, occurred after the release of cis/middle-Golgi proteins from the ER and the assembly of the cis/middle cisternae. PMID:1730750

  17. The carbon mainframe structure of cis-trans-1,3-difluoroacetone

    NASA Astrophysics Data System (ADS)

    Grubbs, G. S.

    2017-01-01

    The carbon-13 backbone structure of 1,3-difluoroacetone has been obtained and reported for the first time. This was achieved through the collection of singly substituted 13C isotopologue rotational spectra at each carbon position. The rotational constants for each isotopologue have been determined and are reported for the first time. Kraitchman coordinates and second moment analysis verify the structure determined by previous literature studies and are presented.

  18. Communication: One-photon phase control of cis-trans isomerization in retinal

    NASA Astrophysics Data System (ADS)

    Arango, Carlos A.; Brumer, Paul

    2013-02-01

    We computationally demonstrate the one-photon phase control of retinal isomerization under conditions of low laser intensity. The calculations, utilizing the multiconfigurational time dependent Hartree method, include coupling between the two modes that are active in isomerization and the background molecular vibrational environment. Noting previously unsuccessful computations highlights the significance of this result.

  1. The SH2 domains of inositol polyphosphate 5-phosphatases SHIP1 and SHIP2 have similar ligand specificity but different binding kinetics.

    PubMed

    Zhang, Yanyan; Wavreille, Anne-Sophie; Kunys, Andrew R; Pei, Dehua

    2009-11-24

    SH2 domain-containing inositol 5-phosphatases 1 (SHIP1) and 2 (SHIP2) are structurally similar proteins that catalyze the degradation of lipid secondary messenger phosphatidylinositol 3,4,5-triphosphate to produce phosphatidylinositol 3,4-diphosphate. Despite their high sequence identity (51%), SHIP1 and SHIP2 share little overlap in their in vivo functions. In this work, the sequence specificity of the SHIP2 SH2 domain was systematically defined through the screening of a combinatorial pY peptide library. Comparison of its specificity profile with that of the SHIP1 SH2 domain showed that the two SH2 domains have similar specificities, both recognizing pY peptides of the consensus sequence pY[S/Y][L/Y/M][L/M/I/V], although there are also subtle differences such as the tolerance of an arginine at the pY + 1 position by the SHIP2 but not SHIP1 SH2 domain. Surface plasmon resonance analysis of their interaction with various pY peptides suggested that the two domains have similar binding affinities but dramatically different binding kinetics, with the SHIP1 SH2 domain having fast association and dissociation rates while the SHIP2 domain showing apparent slow-binding behavior. Site-directed mutagenesis and kinetic studies indicated that the SHIP2 SH2 domain exists as a mixture of two conformational isomers. The major, inactive isomer apparently contains two cis peptidyl-prolyl bonds at positions 88 and 105, whereas the minor, active isomer has both proline residues in their trans configuration. Cis-trans isomerization of the peptidyl-prolyl bonds may provide a potential mechanism for regulating the interaction between SHIP2 and pY proteins. These data suggest that a combination of tissue distribution, specificity, and kinetic differences is likely responsible for their in vivo functional differences.

  2. Protein synthesis factors (RF1, RF2, RF3, RRF, and tmRNA) and peptidyl-tRNA hydrolase rescue stalled ribosomes at sense codons.

    PubMed

    Vivanco-Domínguez, Serafín; Bueno-Martínez, José; León-Avila, Gloria; Iwakura, Nobuhiro; Kaji, Akira; Kaji, Hideko; Guarneros, Gabriel

    2012-04-13

    During translation, ribosomes stall on mRNA when the aminoacyl-tRNA to be read is not readily available. The stalled ribosomes are deleterious to the cell and should be rescued to maintain its viability. To investigate the contribution of some of the cellular translation factors on ribosome rescuing, we provoked stalling at AGA codons in mutants that affected the factors and then analyzed the accumulation of oligopeptidyl (peptides of up to 6 amino acid residues, oligopep-)-tRNA or polypeptidyl (peptides of more than 300 amino acids in length, polypep-)-tRNA associated with ribosomes. Stalling was achieved by starvation for aminoacyl-tRNA(Arg4) upon induced expression of engineered lacZ (β-galactosidase) reporter gene harboring contiguous AGA codons close to the initiation codon or at internal codon positions together with minigene ATGAGATAA accompanied by reduced peptidyl-tRNA hydrolase (Pth). Our results showed accumulations of peptidyl-tRNA associated with ribosomes in mutants for release factors (RF1, RF2, and RF3), ribosome recycling factor (RRF), Pth, and transfer-messenger RNA (tmRNA), implying that each of these factors cooperate in rescuing stalled ribosomes. The role of these factors in ribosome releasing from the stalled complex may vary depending on the length of the peptide in the peptidyl-tRNA. RF3 and RRF rescue stalled ribosomes by "drop-off" of peptidyl-tRNA, while RF1, RF2 (in the absence of termination codon), or Pth may rescue by hydrolyzing the associated peptidyl-tRNA. This is followed by the disassembly of the ribosomal complex of tRNA and mRNA by RRF and elongation factor G.

  3. Peptidyl-tRNA hydrolase screening combined with molecular docking reveals the antibiotic potential of Syzygium johnsonii bark extract.

    PubMed

    Harris, Sarah M; McFeeters, Hana; Ogungbe, Ifedayo V; Cruz-Vera, Luis R; Setzer, William N; Jackes, Betsy R; McFeeters, Robert L

    2011-10-01

    With the rapid rise of antibiotic resistance in pathogenic bacteria, the need for new antibacterial agents is overwhelming. Herein we report the limited screening of tropical plant extracts for inhibitory activity against the essential enzyme peptidyl-tRNA hydrolase (Pth). Initial screening was conducted through an electrophoretic mobility assay and Northern blot detection. The ability of Pth to cleave the peptide-tRNA ester bond was assessed. The ethanol bark extract of Syzygium johnsonii showed strong inhibitory potential. Molecular docking studies point to Syzygium polyphenolics as the potential source of inhibition. This work is the forerunner of activity-directed isolation, purification, and structure elucidation of the inhibitory components from Syzygium johnsonii extracts and studies of compound interaction with Pth.

  4. HIV-1 p6-Another viral interaction partner to the host cellular protein cyclophilin A.

    PubMed

    Solbak, Sara M Ø; Reksten, Tove R; Röder, Rene; Wray, Victor; Horvli, Ole; Raae, Arnt J; Henklein, Petra; Henklein, Peter; Fossen, Torgils

    2012-04-01

    The 52-amino acid human immunodeficiency virus type 1 (HIV-1) p6 protein has previously been recognized as a docking site for several cellular and viral binding factors and is important for the formation of infectious viruses. A particular structural feature of p6 is the notably high relative content of proline residues, located at positions 5, 7, 10, 11, 24, 30, 37 and 49 in the sequence. Proline cis/trans isomerism was detected for all these proline residues to such an extent that more than 40% of all p6 molecules contain at least one proline in a cis conformation. 2D (1)H nuclear magnetic resonance analysis of full-length HIV-1 p6 and p6 peptides established that cyclophilin A (CypA) interacts as a peptidyl-prolyl cis/trans isomerase with all proline residues of p6. Only catalytic amounts of CypA were necessary for the interaction with p6 to occur, strongly suggesting that the observed interaction is highly relevant in vivo. In addition, surface plasmon resonance studies revealed binding of full-length p6 to CypA, and that this binding was significantly stronger than any of its N- or C-terminal peptides. This study demonstrates the first identification of an interaction between HIV-1 p6 and the host cellular protein CypA. The mode of interaction involves both transient enzyme-substrate interactions and a more stable binding. The binding motifs of p6 to Tsg-101, ALIX and Vpr coincide with binding regions and catalytic sites of p6 to CypA, suggesting a potential role of CypA in modulating functional interactions of HIV-1.

  5. A novel binding protein for a member of CyP40-type Cyclophilins: N.crassa CyPBP37, a growth and thiamine regulated protein homolog to yeast Thi4p.

    PubMed

    Faou, Pierre; Tropschug, Maximilian

    2003-10-31

    Cyclophilins belong to the family of peptidyl-prolyl cis/trans isomerases (PPIases), which are ubiquitous and highly conserved enzymes capable of cis/trans isomerizing Xaa-Pro peptide bonds. Members of the CyP40-type cyclophilins have originally been described as components of hormone receptor complexes. Here, we describe NcCyP41, a CyP40 ortholog from Neurospora crassa, its expression in Escherichia coli and subsequent purification. Characterization of NcCyP41 reveals that it is a heat shock protein, which is active as a cyclosporin A-sensitive PPIase. Affinity chromatography using immobilized recombinant NcCyP41 yielded two major NcCyP41-binding proteins: Hsp80 (a Hsp90 ortholog from N.crassa) and CyPBP37. CyPBP37 has not been described. In addition, this is the first record describing an interaction between a member of Cyp40-type cyclophilins and of CyPBP37-type proteins, respectively. CyPBP37 expression is repressed by thiamine and in the stationary phase in N.crassa. CyPBP37 is present in different isoforms. The expression of a CyPBP37 ortholog in yeast, Thi4p, is diminished in a mutant lacking one of the two CyP40 orthologs (Cpr7p). In addition, the DeltaCpr7p deletion mutant shows a thiamine-dependent growth defect. We conclude that, in yeast, Cpr7p and Thi4p interact functionally.

  6. A possible role of prolyl oligopeptidase during Linum usitatissimum (flax) seed development.

    PubMed

    Gutierrez, L; Castelain, M; Verdeil, J-L; Conejero, G; Van Wuytswinkel, O

    2008-05-01

    Involvement of prolyl oligopeptidases (POPs) in the control of several mammalian peptide hormone signalling pathways has been studied extensively in recent years. POPs are ubiquitous enzymes, but little attention has been paid to understanding their function in plants. Using a cDNA-AFLP approach, two flax (Linum usitatissimum) POP ESTs were identified as being specifically expressed in the early stages of flax seed development. This specific expression was confirmed using real time RT-PCR and in situ hybridisation approaches. Seed expression of Arabidopsis POP genes was measured and showed no specificity. Comparison between results obtained with flax and Arabidopsis is discussed in order to address a hypothetic function for POPs during seed formation. These results provide the first insights into POP gene expression and hypothetical function in plants.

  7. Current advances in the novel functions of hypoxia-inducible factor and prolyl hydroxylase in invertebrates.

    PubMed

    Wang, L; Cui, S; Ma, L; Kong, L; Geng, X

    2015-12-01

    Oxygen is essential for aerobic life, and hypoxia has very severe consequences. Organisms need to overcome low oxygen levels to maintain biological functions during normal development and in disease states. The mechanism underlying the hypoxic response has been widely investigated in model animals such as Drosophila melanogaster and Caenorhabditis elegans. Hypoxia-inducible factor (HIF), a key gene product in the response to oxygen deprivation, is primarily regulated by prolyl hydroxylase domain enzymes (PHDs). However, recent findings have uncovered novel HIF-independent functions of PHDs. This review provides an overview of how invertebrates are able to sustain hypoxic damages, and highlights some recent discoveries in the regulation of cellular signalling by PHDs. Given that some core genes and major pathways are evolutionarily conserved, these research findings could provide insight into oxygen-sensitive signalling in mammals, and have biomedical implications for human diseases. © 2015 The Royal Entomological Society.

  8. Pyrithione Zn selectively inhibits hypoxia-inducible factor prolyl hydroxylase PHD3.

    PubMed

    Na, Yu-Ran; Woo, Dustin J; Kim, So Yeon; Yang, Eun Gyeong

    2016-04-01

    Increasing evidence emphasizes the role of the hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD) isoforms in regulating non-HIF substrates, but isoform selective PHD inhibitors under physiological conditions have not yet been reported. Here we have identified pyrithione Zn (PZ) as a potent, isoform-selective PHD3 inhibitor. The IC50 value of PZ was determined as 0.98 μM for PHD3, while it did not show any inhibitory activity toward full length and truncated PHD2 up to 1 mM. The selective efficacy of PZ was further demonstrated at the cellular level by observing inhibition of the PHD3-dependent DNA damage response pathway without stabilization of HIF-1α.

  9. Identification of novel Trypanosoma cruzi prolyl oligopeptidase inhibitors by structure-based virtual screening

    NASA Astrophysics Data System (ADS)

    de Almeida, Hugo; Leroux, Vincent; Motta, Flávia Nader; Grellier, Philippe; Maigret, Bernard; Santana, Jaime M.; Bastos, Izabela Marques Dourado

    2016-12-01

    We have previously demonstrated that the secreted prolyl oligopeptidase of Trypanosoma cruzi (POPTc80) is involved in the infection process by facilitating parasite migration through the extracellular matrix. We have built a 3D structural model where POPTc80 is formed by a catalytic α/β-hydrolase domain and a β-propeller domain, and in which the substrate docks at the inter-domain interface, suggesting a "jaw opening" gating access mechanism. This preliminary model was refined by molecular dynamics simulations and next used for a virtual screening campaign, whose predictions were tested by standard binding assays. This strategy was successful as all 13 tested molecules suggested from the in silico calculations were found out to be active POPTc80 inhibitors in the micromolar range (lowest K i at 667 nM). This work paves the way for future development of innovative drugs against Chagas disease.

  10. Prolyl oligopeptidase inhibition-induced growth arrest of human gastric cancer cells

    SciTech Connect

    Suzuki, Kanayo; Sakaguchi, Minoru; Tanaka, Satoshi; Yoshimoto, Tadashi; Takaoka, Masanori

    2014-01-03

    Highlights: •We examined the effects of prolyl oligopeptidase (POP) inhibition on p53 null gastric cancer cell growth. •POP inhibition-induced cell growth suppression was associated with an increase in a quiescent G{sub 0} state. •POP might regulate the exit from and/or reentry into the cell cycle. -- Abstract: Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyzes post-proline peptide bonds in peptides that are <30 amino acids in length. We recently reported that POP inhibition suppressed the growth of human neuroblastoma cells. The growth suppression was associated with pronounced G{sub 0}/G{sub 1} cell cycle arrest and increased levels of the CDK inhibitor p27{sup kip1} and the tumor suppressor p53. In this study, we investigated the mechanism of POP inhibition-induced cell growth arrest using a human gastric cancer cell line, KATO III cells, which had a p53 gene deletion. POP specific inhibitors, 3-((4-[2-(E)-styrylphenoxy]butanoyl)-L-4-hydroxyprolyl)-thiazolidine (SUAM-14746) and benzyloxycarbonyl-thioprolyl-thioprolinal, or RNAi-mediated POP knockdown inhibited the growth of KATO III cells irrespective of their p53 status. SUAM-14746-induced growth inhibition was associated with G{sub 0}/G{sub 1} cell cycle phase arrest and increased levels of p27{sup kip1} in the nuclei and the pRb2/p130 protein expression. Moreover, SUAM-14746-mediated cell cycle arrest of KATO III cells was associated with an increase in the quiescent G{sub 0} state, defined by low level staining for the proliferation marker, Ki-67. These results indicate that POP may be a positive regulator of cell cycle progression by regulating the exit from and/or reentry into the cell cycle by KATO III cells.

  11. Regulation of Axonal Midline Guidance by Prolyl 4-Hydroxylation in Caenorhabditis elegans

    PubMed Central

    Torpe, Nanna

    2014-01-01

    Neuronal wiring during development requires that the growth cones of axons and dendrites are correctly guided to their appropriate targets. As in other animals, axon growth cones in Caenorhabditis elegans integrate information in their extracellular environment via interactions among transiently expressed cell surface receptors, their ligands, and the extracellular matrix (ECM). Components of the ECM undergo a wide variety of post-translational modifications that may affect efficacy of binding to neuronal guidance molecules. The most common modification of the ECM is prolyl 4-hydroxylation. However, little is known of its importance in the control of axon guidance. In a screen of prolyl 4-hydroxylase (P4H) mutants, we found that genetic removal of a specific P4H subunit, DPY-18, causes dramatic defects in C. elegans neuroanatomy. In dpy-18 mutant animals, the axons of specific ventral nerve cord neurons do not respect the ventral midline boundary and cross over to the contralateral axon fascicle. We found that these defects are independent of the known role of dpy-18 in regulating body size and that dpy-18 acts from multiple tissues to regulate axon guidance. Finally, we found that the neuronal defects in dpy-18 mutant animals are dependent on the expression of muscle-derived basement membrane collagens and motor neuron-derived ephrin ligands. Loss of dpy-18 causes dysregulated ephrin expression and this is at least partially responsible for the neurodevelopmental defects observed. Together, our data suggest that DPY-18 regulates ephrin expression to direct axon guidance, a role for P4Hs that may be conserved in higher organisms. PMID:25471573

  12. Genetic evidence for role of DPP IV in intestinal hydrolysis and assimilation of prolyl peptides.

    PubMed

    Tiruppathi, C; Miyamoto, Y; Ganapathy, V; Leibach, F H

    1993-07-01

    The functional role of dipeptidyl peptidase IV (DPP IV) in the intestinal hydrolysis and assimilation of prolyl peptides was investigated using Japan F344 rats, which genetically lack this enzyme. USA F344 rats possess normal activity of this enzyme and served as matched controls. Intestinal brush-border membranes from the control rats were able to hydrolyze several proline-containing peptides. The hydrolytic ability of the brush-border membranes from the Japan rats against these peptides was markedly low. The difference in the hydrolytic activities between the two groups of rats was solely due to the absence of DPP IV in the Japan rats. There was no difference in the growth rate between the two groups of rats fed a reference diet whose protein constituents were not rich in proline. When the protein source was changed to gliadin, a proline-rich protein, USA F344 rats maintained their body weight for a 4-wk period on this diet, whereas the Japan rats experienced a significant weight loss under similar conditions. In situ perfusion experiments in intact animals revealed that the ability of morphiceptin (a peptide primarily hydrolyzable by DPP IV), when administered into the intestinal lumen, to block the cholera toxin-induced water secretion was significantly greater in Japan F344 rats than in USA F344 rats, indicating the resistance of morphiceptin to hydrolytic breakdown in the intestinal lumen of the Japan rats. It is concluded that the intestinal DPP IV plays a significant role in the hydrolysis of prolyl peptides and assimilation of proline-rich proteins.

  13. Hematological, hepatic, and retinal phenotypes in mice deficient for prolyl hydroxylase domain proteins in the liver.

    PubMed

    Duan, Li-Juan; Takeda, Kotaro; Fong, Guo-Hua

    2014-04-01

    Prolyl hydroxylase domain (PHD) proteins catalyze oxygen-dependent prolyl hydroxylation of hypoxia-inducible factor 1α and 2α, tagging them for pVHL-dependent polyubiquitination and proteasomal degradation. In this study, albumin Cre (Alb(Cre))-mediated, hepatocyte-specific triple disruption of Phd1, Phd2, and Phd3 (Phd(1/2/3)hKO) promoted liver erythropoietin (EPO) expression 1246-fold, whereas renal EPO was down-regulated to 6.7% of normal levels. In Phd(1/2/3)hKO mice, hematocrit levels reached 82.4%, accompanied by severe vascular malformation and steatosis in the liver. In mice double-deficient for hepatic PHD2 and PHD3 (Phd(2/3)hKO), liver EPO increase and renal EPO loss both occurred but were much less dramatic than in Phd(1/2/3)hKO mice. Hematocrit levels, vascular organization, and liver lipid contents all appeared normal in Phd(2/3)hKO mice. In a chronic renal failure model, Phd(2/3)hKO mice maintained normal hematocrit levels throughout the 8-week time course, whereas floxed controls developed severe anemia. Maintenance of normal hematocrit levels in Phd(2/3)hKO mice was accomplished by sensitized induction of liver EPO expression. Consistent with such a mechanism, liver HIF-2α accumulated to higher levels in Phd(2/3)hKO mice in response to conditions causing modest systemic hypoxia. Besides promoting erythropoiesis, EPO is also known to modulate retinal vascular integrity and neovascularization. In Phd(1/2/3)hKO mice, however, neonatal retinas remained sensitive to oxygen-induced retinopathy, suggesting that local EPO may be more important than hepatic and/or renal EPO in mediating protective effects in the retina.

  14. Loss of Prolyl Carboxypeptidase in Two-Kidney, One-Clip Goldblatt Hypertensive Mice

    PubMed Central

    Grobe, Nadja; Leiva, Orly; Morris, Mariana; Elased, Khalid M.

    2015-01-01

    It is well documented that angiotensin (Ang) II contributes to kidney disease progression. The protease prolyl carboxypeptidase (PRCP) is highly expressed in the kidney and may be renoprotective by degrading Ang II to Ang-(1-7). The aim of the study was to investigate whether renal PRCP protein expression and activity are altered in two-kidney, one-clip (2K1C) Goldblatt hypertensive mice. Left renal artery was constricted by using 0.12 mm silver clips. Blood pressure was measured using telemetry over the eleven weeks of study period and revealed an immediate increase in 2K1C animals during the first week of clip placement which was followed by a gradual decrease to baseline blood pressure. Similarly, urinary albumin excretion was significantly increased one week after 2K1C and returned to baseline levels during the following weeks. At 2 weeks and at the end of the study, renal pathologies were exacerbated in the 2K1C model as revealed by a significant increase in mesangial expansion and renal fibrosis. Renal PRCP expression and activity were significantly reduced in clipped kidneys. Immunofluorescence revealed the loss of renal tubular PRCP but not glomerular PRCP. In contrast, expression of prolyl endopeptidase, another enzyme capable of converting Ang II into Ang-(1-7), was not affected, while angiotensin converting enzyme was elevated in unclipped kidneys and renin was increased in clipped kidneys. Results suggest that PRCP is suppressed in 2K1C and that this downregulation may attenuate renoprotective effects via impaired Ang II degradation by PRCP. PMID:25706121

  15. Neuronal apoptosis by prolyl hydroxylation: implication in nervous system tumours and the Warburg conundrum

    PubMed Central

    Schlisio, Susanne

    2009-01-01

    Oxygen-sensing mechanisms are often dysfunctional in tumours. Oxygen sensing is mediated partly via prolyl hydroxylation. The EglN prolyl hydroxylases are well characterized in regulating the hypoxia inducible factor α (HIF-α) hypoxic response, but also are implicated in HIF-independent processes. EglN3 executes apoptosis in neural precursors during development and failure of EglN3 developmental apoptosis can lead to certain forms of sympathetic nervous system tumours. Mutations in metabolic/mitochondrial enzymes (SDH, FH, IDH) impair EglN activity and predisposes to certain cancers. This is because the EglNs not only require molecular oxygen to execute hydroxylation, but also equally require the electron donor α-ketoglutarate, a metabolite from the Krebs cycle. Therefore EglN enzymes are considered oxygen, and also, metabolic sensors. α-Ketoglutarate is crucial for EglN hydroxylation activity, whereas the metabolites succinate and fumarate are inhibitors of the EglN enzymes. Since EglN activity is dependent upon metabolites that take part in the Krebs cycle, these enzymes are directly tied into the cellular metabolic network. Cancer cells tend to convert most glucose to lactate regardless of whether oxygen is present (aerobic glycolysis), an observation that was first made by Otto Warburg in 1924. Despite the striking difference in ATP production, cancer cells might favour aerobic glycolysis to escape from EglN hydroxylation, resulting in the accumulation of oncogenic HIFα and/or resistance to EglN3-mediated apoptosis. PMID:19691672

  16. Prolyl-4-Hydroxylase 3 (PHD3) Expression Is Downregulated during Epithelial-to-Mesenchymal Transition

    PubMed Central

    Place, Trenton L.; Nauseef, Jones T.; Peterson, Maina K.; Henry, Michael D.; Mezhir, James J.; Domann, Frederick E.

    2013-01-01

    Prolyl-4-hydroxylation by the intracellular prolyl-4-hydroxylase enzymes (PHD1-3) serves as a master regulator of environmental oxygen sensing. The activity of these enzymes is tightly tied to tumorigenesis, as they regulate cell metabolism and angiogenesis through their control of hypoxia-inducible factor (HIF) stability. PHD3 specifically, is gaining attention for its broad function and rapidly accumulating array of non-HIF target proteins. Data from several recent studies suggest a role for PHD3 in the regulation of cell morphology and cell migration. In this study, we aimed to investigate this role by closely examining the relationship between PHD3 expression and epithelial-to-mesenchymal transition (EMT); a transcriptional program that plays a major role in controlling cell morphology and migratory capacity. Using human pancreatic ductal adenocarcinoma (PDA) cell lines and Madin-Darby Canine Kidney (MDCK) cells, we examined the correlation between several markers of EMT and PHD3 expression. We demonstrated that loss of PHD3 expression in PDA cell lines is highly correlated with a mesenchymal-like morphology and an increase in cell migratory capacity. We also found that induction of EMT in MDCK cells resulted in the specific downregulation of PHD3, whereas the expression of the other HIF-PHD enzymes was not affected. The results of this study clearly support a model by which the basal expression and hypoxic induction of PHD3 is suppressed by the EMT transcriptional program. This may be a novel mechanism by which migratory or metastasizing cells alter signaling through specific pathways that are sensitive to regulation by O2. The identification of downstream pathways that are affected by the suppression of PHD3 expression during EMT may provide important insight into the crosstalk between O2 and the migratory and metastatic potential of tumor cells. PMID:24367580

  17. Cold sensitivity of TRPA1 is unveiled by the prolyl hydroxylation blockade-induced sensitization to ROS.

    PubMed

    Miyake, Takahito; Nakamura, Saki; Zhao, Meng; So, Kanako; Inoue, Keisuke; Numata, Tomohiro; Takahashi, Nobuaki; Shirakawa, Hisashi; Mori, Yasuo; Nakagawa, Takayuki; Kaneko, Shuji

    2016-09-15

    Mammalian transient receptor potential ankyrin 1 (TRPA1) is a polymodal nociceptor that plays an important role in pain generation, but its role as a cold nociceptor is still controversial. Here, we propose that TRPA1 can sense noxious cold via transduction of reactive oxygen species (ROS) signalling. We show that inhibiting hydroxylation of a proline residue within the N-terminal ankyrin repeat of human TRPA1 by mutation or using a prolyl hydroxylase (PHD) inhibitor potentiates the cold sensitivity of TRPA1 in the presence of hydrogen peroxide. Inhibiting PHD in mice triggers mouse TRPA1 sensitization sufficiently to sense cold-evoked ROS, which causes cold hypersensitivity. Furthermore, this phenomenon underlies the acute cold hypersensitivity induced by the chemotherapeutic agent oxaliplatin or its metabolite oxalate. Thus, our findings provide evidence that blocking prolyl hydroxylation reveals TRPA1 sensitization to ROS, which enables TRPA1 to convert ROS signalling into cold sensitivity.

  18. Cold sensitivity of TRPA1 is unveiled by the prolyl hydroxylation blockade-induced sensitization to ROS

    PubMed Central

    Miyake, Takahito; Nakamura, Saki; Zhao, Meng; So, Kanako; Inoue, Keisuke; Numata, Tomohiro; Takahashi, Nobuaki; Shirakawa, Hisashi; Mori, Yasuo; Nakagawa, Takayuki; Kaneko, Shuji

    2016-01-01

    Mammalian transient receptor potential ankyrin 1 (TRPA1) is a polymodal nociceptor that plays an important role in pain generation, but its role as a cold nociceptor is still controversial. Here, we propose that TRPA1 can sense noxious cold via transduction of reactive oxygen species (ROS) signalling. We show that inhibiting hydroxylation of a proline residue within the N-terminal ankyrin repeat of human TRPA1 by mutation or using a prolyl hydroxylase (PHD) inhibitor potentiates the cold sensitivity of TRPA1 in the presence of hydrogen peroxide. Inhibiting PHD in mice triggers mouse TRPA1 sensitization sufficiently to sense cold-evoked ROS, which causes cold hypersensitivity. Furthermore, this phenomenon underlies the acute cold hypersensitivity induced by the chemotherapeutic agent oxaliplatin or its metabolite oxalate. Thus, our findings provide evidence that blocking prolyl hydroxylation reveals TRPA1 sensitization to ROS, which enables TRPA1 to convert ROS signalling into cold sensitivity. PMID:27628562

  19. A novel profibrotic mechanism mediated by TGF-β-stimulated collagen prolyl hydroxylase expression in fibrotic lung mesenchymal cells

    PubMed Central

    Luo, Yongfeng; Xu, Wei; Chen, Hui; Warburton, David; Dong, Rachel; Qian, Bangping; Selman, Moisés; Gauldie, Jack; Kolb, Martin; Shi, Wei

    2015-01-01

    Idiopathic pulmonary fibrosis is a severe chronic lung disease with a high mortality rate. Excessive TGF-β signaling is recognized as a central player in lung fibrosis. However, the related mechanisms remain unclear. Herein we used a novel Tbx4 lung enhancer-driven Tet-On transgenic system to inhibit TGF-β signaling in mouse lung resident mesenchymal cells at different stages of bleomycin-induced fibrosis by conditionally knocking out TGF-β receptor II or expressing a dominant-negative TGF-β receptor II. Abrogation of mesenchymal TGF-β signaling markedly attenuated bleomycin-induced fibrotic pathology, which was independent of altered early inflammation. Furthermore, a novel TGF-β downstream target gene P4HA3 (an α-subunit of collagen prolyl hydroxylase) was identified, and its expression was significantly increased in fibroblastic foci of both bleomycin-induced fibrotic mouse lungs and idiopathic pulmonary fibrosis patients’ lungs. The relationship between activated TGF-β signaling, upregulation of P4HA3, as well as increased hydroxyproline/collagen production was further verified in cultured lung fibroblasts. Moreover, inhibition of collagen prolyl hydroxylase by pyridine-2,5-dicarboxylate attenuated both TGF-β-stimulated collagen production in cultured fibroblasts and bleomycin-induced mouse lung fibrosis. These data indicate that increased expression and activity of collagen prolyl hydroxylase is one of the important mechanisms underlying TGF-β-mediated profibrotic effects. Inhibition of collagen prolyl hydroxylase may be a new promising approach for preventing and treating pulmonary fibrosis. PMID:25779936

  20. Concomitant inhibition of prolyl hydroxylases and ROCK initiates differentiation of mesenchymal stem cells and PC12 towards the neuronal lineage.

    PubMed

    Pacary, Emilie; Petit, Edwige; Bernaudin, Myriam

    2008-12-12

    This study demonstrates that a prolyl hydroxylase inhibitor, FG-0041, is able, in combination with the ROCK inhibitor, Y-27632, to initiate differentiation of mesenchymal stem cells (MSCs) into neuron-like cells. FG-0041/Y-27632 co-treatment provokes morphological changes into neuron-like cells, increases neuronal marker expression and provokes modifications of cell cycle-related gene expression consistent with a cell cycle arrest of MSC, three events showing the engagement of MSC towards the neuronal lineage. Moreover, as we observed in our previous studies with cobalt chloride and desferroxamine, the activation of HIF-1 by this prolyl hydroxylase inhibitor is potentiated by Y-27632 which could explain at least in part the effect of this co-treatment on MSC neuronal differentiation. In addition, we show that this co-treatment enhances neurite outgrowth and tyrosine hydroxylase expression in PC12 cells. Altogether, these results evidence that concomitant inhibition of prolyl hydroxylases and ROCK represents a relevant protocol to initiate neuronal differentiation.

  1. OsPOP5, a prolyl oligopeptidase family gene from rice confers abiotic stress tolerance in Escherichia coli.

    PubMed

    Tan, Cun-Mei; Chen, Rong-Jun; Zhang, Jian-Hua; Gao, Xiao-Ling; Li, Li-Hua; Wang, Ping-Rong; Deng, Xiao-Jian; Xu, Zheng-Jun

    2013-10-10

    The prolyl oligopeptidase family, which is a group of serine peptidases, can hydrolyze peptides smaller than 30 residues. The prolyl oligopeptidase family in plants includes four members, which are prolyl oligopeptidase (POP, EC3.4.21.26), dipeptidyl peptidase IV (DPPIV, EC3.4.14.5), oligopeptidase B (OPB, EC3.4.21.83), and acylaminoacyl peptidase (ACPH, EC3.4.19.1). POP is found in human and rat, and plays important roles in multiple biological processes, such as protein secretion, maturation and degradation of peptide hormones, and neuropathies, signal transduction and memory and learning. However, the function of POP is unclear in plants. In order to study POP function in plants, we cloned the cDNA of the OsPOP5 gene from rice by nested-PCR. Sequence analysis showed that the cDNA encodes a protein of 596 amino acid residues with Mw ≈ 67.29 kD. In order to analyze the protein function under different abiotic stresses, OsPOP5 was expressed in Escherichia coli. OsPOP5 protein enhanced the tolerance of E. coli to high salinity, high temperature and simulated drought. The results indicate that OsPOP5 is a stress-related gene in rice and it may play an important role in plant tolerance to abiotic stress.

  2. Pyrene Excimer-Based Peptidyl Chemosensors for the Sensitive Detection of Low Levels of Heparin in 100% Aqueous Solutions and Serum Samples.

    PubMed

    Thirupathi, Ponnaboina; Park, Joo-Young; Neupane, Lok Nath; Kishore, Mallela Y L N; Lee, Keun-Hyeung

    2015-07-08

    Fluorescent chemosensors (1 and 2, Py-(Arg)nGlyGlyGly(Arg)nLys(Py)-NH2, n = 2 and 3) bearing two pyrene (Py) labeled heparin-binding peptides were synthesized for the sensitive ratiometric detection of heparin. The peptidyl chemosensors (1 and 2) sensitively detected nanomolar concentrations of heparin in aqueous solutions and in serum samples via a ratiometric response. In 100% aqueous solutions at pH 7.4, both chemosensors exhibited significant excimer emission at 486 nm as well as weak monomer emission in the absence of heparin. Upon the addition of heparin into the solution, excimer emission increased with a blue shift (10 nm) and monomer emission at 376 nm decreased. The chemosensors showed a similar sensitive ratiometric response to heparin independent of the concentration of the chemosensors. The peptidyl chemosensors were applied to the ratiometric detection of heparin over a wide range of pH (1.5-11.5) using the excimer/momomer emission changes. In the presence of serum, 1 and 2 displayed significant monomer emission at 376 nm with relatively weak excimer emission and the addition of heparin induced a significant increase in excimer emission at 480 nm and a concomitant decrease in monomer emission. The enhanced ratiometric response to heparin in the serum sample was due to the interactions between the peptidyl chemosensors and serum albumin in the serum sample. The detection limits of 2 for heparin were less than 1 nM in 100% aqueous solutions and serum samples. The peptidyl chemosensors bearing two heparin-binding sites are a suitable tool for the sensitive ratiometric detection of nanomolar concentrations of heparin in 100% aqueous solutions and serum samples.

  3. Excess of charged tRNALys maintains low levels of peptidyl-tRNA hydrolase in pth(Ts) mutants at a non-permissive temperature

    PubMed Central

    Vivanco-Domínguez, Serafin; Cruz-Vera, Luis Rogelio; Guarneros, Gabriel

    2006-01-01

    Cellular changes have been monitored during the suppression, mediated by the overproduction of tRNALys, of thermosensitivity in Escherichia coli strain AA7852 carrying a mutation in peptidyl-tRNA hydrolase (Pth) encoded by the pth(Ts) gene. The presence in AA7852 cells of a plasmid bearing lysV gene helped to maintain low levels of the unstable Pth(Ts) protein and to preserve the viability of the mutant line at 41°C whereas plasmids bearing other tRNA genes were ineffective. At 32°C the excess of tRNALys did not alter the percentages of the free-, charged- or peptidyl-tRNALys species compared with those found in strains that did not overproduce tRNALys. At 41°C, however, despite increases in the level of peptidyl-tRNALys, the excess tRNALys helped to maintain the concentration of charged-tRNALys at a level comparable with that found in non-overproducer cells grown at a permissive temperature. In addition, the excess tRNALys at 41°C provoked a reduction in the concentrations of various peptidyl-tRNAs, which normally accumulate in pth(Ts) cells, and a proportional increase in the concentrations of the corresponding aminoacyl-tRNAs. The possible mechanism of rescue due to the overexpression of tRNALys and the causes of tRNALys starvation in pth(Ts) strains grown at non-permissive temperatures are considered. PMID:16540595

  4. Reliable semi-synthesis of hydrolysis-resistant 3′-peptidyl-tRNA conjugates containing genuine tRNA modifications

    PubMed Central

    Graber, Dagmar; Moroder, Holger; Steger, Jessica; Trappl, Krista; Polacek, Norbert; Micura, Ronald

    2010-01-01

    The 3′-peptidyl-tRNA conjugates that possess a hydrolysis-resistant ribose-3′-amide linkage instead of the natural ester linkage would represent valuable substrates for ribosomal studies. Up to date, access to these derivatives is severely limited. Here, we present a novel approach for the reliable synthesis of non-hydrolyzable 3′-peptidyl-tRNAs that contain all the respective genuine nucleoside modifications. In short, the approach is based on tRNAs from natural sources that are site-specifically cleaved within the TΨC loop by using DNA enzymes to obtain defined tRNA 5′-fragments carrying the modifications. After dephosphorylation of the 2′,3′-cyclophosphate moieties from these fragments, they are ligated to the respective 3′-peptidylamino-tRNA termini that were prepared following the lines of a recently reported solid-phase synthesis. By this novel concept, non-hydrolyzable 3′-peptidyl-tRNA conjugates possessing all natural nucleoside modifications are accessible in highly efficient manner. PMID:20525967

  5. pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA

    PubMed Central

    Johansson, Magnus; Ieong, Ka-Weng; Trobro, Stefan; Strazewski, Peter; Åqvist, Johan; Pavlov, Michael Y.; Ehrenberg, Måns

    2011-01-01

    We studied the pH-dependence of ribosome catalyzed peptidyl transfer from fMet-tRNAfMet to the aa-tRNAs Phe-tRNAPhe, Ala-tRNAAla, Gly-tRNAGly, Pro-tRNAPro, Asn-tRNAAsn, and Ile-tRNAIle, selected to cover a large range of intrinsic pKa-values for the α-amino group of their amino acids. The peptidyl transfer rates were different at pH 7.5 and displayed different pH-dependence, quantified as the pH-value, , at which the rate was half maximal. The -values were downshifted relative to the intrinsic pKa-value of aa-tRNAs in bulk solution. Gly-tRNAGly had the smallest downshift, while Ile-tRNAIle and Ala-tRNAAla had the largest downshifts. These downshifts correlate strongly with molecular dynamics (MD) estimates of the downshifts in pKa-values of these aa-tRNAs upon A-site binding. Our data show the chemistry of peptide bond formation to be rate limiting for peptidyl transfer at pH 7.5 in the Gly and Pro cases and indicate rate limiting chemistry for all six aa-tRNAs. PMID:21169502

  6. Detection of four oxidation sites in viral prolyl-4-hydroxylase by top-down mass spectrometry

    PubMed Central

    Ge, Ying; Lawhorn, Brian G.; ElNaggar, Mariam; Sze, Siu Kwan; Begley, Tadhg P.; McLafferty, Fred W.

    2003-01-01

    Oxidative inactivation is a common problem for enzymatic reactions that proceed via iron oxo intermediates. In an investigation of the inactivation of a viral prolyl-4-hydroxylase (26 kD), electrospray mass spectrometry (MS) directly shows the degree of oxidation under varying experimental conditions, but indicates the addition at most of three oxygen atoms per molecule. Thus, molecular ion masses (M + nO) of one sample indicate the oxygen atom adducts n = 0, 1, 2, 3, and 4 of 35, 41, 19, 5 ± 3, and <2%, respectively; “top-down” MS/MS of these ions show oxidation at the sites R28–V31, E95–F107, and K216 of 22%, 28%, and 34%, respectively, but with a possible (~4%) fourth site at V125–D150. However, for the doubly oxidized molecular ions (increasing the precursor oxygen content from 0.94 to 2), MS/MS showed an easily observable ~13% oxygen at the V125–D150 site. For the “bottom-up” approach, detection of the ~4% oxidation at the V125–D150 site by MS analysis of a proteolysis mixture would have been very difficult. The unmodified peptide containing this site would represent a few percent of the proteolysis mixture; the oxidized peptide not only would be just ~4% of this, but the uniqueness of its mass value (~1–2 kD) would be far less than the 11,933 Dalton value used here. Using different molecular ion precursors for top-down MS/MS also provides kinetic data from a single sample, that is, from molecular ions with 0.94 and 2 oxygens. Little oxidation occurs at V125–D150 until K216 is oxidized, suggesting that these are competitively catalyzed by the iron center; among several prolyl-4-hydroxylases the K216, H137, and D139 are conserved residues. PMID:14500890

  7. OXYGEN-SENSITIVE RESET OF INDUCIBLE HIF TRANSACTIVATION RESPONSE: PROLYL HYDROXYLASES TUNE THE BIOLOGICAL NORMOXIC SETPOINT

    PubMed Central

    Khanna, Savita; Roy, Sashwati; Maurer, Mariah; Ratan, Rajiv R; Sen, Chandan K

    2006-01-01

    Cellular O2 sensing enables physiological adjustments to variations in tissue pO2. Under basal conditions, cells are adjusted to an O2 environment biologically read as normoxia. Any sharp departure from that state of normoxia triggers O2-sensitive biological responses. The stabilization of hypoxia-inducible factor (HIF) signifies a robust biological read-out of hypoxia. In the presence of sufficient O2, HIF is hydroxylated and degraded. HIF prolyl hydroxylation is catalyzed by prolyl hydroxylase isoenzymes PHD1, 2 and 3. Using HT22 neurons stably transfected with a HIF reporter construct, we tested a novel hypothesis postulating that biological cells are capable of resetting their normoxic set-point by O2-sensitive changes in PHD expression. Results of this study show that the pO2 of the mouse brain cortex was 35 mm Hg or 5% O2. Exposure of HT22, adjusted to growing in 20% O2, to 5% O2 resulted in HIF-driven transcription. However, cells adjusted to growing in 5% O2 did not report hypoxia. Cells adjusted to growing in 30% O2 reported hypoxia when acutely exposed to room air culture conditions. When grown under high O2 conditions, cells reset their normoxic set-point upwards by down-regulating the expression of PHD1–3. When grown under low O2 conditions, cells reset their normoxic set-point downwards by inducing the expression of PHD1–3. Exposure of mice in vivo to a hypoxic 10% O2 environment lowered blood as well as brain pO2. Such hypoxic exposure induced PHD1–3. Exposure of mice to a hyperoxic 50% O2 ambience repressed the expression of PHD1–3 indicating that O2-sensitive regulation of PHD expression is effective in the brain in vivo. siRNA dependent knock-down of PHD expression revealed that O2-sensitive regulation of PHD may contribute to tuning the normoxic set-point in biological cells. PMID:16785028

  8. Prolyl-4-hydroxylase α subunit 2 promotes breast cancer progression and metastasis by regulating collagen deposition.

    PubMed

    Xiong, Gaofeng; Deng, Lei; Zhu, Jieqing; Rychahou, Piotr G; Xu, Ren

    2014-01-02

    Increased collagen deposition provides physical and biochemical signals to support tumor growth and invasion during breast cancer development. Therefore, inhibition of collagen synthesis and deposition has been considered a strategy to suppress breast cancer progression. Collagen prolyl-4-hydroxylase α subunit 2 (P4HA2), an enzyme hydroxylating proline residues in -X-Pro-Gly- sequences, is a potential therapeutic target for the disorders associated with increased collagen deposition. However, expression and function of P4HA2 in breast cancer progression are not well investigated. Gene co-expression analysis was performed in the published microarray datasets to identify potential regulators of collagen I, III, and IV in human breast cancer tissue. Expression of P4HA2 was silenced by shRNAs, and its activity was inhibited by 1, 4-DPCA, a prolyl-4-hydroxylase inhibitor. Three-dimensional culture assay was used to analyze roles of P4HA2 in regulating malignant phenotypes of breast cancer cells. Reduced deposition of collagen I and IV was detected by Western blotting and immunofluorescence. Control and P4HA2-silenced breast cancer cells were injected into fat pad and tail vein of SCID mice to examine effect of P4HA2 on tumor growth and lung metastasis. Using gene co-expression analysis, we showed that P4HA2 was associated with expression of Col1A1, Col3A1, and Col4A1 during breast cancer development and progression. P4HA2 mRNA levels were significantly upregulated in breast cancer compared to normal mammary tissue. Increased mRNA levels of P4HA2 correlated with poor clinical outcome in breast cancer patients, which is independent of estrogen receptor status. Silencing P4HA2 expression or treatment with the P4HA inhibitor significantly inhibited cell proliferation and suppressed aggressive phenotypes of breast cancer cells in 3D culture, accompanied by reduced deposition of collagen I and IV. We also found that knockdown of P4HA2 inhibited mammary tumor growth and

  9. Inhibition of prolyl 4-hydroxylase decreases muscle fibrosis following chronic rotator cuff tear

    PubMed Central

    Gumucio, J. P.; Flood, M. D.; Bedi, A.; Kramer, H. F.; Russell, A. J.

    2017-01-01

    Objectives Rotator cuff tears are among the most frequent upper extremity injuries. Current treatment strategies do not address the poor quality of the muscle and tendon following chronic rotator cuff tears. Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor that activates many genes that are important in skeletal muscle regeneration. HIF-1α is inhibited under normal physiological conditions by the HIF prolyl 4-hydroxylases (PHDs). In this study, we used a pharmacological PHD inhibitor, GSK1120360A, to enhance the activity of HIF-1α following the repair of a chronic cuff tear, and measured muscle fibre contractility, fibrosis, gene expression, and enthesis mechanics. Methods Chronic supraspinatus tears were induced in adult rats, and repaired 28 days later. Rats received 0 mg/kg, 3 mg/kg, or 10 mg/kg GSK1120360A daily. Collagen content, contractility, fibre type distribution and size, the expression of genes involved in fibrosis, lipid accumulation, atrophy and inflammation, and the mechanical properties of the enthesis were then assessed two weeks following surgical repair. Results At two weeks following repair, treatment groups showed increased muscle mass but there was a 15% decrease in force production in the 10 mg/kg group from controls, and no difference between the 0 mg/kg and the 3 mg/kg groups. There was a decrease in the expression of several gene transcripts related to matrix accumulation and fibrosis, and a 50% decrease in collagen content in both treated groups compared with controls. Additionally, the expression of inflammatory genes was reduced in the treated groups compared with controls. Finally, PHD inhibition improved the maximum stress and displacement to failure in repaired tendons. Conclusions GSK1120360A resulted in improved enthesis mechanics with variable effects on muscle function. PHD inhibition may be beneficial for connective tissue injuries in which muscle atrophy has not occurred. Cite this article: J. P. Gumucio

  10. Inhibition of prolyl 4-hydroxylase decreases muscle fibrosis following chronic rotator cuff tear.

    PubMed

    Gumucio, J P; Flood, M D; Bedi, A; Kramer, H F; Russell, A J; Mendias, C L

    2017-01-01

    Rotator cuff tears are among the most frequent upper extremity injuries. Current treatment strategies do not address the poor quality of the muscle and tendon following chronic rotator cuff tears. Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor that activates many genes that are important in skeletal muscle regeneration. HIF-1α is inhibited under normal physiological conditions by the HIF prolyl 4-hydroxylases (PHDs). In this study, we used a pharmacological PHD inhibitor, GSK1120360A, to enhance the activity of HIF-1α following the repair of a chronic cuff tear, and measured muscle fibre contractility, fibrosis, gene expression, and enthesis mechanics. Chronic supraspinatus tears were induced in adult rats, and repaired 28 days later. Rats received 0 mg/kg, 3 mg/kg, or 10 mg/kg GSK1120360A daily. Collagen content, contractility, fibre type distribution and size, the expression of genes involved in fibrosis, lipid accumulation, atrophy and inflammation, and the mechanical properties of the enthesis were then assessed two weeks following surgical repair. At two weeks following repair, treatment groups showed increased muscle mass but there was a 15% decrease in force production in the 10 mg/kg group from controls, and no difference between the 0 mg/kg and the 3 mg/kg groups. There was a decrease in the expression of several gene transcripts related to matrix accumulation and fibrosis, and a 50% decrease in collagen content in both treated groups compared with controls. Additionally, the expression of inflammatory genes was reduced in the treated groups compared with controls. Finally, PHD inhibition improved the maximum stress and displacement to failure in repaired tendons. GSK1120360A resulted in improved enthesis mechanics with variable effects on muscle function. PHD inhibition may be beneficial for connective tissue injuries in which muscle atrophy has not occurred.Cite this article: J. P. Gumucio, M. D. Flood, A. Bedi, H. F. Kramer, A. J

  11. Recombinant production, crystallization and X-ray crystallographic structure determination of the peptidyl-tRNA hydrolase of Pseudomonas aeruginosa

    SciTech Connect

    Hughes, Ronny C.; McFeeters, Hana; Coates, Leighton; McFeeters, Robert L.

    2014-10-15

    The peptidyl-tRNA hydrolase enzyme from the pathogenic bacterium Pseudomonas aeruginosa (Pth; EC 3.1.1.29) has been cloned, expressed in Escherichia coli and crystallized for X-ray structural analysis. Suitable crystals were grown using the sitting-drop vapour-diffusion method after one week of incubation against a reservoir solution consisting of 20% polyethylene glycol 4000, 100 mM Tris pH 7.5, 10%(v/v) isopropyl alcohol. The crystals were used to obtain the three-dimensional structure of the native protein at 1.77 Å resolution. The structure was determined by molecular replacement of the crystallographic data processed in space group P6122 with unit-cell parameters a = b = 63.62,c = 155.20 Å, α = β = 90, γ = 120°. The asymmetric unit of the crystallographic lattice was composed of a single copy of the enzyme molecule with a 43% solvent fraction, corresponding to a Matthews coefficient of 2.43 Å3 Da-1. The crystallographic structure reported here will serve as the foundation for future structure-guided efforts towards the development of novel small-molecule inhibitors specific to bacterial Pths.

  12. Nascent peptide in the ribosome exit tunnel affects functional properties of the A-site of the peptidyl transferase center.

    PubMed

    Ramu, Haripriya; Vázquez-Laslop, Nora; Klepacki, Dorota; Dai, Qing; Piccirilli, Joseph; Micura, Ronald; Mankin, Alexander S

    2011-02-04

    The ability to monitor the nascent peptide structure and to respond functionally to specific nascent peptide sequences is a fundamental property of the ribosome. An extreme manifestation of such response is nascent peptide-dependent ribosome stalling, involved in the regulation of gene expression. The molecular mechanisms of programmed translation arrest are unclear. By analyzing ribosome stalling at the regulatory cistron of the antibiotic resistance gene ermA, we uncovered a carefully orchestrated cooperation between the ribosomal exit tunnel and the A-site of the peptidyl transferase center (PTC) in halting translation. The presence of an inducing antibiotic and a specific nascent peptide in the exit tunnel abrogate the ability of the PTC to catalyze peptide bond formation with a particular subset of amino acids. The extent of the conferred A-site selectivity is modulated by the C-terminal segment of the nascent peptide, where the third-from-last residue plays a critical role. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Peptidyl Aldehyde NK-1.8k Suppresses Enterovirus 71 and Enterovirus 68 Infection by Targeting Protease 3C

    PubMed Central

    Wang, Yaxin; Yang, Ben; Zhai, Yangyang; Rao, Zihe

    2015-01-01

    Enterovirus (EV) is one of the major causative agents of hand, foot, and mouth disease in the Pacific-Asia region. In particular, EV71 causes severe central nervous system infections, and the fatality rates from EV71 infection are high. Moreover, an outbreak of respiratory illnesses caused by an emerging EV, EV68, recently occurred among over 1,000 young children in the United States and was also associated with neurological infections. Although enterovirus has emerged as a considerable global public health threat, no antiviral drug for clinical use is available. In the present work, we screened our compound library for agents targeting viral protease and identified a peptidyl aldehyde, NK-1.8k, that inhibits the proliferation of different EV71 strains and one EV68 strain and that had a 50% effective concentration of 90 nM. Low cytotoxicity (50% cytotoxic concentration, >200 μM) indicated a high selective index of over 2,000. We further characterized a single amino acid substitution inside protease 3C (3Cpro), N69S, which conferred EV71 resistance to NK-1.8k, possibly by increasing the flexibility of the substrate binding pocket of 3Cpro. The combination of NK-1.8k and an EV71 RNA-dependent RNA polymerase inhibitor or entry inhibitor exhibited a strong synergistic anti-EV71 effect. Our findings suggest that NK-1.8k could potentially be developed for anti-EV therapy. PMID:25691647

  14. Structure of the adenylation-peptidyl carrier protein didomain of the Microcystis aeruginosa microcystin synthetase McyG.

    PubMed

    Tan, Xiao-Feng; Dai, Ya-Nan; Zhou, Kang; Jiang, Yong-Liang; Ren, Yan-Min; Chen, Yuxing; Zhou, Cong-Zhao

    2015-04-01

    Microcystins, which are the most common cause of hepatotoxicity associated with cyanobacterial water blooms, are assembled in vivo on a large multienzyme complex via a mixed nonribosomal peptide synthetase/polyketide synthetase (NRPS/PKS). The biosynthesis of microcystin in Microcystis aeruginosa PCC 7806 starts with the enzyme McyG, which contains an adenylation-peptidyl carrier protein (A-PCP) didomain for loading the starter unit to assemble the side chain of an Adda residue. However, the catalytic mechanism remains unclear. Here, the 2.45 Å resolution crystal structure of the McyG A-PCP didomain complexed with the catalytic intermediate L-phenylalanyl-adenylate (L-Phe-AMP) is reported. Each asymmetric unit contains two protein molecules, one of which consists of the A-PCP didomain and the other of which comprises only the A domain. Structural analyses suggest that Val227 is likely to be critical for the selection of hydrophobic substrates. Moreover, two distinct interfaces demonstrating variable crosstalk between the PCP domain and the A domain were observed. A catalytic cycle for the adenylation and peptide transfer of the A-PCP didomain is proposed.

  15. Induced fit of the peptidyl-transferase center of the ribosome and conformational freedom of the esterified amino acids.

    PubMed

    Lehmann, Jean

    2017-02-01

    The catalytic site of most enzymes can efficiently handle only one substrate. In contrast, the ribosome is capable of polymerizing at a similar rate at least 20 different kinds of amino acids from aminoacyl-tRNA carriers while using just one catalytic site, the peptidyl-transferase center (PTC). An induced-fit mechanism has been uncovered in the PTC, but a possible connection between this mechanism and the uniform handling of the substrates has not been investigated. We present an analysis of published ribosome structures supporting the hypothesis that the induced fit eliminates unreactive rotamers predominantly populated for some A-site aminoacyl esters before induction. We show that this hypothesis is fully consistent with the wealth of kinetic data obtained with these substrates. Our analysis reveals that induction constrains the amino acids into a reactive conformation in a side-chain independent manner. It allows us to highlight the rationale of the PTC structural organization, which confers to the ribosome the very unusual ability to handle large as well as small substrates. © 2017 Lehmann; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  16. Membrane-anchored prolyl hydroxylase with an export signal from the endoplasmic reticulum.

    PubMed

    Yuasa, Koji; Toyooka, Kiminori; Fukuda, Hiroo; Matsuoka, Ken

    2005-01-01

    We cloned a novel prolyl 4-hydroxylase (PH; EC 1.14.11.2) homolog cDNA from tobacco (Nicotiana tabacum) BY-2 cells based on expression sequence tag information. Like other PHs, this tobacco PH polypeptide has two conserved histidine residues, and it comprises 286 amino acids with a calculated molecular mass of 32 kDa. Interestingly, this protein and homologs in Arabidopsis and rice have predicted transmembrane sequences in their N-terminal regions. This PH homolog was expressed in BY-2 cells as a His-tagged protein, and the expressed protein showed PH activity. Incubation of membranes with high salt, urea, and protease with or without detergents indicated that this protein is an integral membrane protein with a type II configuration. Its membrane-anchored nature is specific for plants because no integral membrane PH has been found in animals. A membrane fractionation study and immunocytochemical studies indicate that this protein localizes in both the endoplasmic reticulum (ER) and Golgi apparatus. Analysis of this protein fused to green fluorescent protein indicated that basic amino acids in the cytoplasmic, N-terminal region of the PH play a role in its export from the ER.

  17. Sequential expression, activity and nuclear localization of prolyl oligopeptidase protein in the developing rat brain.

    PubMed

    Hannula, Mirva J; Männistö, Pekka T; Myöhänen, Timo T

    2011-01-01

    Prolyl oligopeptidase (POP) is a serine protease that hydrolyzes peptides shorter than 30-mer. Some evidence has recently been obtained that POP can generate protein-protein interactions and therefore participate in various physiological and pathological events. Several studies have reported that POP may be involved in neurogenesis since its activity increases during development and can be found in the nucleus of proliferating tissues. In cell cultures, POP has been shown to be localized in the nucleus, but only early in the development, since during maturation it is moved to the cytosol. We have now studied for the first time the expression of POP protein, its enzymatic activity and nuclear localization in vivo in the developing rat brain. We observed that enzymatic activity of POP is highest on embryonic day 18 while the protein amounts reach their peak at birth. Furthermore, POP is located in the nucleus only early in the development but is transferred to the cytosol already before parturition. Our in vivo results confirm the previous cell culture results supporting the role of POP in neurogenesis. A discordance of antenatal protein amounts and enzymatic activities is suggesting a tight regulation of POP activity and possibly even a nonhydrolytic role at that stage.

  18. Molecular dynamics study of prolyl oligopeptidase with inhibitor in binding cavity.

    PubMed

    Kaszuba, K; Rog, T; St Pierre, J-F; Mannisto, P T; Karttunen, M; Bunker, A

    2009-10-01

    We used the crystal structure of prolyl oligopeptidase (POP) with bound Z-pro-prolinal (ZPP) inhibitor (Protein Data Bank (PDB) structure 1QFS) to perform an intensive molecular dynamics study of the POP-ZPP complex. We performed 100 ns of simulation with the hemiacetal bond, through which the ZPP is bound to the POP, removed in order to better investigate the binding cavity environment. From basic analysis, measuring the radius of gyration, root mean square deviation, solvent accessible surface area and definition of the secondary structure of protein, we determined that the protein structure is highly stable and maintains its structure over the entire simulation time. This demonstrates that such long time simulations can be performed without the protein structure losing stability. We found that water bridges and hydrogen bonds play a negligible role in binding the ZPP thus indicating the importance of the hemiacetal bond. The two domains of the protein are bound by a set of approximately 12 hydrogen bonds, specific to the particular POP protein.

  19. Colocalization of Amanitin and a Candidate Toxin-Processing Prolyl Oligopeptidase in Amanita Basidiocarps ▿

    PubMed Central

    Luo, Hong; Hallen-Adams, Heather E.; Scott-Craig, John S.; Walton, Jonathan D.

    2010-01-01

    Fungi in the basidiomycetous genus Amanita owe their high mammalian toxicity to the bicyclic octapeptide amatoxins such as α-amanitin. Amatoxins and the related phallotoxins (such as the heptapeptide phalloidin) are encoded by members of the “MSDIN” gene family and are synthesized on ribosomes as short (34- to 35-amino-acid) proproteins. Antiamanitin antibodies and confocal microscopy were used to determine the cellular and subcellular localizations of amanitin accumulation in basidiocarps (mushrooms) of the Eastern North American destroying angel (Amanita bisporigera). Consistent with previous studies, amanitin is present throughout the basidiocarp (stipe, pileus, lamellae, trama, and universal veil), but it is present in only a subset of cells within these tissues. Restriction of amanitin to certain cells is especially marked in the hymenium. Several lines of evidence implicate a specific prolyl oligopeptidase, A. bisporigera POPB (AbPOPB), in the initial processing of the amanitin and phallotoxin proproteins. The gene for AbPOPB is restricted taxonomically to the amatoxin-producing species of Amanita and is clustered in the genome with at least one expressed member of the MSDIN gene family. Immunologically, amanitin and AbPOPB show a high degree of colocalization, indicating that toxin biosynthesis and accumulation occur in the same cells and possibly in the same subcellular compartments. PMID:20889720

  20. Remodeling of rat stromal-vascular cells to brite/beige adipocytes by prolyl-hydroxyproline

    PubMed Central

    MINAGUCHI, Jun A.; OGATA, Sakino; TAKAHASHI, Naoki; HIROSE, Takuya; UEDA, Hiromi; TAKEHANA, Kazushige

    2017-01-01

    The aim of this study was to determine the effects of prolyl-hydroxyproline (Pro-Hyp) on the proliferation and differentiation of rat stromal-vascular cells (SVCs) being cultured in a medium with (Pro-Hyp group) or without Pro-Hyp (control group). The results showed that there was no significant difference in proliferation rate of SVCs, lipid droplet (LD) diameter or intracellular concentration of triglycerides between two groups. However, the diameter range of LDs in the Pro-Hyp group tended to be smaller than that in the control group. Transmission electron microscopy showed a tendency for increase in the area of mitochondria and decrease in the number of mitochondria in the Pro-Hyp-treated SVCs. The mRNA expression levels of white adipose tissue differentiation markers (Cbp, Fabp and Serpina3k) were significantly lower, but those of the brown adipose tissue differentiation markers (Dio2, Ucp1 and Ucp3) were significantly higher in the Pro-Hyp group than in the control group. Our results suggested that Pro-Hyp can facilitate SVCs to differentiate into “brite/beige” adipocytes. PMID:28123139

  1. Prolyl hydroxylase domain-2 (PHD2) inhibition may be a better therapeutic strategy in renal anemia.

    PubMed

    Soni, Hitesh

    2014-05-01

    Recombinant human erythropoietin (rHuEPO) has revolutionized the life of dialysis patients with anemia of chronic kidney disease (CKD). Newer erythropoietin analogues with improved profile have been introduced recently. However, there are many concerns such as safety, economy and patient compliance with these newer rHuEPo analogues. Small molecules aimed to inhibit prolyl hydroxylase domain-2 (PHD2) may prevent degradation of hypoxia inducible factor-2 (HIF2) which leads to endogenous erythropoietin production. This therapeutic intervention may not only overcome the patient compliance and economic burden but also possibly overcome the safety issues related to rHuEPO and its analogues. Moreover, PHD2 inhibitors may increase the endogenous circulating iron availability via suppression of hepcidin, a master regulator of iron homeostasis which further reduces the need for exogenous intravenous iron administration for effective erythropoiesis in renal anemia patients. In conclusion, small molecule PHD2 inhibitors may have better therapeutic efficacy and potential to address clinical concerns associated with rHuEPO and rHuEPO mimetic peptides.

  2. Failure to prolyl hydroxylate hypoxia-inducible factor α phenocopies VHL inactivation in vivo

    PubMed Central

    Kim, William Y; Safran, Michal; Buckley, Marshall R M; Ebert, Benjamin L; Glickman, Jonathan; Bosenberg, Marcus; Regan, Meredith; Kaelin, William G

    2006-01-01

    Many functions have been assigned to the von Hippel-Lindau tumor suppressor gene product (pVHL), including targeting the alpha subunits of the heterodimeric transcription factor HIF (hypoxia-inducible factor) for destruction. The binding of pVHL to HIFα requires that HIFα be hydroxylated on one of two prolyl residues. We introduced HIF1α and HIF2α variants that cannot be hydroxylated on these sites into the ubiquitously expressed ROSA26 locus along with a Lox-stop-Lox cassette that renders their expression Cre-dependent. Expression of the HIF2α variant in the skin and liver induced changes that were highly similar to those seen when pVHL is lost in these organs. Dual expression of the HIF1α and HIF2α variants in liver, however, more closely phenocopied the changes seen after pVHL inactivation than did the HIF2α variant alone. Moreover, gene expression profiling confirmed that the genes regulated by HIF1α and HIF2α in the liver are overlapping but non-identical. Therefore, the pathological changes caused by pVHL inactivation in skin and liver are due largely to dysregulation of HIF target genes. PMID:16977322

  3. Reactivating HIF prolyl hydroxylases under hypoxia results in metabolic catastrophe and cell death.

    PubMed

    Tennant, D A; Frezza, C; MacKenzie, E D; Nguyen, Q D; Zheng, L; Selak, M A; Roberts, D L; Dive, C; Watson, D G; Aboagye, E O; Gottlieb, E

    2009-11-12

    Cells exposed to low-oxygen conditions (hypoxia) alter their metabolism to survive. This response, although vital during development and high-altitude survival, is now known to be a major factor in the selection of cells with a transformed metabolic phenotype during tumorigenesis. It is thought that hypoxia-selected cells have increased invasive capacity and resistance to both chemo- and radiotherapies, and therefore represent an attractive target for antitumor therapy. Hypoxia inducible factors (HIFs) are responsible for the majority of gene expression changes under hypoxia, and are themselves controlled by the oxygen-sensing HIF prolyl hydroxylases (PHDs). It was previously shown that mutations in succinate dehydrogenase lead to the inactivation PHDs under normoxic conditions, which can be overcome by treatment with alpha-ketoglutarate derivatives. Given that solid tumors contain large regions of hypoxia, the reactivation of PHDs in these conditions could induce metabolic catastrophe and therefore prove an effective antitumor therapy. In this report we demonstrate that derivatized alpha-ketoglutarate can be used as a strategy for maintaining PHD activity under hypoxia. By increasing intracellular alpha-ketoglutarate and activating PHDs we trigger PHD-dependent reversal of HIF1 activation, and PHD-dependent hypoxic cell death. We also show that derivatized alpha-ketoglutarate can permeate multiple layers of cells, reducing HIF1alpha levels and its target genes in vivo.

  4. Hypothalamic prolyl endopeptidase (PREP) regulates pancreatic insulin and glucagon secretion in mice

    PubMed Central

    Kim, Jung Dae; Toda, Chitoku; D’Agostino, Giuseppe; Zeiss, Caroline J.; DiLeone, Ralph J.; Elsworth, John D.; Kibbey, Richard G.; Chan, Owen; Harvey, Brandon K.; Richie, Christopher T.; Savolainen, Mari; Myöhänen, Timo; Jeong, Jin Kwon; Diano, Sabrina

    2014-01-01

    Prolyl endopeptidase (PREP) has been implicated in neuronal functions. Here we report that hypothalamic PREP is predominantly expressed in the ventromedial nucleus (VMH), where it regulates glucose-induced neuronal activation. PREP knockdown mice (Prepgt/gt) exhibited glucose intolerance, decreased fasting insulin, increased fasting glucagon levels, and reduced glucose-induced insulin secretion compared with wild-type controls. Consistent with this, central infusion of a specific PREP inhibitor, S17092, impaired glucose tolerance and decreased insulin levels in wild-type mice. Arguing further for a central mode of action of PREP, isolated pancreatic islets showed no difference in glucose-induced insulin release between Prepgt/gt and wild-type mice. Furthermore, hyperinsulinemic euglycemic clamp studies showed no difference between Prepgt/gt and wild-type control mice. Central PREP regulation of insulin and glucagon secretion appears to be mediated by the autonomic nervous system because Prepgt/gt mice have elevated sympathetic outflow and norepinephrine levels in the pancreas, and propranolol treatment reversed glucose intolerance in these mice. Finally, re-expression of PREP by bilateral VMH injection of adeno-associated virus–PREP reversed the glucose-intolerant phenotype of the Prepgt/gt mice. Taken together, our results unmask a previously unknown player in central regulation of glucose metabolism and pancreatic function. PMID:25071172

  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. Loss of Epithelial Hypoxia-Inducible Factor Prolyl Hydroxylase 2 Accelerates Skin Wound Healing in Mice

    PubMed Central

    Kalucka, Joanna; Ettinger, Andreas; Franke, Kristin; Mamlouk, Soulafa; Singh, Rashim Pal; Farhat, Katja; Muschter, Antje; Olbrich, Susanne; Breier, Georg; Katschinski, Dörthe M.; Huttner, Wieland; Weidemann, Alexander

    2013-01-01

    Skin wound healing in mammals is a complex, multicellular process that depends on the precise supply of oxygen. Hypoxia-inducible factor (HIF) prolyl hydroxylase 2 (PHD2) serves as a crucial oxygen sensor and may therefore play an important role during reepithelialization. Hence, this study was aimed at understanding the role of PHD2 in cutaneous wound healing using different lines of conditionally deficient mice specifically lacking PHD2 in inflammatory, vascular, or epidermal cells. Interestingly, PHD2 deficiency only in keratinocytes and not in myeloid or endothelial cells was found to lead to faster wound closure, which involved enhanced migration of the hyperproliferating epithelium. We demonstrate that this effect relies on the unique expression of β3-integrin in the keratinocytes around the tip of the migrating tongue in an HIF1α-dependent manner. Furthermore, we show enhanced proliferation of these cells in the stratum basale, which is directly related to their attenuated transforming growth factor β signaling. Thus, loss of the central oxygen sensor PHD2 in keratinocytes stimulates wound closure by prompting skin epithelial cells to migrate and proliferate. Inhibition of PHD2 could therefore offer novel therapeutic opportunities for the local treatment of cutaneous wounds. PMID:23798557

  7. Prolyl hydroxylase domain proteins regulate bone mass through their expression in osteoblasts.

    PubMed

    Zhu, Ke; Song, Pingping; Lai, Yumei; Liu, Chuanju; Xiao, Guozhi

    2016-12-05

    The roles of prolyl hydroxylase domain proteins (PHDs) in bone are incompletely understood. Here we deleted the expression of genes encoding PHD1, PHD2, and PHD3 in osteoblasts in mice by breeding the floxed Phd1-3 mice with Col1a1-Cre transgenic mice. Results showed that mice lacking PHD1-3 in osteoblasts (Phd1-3ob-/-) had increased bone mass. Bone parameters such as bone volume/tissue volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) were increased, while trabecular spacing (Tb.Sp) was decreased in Phd1-3ob-/- relative to wild-type (WT) femurs. In contrast, loss of PHD1-3 in osteoblasts did not alter cortical thickness (Cort.Th). The mineralization apposition rate (MAR) was increased in Phd1-3ob-/- bone compared to that of wild-type (WT) bone, demonstrating an enhancement of osteoblast function. Loss of PHD1-3 increased the number of osteoblast progenitors (CFU-OBs) in bone marrow cultures. Interestingly, deleting Phd1-3 genes in osteoblasts increased osteoclast formation in vitro and in bone.

  8. Inhibition of prolyl hydroxylase domain-containing protein downregulates vascular angiotensin II type 1 receptor.

    PubMed

    Matsuura, Hirohide; Ichiki, Toshihiro; Ikeda, Jiro; Takeda, Kotaro; Miyazaki, Ryohei; Hashimoto, Toru; Narabayashi, Eriko; Kitamoto, Shiro; Tokunou, Tomotake; Sunagawa, Kenji

    2011-09-01

    Inhibition of prolyl hydroxylase domain-containing protein (PHD) by hypoxia stabilizes hypoxia-inducible factor 1 and increases the expression of target genes, such as vascular endothelial growth factor. Although the systemic renin-angiotensin system is activated by hypoxia, the role of PHD in the regulation of the renin-angiotensin system remains unknown. We examined the effect of PHD inhibition on the expression of angiotensin II type 1 receptor (AT(1)R). Hypoxia, cobalt chloride, and dimethyloxalylglycine, all known to inhibit PHD, reduced AT(1)R expression in vascular smooth muscle cells. Knockdown of PHD2, a major isoform of PHDs, by RNA interference also reduced AT(1)R expression. Cobalt chloride diminished angiotensin II-induced extracellular signal-regulated kinase phosphorylation. Cobalt chloride decreased AT(1)R mRNA through transcriptional and posttranscriptional mechanisms. Oral administration of cobalt chloride (14 mg/kg per day) to C57BL/6J mice receiving angiotensin II infusion (490 ng/kg per minute) for 4 weeks significantly attenuated perivascular fibrosis of the coronary arteries without affecting blood pressure level. These data suggest that PHD inhibition may be beneficial for the treatment of cardiovascular diseases by inhibiting renin-angiotensin system via AT(1)R downregulation.

  9. Mycobacterium tuberculosis Prolyl Oligopeptidase Induces In vitro Secretion of Proinflammatory Cytokines by Peritoneal Macrophages

    PubMed Central

    Portugal, Brina; Motta, Flávia N.; Correa, Andre F.; Nolasco, Diego O.; de Almeida, Hugo; Magalhães, Kelly G.; Atta, Ana L. V.; Vieira, Francisco D.; Bastos, Izabela M. D.; Santana, Jaime M.

    2017-01-01

    Tuberculosis (TB) is a disease that leads to death over 1 million people per year worldwide and the biological mediators of this pathology are poorly established, preventing the implementation of effective therapies to improve outcomes in TB. Host–bacterium interaction is a key step to TB establishment and the proteases produced by these microorganisms seem to facilitate bacteria invasion, migration and host immune response evasion. We presented, for the first time, the identification, biochemical characterization, molecular dynamics (MDs) and immunomodulatory properties of a prolyl oligopeptidase (POP) from Mycobacterium tuberculosis (POPMt). POP is a serine protease that hydrolyzes substrates with high specificity for proline residues and has already been characterized as virulence factor in infectious diseases. POPMt reveals catalytic activity upon N-Suc-Gly-Pro-Leu-Gly-Pro-AMC, a recognized POP substrate, with optimal activity at pH 7.5 and 37°C. The enzyme presents KM and Kcat/KM values of 108 μM and 21.838 mM-1 s-1, respectively. MDs showed that POPMt structure is similar to that of others POPs, which consists of a cylindrical architecture divided into an α/β hydrolase catalytic domain and a β-propeller domain. Finally, POPMt was capable of triggering in vitro secretion of proinflammatory cytokines by peritoneal macrophages, an event dependent on POPMt intact structure. Our data suggests that POPMt may contribute to an inflammatory response during M. tuberculosis infection. PMID:28223969

  10. Structure of proline iminopeptidase from Xanthomonas campestris pv. citri: a prototype for the prolyl oligopeptidase family.

    PubMed

    Medrano, F J; Alonso, J; García, J L; Romero, A; Bode, W; Gomis-Rüth, F X

    1998-01-02

    The proline iminopeptidase from Xanthomonas campestris pv. citri is a serine peptidase that catalyses the removal of N-terminal proline residues from peptides with high specificity. We have solved its three-dimensional structure by multiple isomorphous replacement and refined it to a crystallographic R-factor of 19.2% using X-ray data to 2.7 A resolution. The protein is folded into two contiguous domains. The larger domain shows the general topology of the alpha/beta hydrolase fold, with a central eight-stranded beta-sheet flanked by two helices and the 11 N-terminal residues on one side, and by four helices on the other side. The smaller domain is placed on top of the larger domain and essentially consists of six helices. The active site, located at the end of a deep pocket at the interface between both domains, includes a catalytic triad of Ser110, Asp266 and His294. Cys269, located at the bottom of the active site very close to the catalytic triad, presumably accounts for the inhibition by thiol-specific reagents. The overall topology of this iminopeptidase is very similar to that of yeast serine carboxypeptidase. The striking secondary structure similarity to human lymphocytic prolyl oligopeptidase and dipeptidyl peptidase IV makes this proline iminopeptidase structure a suitable model for the three-dimensional structure of other peptidases of this family.

  11. Structure of proline iminopeptidase from Xanthomonas campestris pv. citri: a prototype for the prolyl oligopeptidase family.

    PubMed Central

    Medrano, F J; Alonso, J; García, J L; Romero, A; Bode, W; Gomis-Rüth, F X

    1998-01-01

    The proline iminopeptidase from Xanthomonas campestris pv. citri is a serine peptidase that catalyses the removal of N-terminal proline residues from peptides with high specificity. We have solved its three-dimensional structure by multiple isomorphous replacement and refined it to a crystallographic R-factor of 19.2% using X-ray data to 2.7 A resolution. The protein is folded into two contiguous domains. The larger domain shows the general topology of the alpha/beta hydrolase fold, with a central eight-stranded beta-sheet flanked by two helices and the 11 N-terminal residues on one side, and by four helices on the other side. The smaller domain is placed on top of the larger domain and essentially consists of six helices. The active site, located at the end of a deep pocket at the interface between both domains, includes a catalytic triad of Ser110, Asp266 and His294. Cys269, located at the bottom of the active site very close to the catalytic triad, presumably accounts for the inhibition by thiol-specific reagents. The overall topology of this iminopeptidase is very similar to that of yeast serine carboxypeptidase. The striking secondary structure similarity to human lymphocytic prolyl oligopeptidase and dipeptidyl peptidase IV makes this proline iminopeptidase structure a suitable model for the three-dimensional structure of other peptidases of this family. PMID:9427736

  12. Role of NonO-histone interaction in TNFalpha-suppressed prolyl-4-hydroxylase alpha1.

    PubMed

    Zhang, Cheng; Zhang, Ming-Xiang; Shen, Ying H; Burks, Jared K; Li, Xiao-Nan; LeMaire, Scott A; Yoshimura, Koichi; Aoki, Hiroki; Matsuzaki, Masunori; An, Feng-Shuang; Engler, David A; Matsunami, Risë K; Coselli, Joseph S; Zhang, Yun; Wang, Xing Li

    2008-08-01

    Inflammation is a key process in cardiovascular diseases. The extracellular matrix (ECM) of the vasculature is a major target of inflammatory cytokines, and TNFalpha regulates ECM metabolism by affecting collagen production. In this study, we have examined the pathways mediating TNFalpha-induced suppression of prolyl-4 hydroxylase alpha1 (P4Halpha1), the rate-limiting isoform of P4H responsible for procollagen hydroxylation, maturation, and organization. Using human aortic smooth muscle cells, we found that TNFalpha activated the MKK4-JNK1 pathway, which induced histone (H) 4 lysine 12 acetylation within the TNFalpha response element in the P4Halpha1 promoter. The acetylated-H4 then recruited a transcription factor, NonO, which, in turn, recruited HDACs and induced H3 lysine 9 deacetylation, thereby inhibiting transcription of the P4Halpha1 promoter. Furthermore, we found that TNFalpha oxidized DJ-1, which may be essential for the NonO-P4Halpha1 interaction because treatment with gene specific siRNA to knockout DJ-1 eliminated the TNFalpha-induced NonO-P4Halpha1 interaction and its suppression. Our findings may be relevant to aortic aneurysm and dissection and the stability of the fibrous cap of atherosclerotic plaque in which collagen metabolism is important in arterial remodeling. Defining this cytokine-mediated regulatory pathway may provide novel molecular targets for therapeutic intervention in preventing plaque rupture and acute coronary occlusion.

  13. Prolyl hydroxylase 2: a novel regulator of β2 -adrenoceptor internalization.

    PubMed

    Yan, Biao; Huo, Zhaoxia; Liu, Ying; Lin, Xiaoping; Li, Jun; Peng, Luying; Zhao, Hong; Zhou, Zhao-Nian; Liang, Xingqun; Liu, Yi; Zhu, Weidong; Liang, Dandan; Li, Li; Sun, Yunfu; Cui, Jianmin; Chen, Yi-Han

    2011-12-01

    Adrenergic receptor (AR)-mediated signalling is modulated by oxygen levels. Prolyl hydroxylases (PHDs) are crucial for intracellular oxygen sensing and organism survival. However, it remains to be clarified whether or how PHDs are involved in the regulation of β(2) -adrenoceptor (β(2) -AR) signalling. Here we show that PHD2 can modulate the rate of β(2) -AR internalization through interactions with β-arrestin 2. PHD2 hydroxylates β-arrestin 2 at the proline (Pro)(176), Pro(179) and Pro(181) sites, which retards the recruitment of β-arrestin 2 to the plasma membrane and inhibits subsequent co-internalization with β(2) -AR into the cytosol. β(2) -AR internalization is critical to control the temporal and spatial aspects of β(2) -AR signalling. Identifying novel regulators of β(2) -AR internalization will enable us to develop new strategies to manipulate receptor signalling and provide potential targets for drug development in the prevention and treatment of diseases associated with β(2) -AR signalling dysregulation.

  14. Purification and Characterization of an X-Prolyl-Dipeptidyl Peptidase from Lactobacillus sakei

    PubMed Central

    Sanz, Yolanda; Toldrá, Fidel

    2001-01-01

    An X-prolyl-dipeptidyl peptidase has been purified from Lactobacillus sakei by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55°C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu2+, Hg2+, and Zn2+). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates. Km s for Gly-Pro- and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 μM, respectively; those for Gly-Pro- and Ala-Pro-p-nitroanilide were 192 and 50 μM, respectively. Among peptides, β-casomorphin 1-3 was hydrolyzed at the highest rates, while the relative hydrolysis of the other tested peptides was only 1 to 12%. The potential role of the purified enzyme in the proteolytic pathway by catalyzing the hydrolysis of peptide bonds involving proline is discussed. PMID:11282638

  15. Generation of food-grade recombinant Lactobacillus casei delivering Myxococcus xanthus prolyl endopeptidase.

    PubMed

    Alvarez-Sieiro, Patricia; Martin, Maria Cruz; Redruello, Begoña; Del Rio, Beatriz; Ladero, Victor; Palanski, Brad A; Khosla, Chaitan; Fernandez, Maria; Alvarez, Miguel A

    2014-08-01

    Prolyl endopeptidases (PEP) (EC 3.4.21.26), a family of serine proteases with the ability to hydrolyze the peptide bond on the carboxyl side of an internal proline residue, are able to degrade immunotoxic peptides responsible for celiac disease (CD), such as a 33-residue gluten peptide (33-mer). Oral administration of PEP has been suggested as a potential therapeutic approach for CD, although delivery of the enzyme to the small intestine requires intrinsic gastric stability or advanced formulation technologies. We have engineered two food-grade Lactobacillus casei strains to deliver PEP in an in vitro model of small intestine environment. One strain secretes PEP into the extracellular medium, whereas the other retains PEP in the intracellular environment. The strain that secretes PEP into the extracellular medium is the most effective to degrade the 33-mer and is resistant to simulated gastrointestinal stress. Our results suggest that in the future, after more studies and clinical trials, an engineered food-grade Lactobacillus strain may be useful as a vector for in situ production of PEP in the upper small intestine of CD patients.

  16. No genetic association of the human prolyl endopeptidase gene in the Dutch celiac disease population.

    PubMed

    Diosdado, Begoña; Stepniak, Dariusz T; Monsuur, Alienke J; Franke, Lude; Wapenaar, Martin C; Mearin, Maria Luisa; Koning, Frits; Wijmenga, Cisca

    2005-09-01

    Celiac disease (CD) is a complex genetic disorder of the small intestine. The DQ2/DQ8 human leucocyte antigen (HLA) genes explain approximately 40% of the genetic component of the disease, but the remaining non-HLA genes have not yet been identified. The key environmental factor known to be involved in the disease is gluten, a major protein present in wheat, barley, and rye. Integrating microarray data and linkage data from chromosome 6q21-22 revealed the prolyl endopeptidase (PREP) gene as a potential CD candidate in the Dutch population. Interestingly, this gene encodes for the only enzyme that is able to cleave the proline-rich gluten peptides. To investigate the role of the human PREP gene as a primary genetic factor in CD, we conducted gene expression, sequence analysis, and genetic association studies of the PREP gene and determined PREP enzyme activity in biopsies from CD patients and controls. Sequence analysis of the coding region of the PREP gene revealed two novel polymorphisms. Genetic association studies using two novel polymorphisms and three known PREP variants excluded a genetic association between PREP and CD. Determination of PREP activity revealed weak but significant differences between treated and untreated CD biopsies (P < 0.05). Our results from the association study indicate that PREP is not a causative gene for CD in the Dutch population. These are further supported by the activity determinations in which we observed no differences in PREP activity between CD patients and controls.

  17. Efficient degradation of gluten by a prolyl endoprotease in a gastrointestinal model: implications for coeliac disease.

    PubMed

    Mitea, C; Havenaar, R; Drijfhout, J Wouter; Edens, L; Dekking, L; Koning, F

    2008-01-01

    Coeliac disease is caused by an immune response to gluten. As gluten proteins are proline rich they are resistant to enzymatic digestion in the gastrointestinal tract, a property that probably contributes to the immunogenic nature of gluten. This study determined the efficiency of gluten degradation by a post-proline cutting enzyme, Aspergillus niger prolyl endoprotease (AN-PEP), in a dynamic system that closely mimics the human gastrointestinal tract (TIM system). Two experiments were performed. In the first, a slice of bread was processed in the TIM system with and without co-administration of AN-PEP. In the second, a standard fast food menu was used. Samples of the digesting meals were taken from the stomach, duodenum, jejunum and ileum compartments at time zero until 4 hours after the start of the experiment. In these samples the levels of immunogenic peptides from gliadins and glutenins were assessed by monoclonal antibody-based competition assays, Western blot analysis and proliferation T-cell assays. AN-PEP accelerated the degradation of gluten in the stomach compartment to such an extent that hardly any gluten reached the duodenum compartment. AN-PEP is capable of accelerating the degradation of gluten in a gastrointestinal system that closely mimics in-vivo digestion. This implies that the co-administration of AN-PEP with a gluten-containing meal might eliminate gluten toxicity, thus offering patients the possibility of abandoning (occasionally) their strict gluten-free diet.

  18. 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

  19. Generation of food-grade recombinant Lactobacillus casei delivering Myxococcus xanthus prolyl endopeptidase

    PubMed Central

    Alvarez-Sieiro, Patricia; Martin, Maria Cruz; Redruello, Begoña; del Rio, Beatriz; Ladero, Victor; Palanski, Brad A.; Khosla, Chaitan; Fernandez, Maria; Alvarez, Miguel A.

    2015-01-01

    Prolyl endopeptidases (PEP), a family of serine proteases with the ability to hydrolyze the peptide bond on the carboxyl side of an internal proline residue, are able to degrade immunotoxic peptides responsible for celiac disease (CD), such as a 33-residue gluten peptide (33-mer). Oral administration of PEP has been suggested as a potential therapeutic approach for CD, although delivery of the enzyme to the small intestine requires intrinsic gastric stability or advanced formulation technologies. We have engineered two food-grade Lactobacillus casei strains to deliver PEP in an in vitro model of small intestine environment. One strain secretes PEP into the extracellular medium, whereas the other retains PEP in the intracellular environment. The strain that secretes PEP into the extracellular medium is the most effective to degrade the 33-mer and is resistant to simulated gastrointestinal stress. Our results suggest that in a future, after more studies and clinical trials, an engineered food-grade Lactobacillus strain may be useful as a vector for in situ production of PEP in the upper small intestine of CD patients. PMID:24752841

  20. 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

  1. High-resolution solid-state NMR structure of Alanyl-Prolyl-Glycine

    PubMed Central

    Barnes, Alexander B.; Andreas, Loren B.; Huber, Matthias; Ramachandran, Ramesh; van der Wel, Patrick C.A.; Veshtort, Mikhail; Griffin, Robert G.; Mehta, Manish A.

    2014-01-01

    We present a de novo high-resolution structure of the peptide Alanyl-Prolyl-Glycine using a combination of sensitive solid-state NMR techniques that each yield precise structural constraints. High-quality 13C–13C distance constraints are extracted by fitting rotational resonance width (R2W) experiments using Multimode Multipole Floquet Theory and experimental chemical shift anisotropy (CSA) orientations. In this strategy, a structure is first calculated using DANTE-REDOR and torsion angle measurements and the resulting relative CSA orientations are used as an input parameter in the 13C–13C distance calculations. Finally, a refined structure is calculated using all the constraints. We investigate the effect of different structural constraints on structure quality, as determined by comparison to the crystal structure and also self-consistency of the calculated structures. Inclusion of all or subsets of these constraints into CNS calculations resulted in high-quality structures (0.02 Å backbone RMSD using all 11 constraints). PMID:19596601

  2. Prolyl endopeptidase-mediated destruction of T cell epitopes in whole gluten: chemical and immunological characterization.

    PubMed

    Marti, Thomas; Molberg, Oyvind; Li, Qing; Gray, Gary M; Khosla, Chaitan; Sollid, Ludvig M

    2005-01-01

    Celiac Sprue is a widely prevalent immune disease of the small intestine induced by dietary gluten intake in genetically susceptible individuals. It has been suggested that prolyl endopeptidases (PEPs) may be useful catalysts for gluten detoxification. We have investigated this hypothesis using food-grade gluten as the target antigen, and a combination of mass spectrometry and patient-derived T cells as quantitative assay systems. Spectrometric characterization of physiologically proteolyzed gluten revealed a number of 10 to 50 residue peptides containing known T cell epitopes involved in Celiac Sprue pathogenesis. Several of these peptides were multivalent, suggesting they may be potent triggers of the inflammatory response to gluten in celiac patients. Treatment of proteolyzed gluten with recombinant bacterial PEP decreased the number of potentially immunostimulatory peptides. Substantially reduced immunogenicity was also quantified in 12 of 14 intestinal polyclonal T cell lines from celiac patients. Kinetic investigations using eight T cell clones showed rapid destruction of alpha-gliadin epitopes, but less complete processing of gamma-gliadin epitopes. Given the difficulty associated with a strict lifelong gluten-exclusion diet, the ability of a single enzyme to greatly reduce the antigenic burden of grocery store gluten reinforces the case for developing oral peptidase therapy against Celiac Sprue.

  3. Toll-Like Receptor 4 Engagement Mediates Prolyl Endopeptidase Release from Airway Epithelia via Exosomes.

    PubMed

    Szul, Tomasz; Bratcher, Preston E; Fraser, Kyle B; Kong, Michele; Tirouvanziam, Rabindra; Ingersoll, Sarah; Sztul, Elizabeth; Rangarajan, Sunil; Blalock, J Edwin; Xu, Xin; Gaggar, Amit

    2016-03-01

    Proteases are important regulators of pulmonary remodeling and airway inflammation. Recently, we have characterized the enzyme prolyl endopeptidase (PE), a serine peptidase, as a critical protease in the generation of the neutrophil chemoattractant tripeptide Pro-Gly-Pro (PGP) from collagen. However, PE has been characterized as a cytosolic enzyme, and the mechanism mediating PE release extracellularly remains unknown. We examined the role of exosomes derived from airway epithelia as a mechanism for PE release and the potential extracellular signals that regulate the release of these exosomes. We demonstrate a specific regulatory pathway of exosome release from airway epithelia and identify PE as novel exosome cargo. LPS stimulation of airway epithelial cells induces release of PE-containing exosomes, which is significantly attenuated by small interfering RNA depletion of Toll-like receptor 4 (TLR4). These differences were recapitulated upon intratracheal LPS administration in mice competent versus deficient for TLR4 signaling. Finally, sputum samples from subjects with cystic fibrosis colonized with Pseudomonas aeruginosa demonstrate elevated exosome content and increased PE levels. This TLR4-based mechanism highlights the first report of nonstochastic release of exosomes in the lung and couples TLR4 activation with matrikine generation. The increased quantity of these proteolytic exosomes in the airways of subjects with chronic lung disease highlights a new mechanism of injury and inflammation in the pathogenesis of pulmonary disorders.

  4. Loss of prolyl hydroxylase-2 in myeloid cells and T-lymphocytes impairs tumor development.

    PubMed

    Mamlouk, Soulafa; Kalucka, Joanna; Singh, Rashim Pal; Franke, Kristin; Muschter, Antje; Langer, Anika; Jakob, Christiane; Gassmann, Max; Baretton, Gustavo B; Wielockx, Ben

    2014-02-15

    The tumor microenvironment plays a pivotal role during cancer development and progression. The balance between suppressive and cytotoxic responses of the tumor immune microenvironment has been shown to have a direct effect on the final outcome in various human and experimental tumors. Recently, we demonstrated that the oxygen sensor HIF-prolyl hydroxylase-2 (PHD2) plays a detrimental role in tumor cells, stimulating systemic growth and metastasis in mice. In our current study, we show that the conditional ablation of PHD2 in the hematopoietic system also leads to reduced tumor volume, intriguingly generated by an imbalance between enhanced cell death and improved proliferation of tumor cells. This effect seems to rely on the overall downregulation of protumoral as well as antitumoral cytokines. Using different genetic approaches, we were able to confine this complex phenotype to the crosstalk of PHD2-deficient myeloid cells and T-lymphocytes. Taken together, our findings reveal a multifaceted role for PHD2 in several hematopoietic lineages during tumor development and might have important implications for the development of tumor therapies in the future.

  5. Loss of epithelial hypoxia-inducible factor prolyl hydroxylase 2 accelerates skin wound healing in mice.

    PubMed

    Kalucka, Joanna; Ettinger, Andreas; Franke, Kristin; Mamlouk, Soulafa; Singh, Rashim Pal; Farhat, Katja; Muschter, Antje; Olbrich, Susanne; Breier, Georg; Katschinski, Dörthe M; Huttner, Wieland; Weidemann, Alexander; Wielockx, Ben

    2013-09-01

    Skin wound healing in mammals is a complex, multicellular process that depends on the precise supply of oxygen. Hypoxia-inducible factor (HIF) prolyl hydroxylase 2 (PHD2) serves as a crucial oxygen sensor and may therefore play an important role during reepithelialization. Hence, this study was aimed at understanding the role of PHD2 in cutaneous wound healing using different lines of conditionally deficient mice specifically lacking PHD2 in inflammatory, vascular, or epidermal cells. Interestingly, PHD2 deficiency only in keratinocytes and not in myeloid or endothelial cells was found to lead to faster wound closure, which involved enhanced migration of the hyperproliferating epithelium. We demonstrate that this effect relies on the unique expression of β3-integrin in the keratinocytes around the tip of the migrating tongue in an HIF1α-dependent manner. Furthermore, we show enhanced proliferation of these cells in the stratum basale, which is directly related to their attenuated transforming growth factor β signaling. Thus, loss of the central oxygen sensor PHD2 in keratinocytes stimulates wound closure by prompting skin epithelial cells to migrate and proliferate. Inhibition of PHD2 could therefore offer novel therapeutic opportunities for the local treatment of cutaneous wounds.

  6. Parasite prolyl oligopeptidases and the challenge of designing chemotherapeuticals for Chagas disease, leishmaniasis and African trypanosomiasis.

    PubMed

    Bastos, I M D; Motta, F N; Grellier, P; Santana, J M

    2013-01-01

    The trypanosomatids Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp. cause Chagas disease, leishmaniasis and human African trypanosomiasis, respectively. It is estimated that over 10 million people worldwide suffer from these neglected diseases, posing enormous social and economic problems in endemic areas. There are no vaccines to prevent these infections and chemotherapies are not adequate. This picture indicates that new chemotherapeutic agents must be developed to treat these illnesses. For this purpose, understanding the biology of the pathogenic trypanosomatid- host cell interface is fundamental for molecular and functional characterization of virulence factors that may be used as targets for the development of inhibitors to be used for effective chemotherapy. In this context, it is well known that proteases have crucial functions for both metabolism and infectivity of pathogens and are thus potential drug targets. In this regard, prolyl oligopeptidase and oligopeptidase B, both members of the S9 serine protease family, have been shown to play important roles in the interactions of pathogenic protozoa with their mammalian hosts and may thus be considered targets for drug design. This review aims to discuss structural and functional properties of these intriguing enzymes and their potential as targets for the development of drugs against Chagas disease, leishmaniasis and African trypanosomiasis.

  7. The prolyl oligopeptidase inhibitor IPR19 ameliorates cognitive deficits in mouse models of schizophrenia.

    PubMed

    Prades, Roger; Munarriz-Cuezva, Eva; Urigüen, Leyre; Gil-Pisa, Itziar; Gómez, Lídia; Mendieta, Laura; Royo, Soledad; Giralt, Ernest; Tarragó, Teresa; Meana, J Javier

    2017-02-01

    Cognitive deficits are considered a key feature of schizophrenia, and they usually precede the onset of the illness and continue after psychotic symptoms appear. Current antipsychotic drugs have little or no effect on the cognitive deficits of this disorder. Prolyl oligopeptidase (POP) is an 81-kDa monomeric serine protease that is expressed in brain and other tissues. POP inhibitors have shown neuroprotective, anti-amnesic and cognition-enhancing properties. Here we studied the potential of IPR19, a new POP inhibitor, for the treatment of the cognitive symptoms related to schizophrenia. The efficacy of the inhibitor was evaluated in mouse models based on subchronic phencyclidine and acute dizocilpine administration, and in adult offspring from mothers with immune reaction induced by polyinosinic:polycytidylic acid administration during pregnancy. Acute IPR19 administration (5mg/kg, i.p.) reversed the cognitive performance deficits of the three mouse models in the novel object recognition test, T-maze, and eight-arm radial maze. The compound also ameliorates deficits of the prepulse inhibition response. The in vitro inhibitory efficacy and selectivity, brain penetration and exposure time after injection of IPR19 were also addressed. Our results indicate that the inhibition of POP using IPR19 may offer a promising strategy to develop drugs to ameliorate the cognitive deficits of schizophrenia.

  8. Dual-Action Inhibitors of HIF Prolyl Hydroxylases That Induce Binding of a Second Iron Ion

    PubMed Central

    Thalhammer, Armin; Demetriades, Marina; Chowdhury, Rasheduzzaman; Tian, Ya-Min; Stolze, Ineke; McNeill, Luke A.; Lee, Myung Kyu; Woon, Esther C. Y.; Mackeen, Mukram M.; Kawamura, Akane; Ratcliffe, Peter J.; Mecinović, Jasmin; Schofield, Christopher J.

    2015-01-01

    Inhibition of the hypoxia-inducible factor (HIF) prolyl-hydroxylases (PHD or EGLN enzymes) is of interest for the treatment of anemia and ischemia-related diseases. Most PHD inhibitors work by binding to the single ferrous ion and competing with 2-oxoglutarate (2OG) co-substrate for binding at the PHD active site. Non-specific iron chelators also inhibit the PHDs, both in vitro and in cells. We report the identification of dual action PHD inhibitors, which bind to the active site iron and also induce the binding of a second iron ion at the active site. Following analysis of small-molecule iron complexes and application of non-denaturing protein mass spectrometry to assess PHD2·iron·inhibitor stoichimetry, selected diacylhydrazines were identified as PHD2 inhibitors that induce the binding of a second iron ion. Some compounds were shown to inhibit the HIF hydroxylases in human hepatoma and renal carcinoma cell lines. PMID:23151668

  9. Hyperplasia of pulmonary neuroepithelial bodies (NEB) in lungs of prolyl hydroxylase -1(PHD-1) deficient mice.

    PubMed

    Pan, Jie; Yeger, Herman; Ratcliffe, Peter; Bishop, Tammie; Cutz, Ernest

    2012-01-01

    Pulmonary NEB, widely distributed within the airway mucosa of mammalian lungs, are presumed hypoxia sensitive airway O(2) sensors responding to changes in airway gas concentration. NEB cell hyperplasia has been reported after exposure to chronic hypoxia and in a variety of paediatric and adult lung disorders. Prolyl hydroxylases (PHD 1-3) regulate the stability of hypoxia-inducible factors (HIF's) in an O(2)-dependent manner and function as intrinsic oxygen sensors. To determine a possible role of PHD-1in NEB cells we have quantitated NEB's in lungs of neonatal (P2) and adult (2 months) PHD-1-deficient mice and compared them to wild type (WT) control mice. Lung tissues fixed in formalin and embedded in paraffin were processed for immunoperoxidase method and frozen sections for multilabel immunoflourescence using antibodies for NEB markers synaptophysin, synaptic vesicle protein 2 and the peptide CGRP. The frequency and size of NEB in lungs of PHD-1 deficient neonatal mice (P2) and at 2 months was increased significantly compared to WT controls (p < 0.01). The present data suggests an important role for PHD enzymes in NEB cell biology deserving further studies. Since the PHD-1 deficient mouse appears to be the first animal model showing NEB cell hyperplasia it may be useful for studies of NEB physiology and pathobiology.

  10. Notch Downregulation and Extramedullary Erythrocytosis in Hypoxia-Inducible Factor Prolyl 4-Hydroxylase 2-Deficient Mice.

    PubMed

    Myllymäki, Mikko N M; Määttä, Jenni; Dimova, Elitsa Y; Izzi, Valerio; Väisänen, Timo; Myllyharju, Johanna; Koivunen, Peppi; Serpi, Raisa

    2017-01-15

    Erythrocytosis is driven mainly by erythropoietin, which is regulated by hypoxia-inducible factor (HIF). Mutations in HIF prolyl 4-hydroxylase 2 (HIF-P4H-2) (PHD2/EGLN1), the major downregulator of HIFα subunits, are found in familiar erythrocytosis, and large-spectrum conditional inactivation of HIF-P4H-2 in mice leads to severe erythrocytosis. Although bone marrow is the primary site for erythropoiesis, spleen remains capable of extramedullary erythropoiesis. We studied HIF-P4H-2-deficient (Hif-p4h-2(gt/gt)) mice, which show slightly induced erythropoiesis upon aging despite nonincreased erythropoietin levels, and identified spleen as the site of extramedullary erythropoiesis. Splenic hematopoietic stem cells (HSCs) of these mice exhibited increased erythroid burst-forming unit (BFU-E) growth, and the mice were protected against anemia. HIF-1α and HIF-2α were stabilized in the spleens, while the Notch ligand genes Jag1, Jag2, and Dll1 and target Hes1 became downregulated upon aging HIF-2α dependently. Inhibition of Notch signaling in wild-type spleen HSCs phenocopied the increased BFU-E growth. HIFα stabilization can thus mediate non-erythropoietin-driven splenic erythropoiesis via altered Notch signaling.

  11. Peptide Macrocyclization Catalyzed by a Prolyl Oligopeptidase Involved in α-Amanitin Biosynthesis

    DOE PAGES

    Luo, Hong; Hong, Sung-Yong; Sgambelluri, R.  Michael; ...

    2014-12-04

    Amatoxins are ribosomally encoded and posttranslationally modified peptides that account for the majority of fatal mushroom poisonings of humans. A representative amatoxin is the bicyclic octapeptide α-amanitin, formed via head-to-tail macrocyclization, which is ribosomally biosynthesized as a 35-amino acid propeptide in Amanita bisporigera and in the distantly related mushroom Galerina marginata. Although members of the prolyl oligopeptidase (POP) family of serine proteases have been proposed to play a role in α-amanitin posttranslational processing, the exact mechanistic details are not known. In this paper, we show that a specific POP (GmPOPB) is required for toxin maturation in G. marginata. Recombinant GmPOPBmore » catalyzed two nonprocessive reactions: hydrolysis at an internal Pro to release the C-terminal 25-mer from the 35-mer propeptide and transpeptidation at the second Pro to produce the cyclic octamer. Finally on the other hand, we show that GmPOPA, the putative housekeeping POP of G. marginata, behaves like a conventional POP.« less

  12. Hsp70 facilitates trans-membrane transport of bacterial ADP-ribosylating toxins into the cytosol of mammalian cells.

    PubMed

    Ernst, Katharina; Schmid, Johannes; Beck, Matthias; Hägele, Marlen; Hohwieler, Meike; Hauff, Patricia; Ückert, Anna Katharina; Anastasia, Anna; Fauler, Michael; Jank, Thomas; Aktories, Klaus; Popoff, Michel R; Schiene-Fischer, Cordelia; Kleger, Alexander; Müller, Martin; Frick, Manfred; Barth, Holger

    2017-06-02

    Binary enterotoxins Clostridium (C.) botulinum C2 toxin, C. perfringens iota toxin and C. difficile toxin CDT are composed of a transport (B) and a separate non-linked enzyme (A) component. Their B-components mediate endocytic uptake into mammalian cells and subsequently transport of the A-components from acidic endosomes into the cytosol, where the latter ADP-ribosylate G-actin resulting in cell rounding and cell death causing clinical symptoms. Protein folding enzymes, including Hsp90 and peptidyl-prolyl cis/trans isomerases facilitate transport of the A-components across endosomal membranes. Here, we identified Hsp70 as a novel host cell factor specifically interacting with A-components of C2, iota and CDT toxins to facilitate their transport into the cell cytosol. Pharmacological Hsp70-inhibition specifically prevented pH-dependent trans-membrane transport of A-components into the cytosol thereby protecting living cells and stem cell-derived human miniguts from intoxication. Thus, Hsp70-inhibition might lead to development of novel therapeutic strategies to treat diseases associated with bacterial ADP-ribosylating toxins.

  13. The Hsp90 machinery facilitates the transport of diphtheria toxin into human cells.

    PubMed

    Schuster, Manuel; Schnell, Leonie; Feigl, Peter; Birkhofer, Carina; Mohr, Katharina; Roeder, Maurice; Carle, Stefan; Langer, Simon; Tippel, Franziska; Buchner, Johannes; Fischer, Gunter; Hausch, Felix; Frick, Manfred; Schwan, Carsten; Aktories, Klaus; Schiene-Fischer, Cordelia; Barth, Holger

    2017-04-04

    Diphtheria toxin kills human cells because it delivers its enzyme domain DTA into their cytosol where it inhibits protein synthesis. After receptor-mediated uptake of the toxin, DTA translocates from acidic endosomes into the cytosol, which might be assisted by host cell factors. Here we investigated the role of Hsp90 and its co-chaperones during the uptake of native diphtheria toxin into human cells and identified the components of the Hsp90 machinery including Hsp90, Hsp70, Cyp40 and the FK506 binding proteins FKBP51 and FKBP52 as DTA binding partners. Moreover, pharmacological inhibition of the chaperone activity of Hsp90 and Hsp70 and of the peptidyl-prolyl cis/trans isomerase (PPIase) activity of Cyps and FKBPs protected cells from intoxication with diphtheria toxin and inhibited the pH-dependent trans-membrane transport of DTA into the cytosol. In conclusion, these host cell factors facilitate toxin uptake into human cells, which might lead to development of novel therapeutic strategies against diphtheria.

  14. Physiological and comparative genomic analysis of Acidithiobacillus ferrivorans PQ33 provides psychrotolerant fitness evidence for oxidation at low temperature.

    PubMed

    Ccorahua-Santo, Robert; Eca, Anika; Abanto, Michel; Guerra, Gregory; Ramírez, Pablo

    2017-06-01

    Friendly environmental hydrometallurgy at low temperatures is principally promoted by Acidithiobacillus ferrivorans. Until recently, the synergy between cold tolerance and the molecular mechanism of ferrous iron (Fe(2+)) oxidation was unknown. In the present paper, we conducted a physiological and comparative genomics analysis of the new strain A. ferrivorans PQ33 to elucidate the oxidation mechanism at low temperatures, with emphasis placed on trehalose and the Rus operon. PQ33 exhibited a doubling time of 66.6 h in Fe(2+) at pH 1.6 and 63.6 h in CuS at 5 °C. Genomic island (GI) identification and comparative genome analysis were performed with four available genomes of Acidithiobacillus sp. The genome comprised 3,298,172 bp and 56.55% GC content. In contrast to ATCC Acidithiobacillus ferrooxidans strains, the genome of A. ferrivorans PQ33 harbors one GI, which contains a RusB gene. Moreover, five genes of peptidyl-prolyl cis-trans isomerase (PPIases) were observed. Furthermore, comparative analysis of the trehalose operon suggested the presence of a horizontal transfer event. In addition, comparison of rusticyanin proteins revealed that RusB has better intrinsic flexibility than RusA. This comparison suggests psychrotolerant fitness and supports the genetic canalization of A. ferrivorans PQ33 for oxidation at low temperature. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  15. Cyclophilin D Is Involved in the Regulation of Autophagy and Affects the Lifespan of P. anserina in Response to Mitochondrial Oxidative Stress

    PubMed Central

    Kramer, Piet; Jung, Alexander T.; Hamann, Andrea; Osiewacz, Heinz D.

    2016-01-01

    The mitochondrial permeability transition pore plays a key role in programmed cell death and the induction of autophagy. Opening of the pore is regulated by the mitochondrial peptidyl prolyl-cis, trans-isomerase cyclophilin D (CYPD). Previously it was shown in the aging model organism Podospora anserina that PaCYPD abundance increases during aging and that PaCypD overexpressors are characterized by accelerated aging. Here, we describe a role of PaCYPD in the regulation of autophagy. We found that the accelerated aging phenotype observed in a strain overexpressing PaCypD is not metacaspase-dependent but is accompanied by an increase of general autophagy and mitophagy, the selective autophagic degradation of mitochondria. It thus is linked to what has been defined as “autophagic cell death” or “type II” programmed cell death. Moreover, we found that the previously demonstrated age-related induction of autophagy in wild-type aging depends on the presence of PaCYPD. Deletion of PaCypD leads to a decrease in autophagy in later stages of age and under paraquat-mediated oxidative stress. Finally, we report that PaCYPD is also required for mitohormesis, the beneficial effect of mild mitochondrial stress. Thus, PaCYPD plays a key role in the context-dependent regulation of pathways leading to pro-survival and pro-death effects of autophagy. PMID:27683587

  16. Effect of Cu2+ on the oxidative folding of synthetic maurotoxin in vitro

    PubMed Central

    Regaya, Imed; Andreotti, Nicolas; Di Luccio, Eric; De Waard, Michel; Sabatier, Jean-Marc

    2008-01-01

    Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulphide bridges that acts on various K+ channel types. It folds according to an α/β scaffold, i.e. a helix connected to a two stranded β-sheet by two disulphide bridges. In a former study, various parameters that affect the oxidation and folding of the reduced form of synthetic MTX were investigated in vitro. It was found that MTX achieves its final 3-D structure by evolving over time through a series of oxidation intermediates, from the least to the most oxidised species. MTX oxidative intermediates can be studied by iodoacetamide alkylation of free cysteine residues followed by mass spectrometry analysis. Here, we have analysed the effect of Cu2+ on the kinetics of MTX oxidative folding and found that it dramatically speeds up the formation of the four-disulphide bridged, native-like, MTX (maximal production within 30 minutes instead of > 60 hours). Cu2+ was also found to prevent the slow transition of a three disulphide-bridged MTX intermediate towards the final four disulphide-bridged product (12% of total MTX). The data are discussed in light of the potential effects of Cu2+ on MTX secondary structure formation, disulphide bridging and peptidyl prolyl cis-trans isomerisation. PMID:18533728

  17. Novel IEF Peptide Fractionation Method Reveals a Detailed Profile of N-Terminal Acetylation in Chemotherapy-Responsive and -Resistant Ovarian Cancer Cells.

    PubMed

    Weiland, Florian; Arentz, Georgia; Klingler-Hoffmann, Manuela; McCarthy, Peter; Lokman, Noor A; Kaur, Gurjeet; Oehler, Martin K; Hoffmann, Peter

    2016-11-04

    Although acetylation is regarded as a common protein modification, a detailed proteome-wide profile of this post-translational modification may reveal important biological insight regarding differential acetylation of individual proteins. Here we optimized a novel peptide IEF fractionation method for use prior to LC-MS/MS analysis to obtain a more in depth coverage of N-terminally acetylated proteins from complex samples. Application of the method to the analysis of the serous ovarian cancer cell line OVCAR-5 identified 344 N-terminally acetylated proteins, 12 of which are previously unreported. The protein peptidyl-prolyl cis-trans isomerase A (PPIA) was detected in both the N-terminally acetylated and unmodified forms and was further analyzed by data-independent acquisition in carboplatin-responsive parental OVCAR-5 cells and carboplatin-resistant OVCAR-5 cells. This revealed a higher ratio of unacetylated to acetylated N-terminal PPIA in the parental compared with the carboplatin-resistant OVCAR-5 cells and a 4.1-fold increase in PPIA abundance overall in the parental cells relative to carboplatin-resistant OVCAR-5 cells (P = 0.015). In summary, the novel IEF peptide fractionation method presented here is robust, reproducible, and can be applied to the profiling of N-terminally acetylated proteins. All mass spectrometry data is available as a ProteomeXchange repository (PXD003547).

  18. A novel multi-functional chloroplast protein: identification of a 40 kDa immunophilin-like protein located in the thylakoid lumen.

    PubMed Central

    Fulgosi, H; Vener, A V; Altschmied, L; Herrmann, R G; Andersson, B

    1998-01-01

    We describe the identification of the first immunophilin associated with the photosynthetic membrane of chloroplasts. This complex 40 kDa immunophilin, designated TLP40 (thylakoid lumen PPIase), located in the lumen of the thylakoids, was found to play a dual role in photosynthesis involving both biogenesis and intraorganelle signalling. It originates in a single-copy nuclear gene, is made as a precursor of 49.2 kDa with a bipartite lumenal targeting transit peptide, and is characterized by a structure including a cyclophilin-like C-terminal segment of 20 kDa, a predicted N-terminal leucine zipper and a potential phosphatase-binding domain. It can exist in different oligomeric conformations and attach to the inner membrane surface. It is confined predominantly to the non-appressed thylakoid regions, the site of protein integration into the photosynthetic membrane. The isolated protein possesses peptidyl-prolyl cis-trans isomerase protein folding activity characteristic of immunophilins, but is not inhibited by cyclosporin A. TLP40 also exerts an effect on dephosphorylation of several key proteins of photosystem II, probably as a constituent of a transmembrane signal transduction chain. This first evidence for a direct role of immunophilins in a photoautotrophic process suggests that light-mediated protein phosphorylation in photosynthetic membranes and the role of the thylakoid lumen are substantially more complex than anticipated. PMID:9501079

  19. PPIase catalysis by human FK506-binding protein proceeds through a conformational twist mechanism.

    PubMed

    Park, S T; Aldape, R A; Futer, O; DeCenzo, M T; Livingston, D J

    1992-02-15

    FK506-binding protein (FKBP) catalyzes the cis-trans isomerization of the peptidyl-prolyl amide bond (the PPIase reaction) and is the major intracellular receptor for the immunosuppressive drugs FK506 and rapamycin. One mechanism proposed for catalysis of the PPIase reaction requires attack of an enzyme nucleophile on the carbonyl carbon of the isomerized peptide bond. An alternative mechanism requires conformational distortion of the peptide bond with or without assistance by an enzyme hydrogen bond donor. We have determined the kinetic parameters of the human FKBP-catalyzed PPIase reaction. At 5 degrees C, the isomerization of Suc-Ala-Leu-Pro-Phe-pNA proceeds in 2.5% trifluorethanol with kcat = 600 s-1, Km = 0.5 mM and kcat/Km = 1.2 x 10(6) M-1s-1. The kcat/Km shows little pH dependence between 5 and 10. A normal secondary deuterium isotope effect is observed on both kcat and kcat/Km. To investigate dependence on enzyme nucleophiles and proton donors, we have replaced eight potential catalytic residues with alanine by site-directed mutagenesis. Each FKBP variant efficiently catalyzes the PPIase reaction. Taken together, these data support an unassisted conformational twist mechanism with rate enhancement due in part to desolvation of the peptide bond at the active site. Fluorescence quenching of the buried tryptophan 59 residue by peptide substrate suggests that isomerization occurs in a hydrophobic environment.

  20. Catalytic and ligand binding properties of the FK506 binding protein FKBP12: effects of the single amino acid substitution of Tyr82 to Leu.

    PubMed Central

    Bossard, M J; Bergsma, D J; Brandt, M; Livi, G P; Eng, W K; Johnson, R K; Levy, M A

    1994-01-01

    The binding of FK506 and rapamycin to their cytosolic receptor FKBP12 is an intermediate step in the paths leading to their potent immunosuppressive properties. One of the amino acids defining the hydrophobic binding cleft for the macrocycles is Tyr82, which is thought to form a hydrogen bond with the amide oxygens of the common pipecolyl structural element within the two macrolides. To understand better the influence of this amino acid residue in catalytic activity (cis-trans peptidyl prolyl isomerization) and ligand binding properties, a Tyr82 to Leu site-specific modification of FKBP12 was prepared, purified and characterized. Kinetic experiments have demonstrated that the Tyr82 to Leu modification has a greater effect on catalytic properties than on ligand binding affinities, a result which indicates that these inhibitors may not be binding as true transition-state analogues. In an additional test for cellular function, expression of both wild-type and mutant human FKBP12 in a strain of Saccharomyces cerevisiae rendered resistant to rapamycin by deletion of the gene encoding a cytosolic rapamycin binding protein (RPB1), the yeast homologue of FKBP12, restored wild-type drug sensitivity. PMID:7507662

  1. Comparative proteomic analysis of paclitaxel resistance-related proteins in human breast cancer cell lines

    PubMed Central

    Fujioka, Hiroya; Sakai, Akiko; Tanaka, Satoru; Kimura, Kosei; Miyamoto, Akiko; Iwamoto, Mitsuhiko; Uchiyama, Kazuhisa

    2017-01-01

    Paclitaxel is widely used to treat various cancers; however, resistance to this drug is a major obstacle to breast cancer chemotherapy. To identify the proteins involved in paclitaxel resistance, the present study compared the proteomes of MCF-7 human breast cancer cells and its paclitaxel-resistant subclone MCF-7/PTX. Using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry, 11 upregulated and 12 downregulated proteins were identified in MCF-7/PTX cells compared with the parental cell line. These 23 proteins were functionally classified as stress-induced chaperones, metabolic enzymes and cytoskeletal proteins. The anti-apoptotic proteins, stress-70 protein, 78-kD glucose-regulated protein, peptidyl-prolyl cis-trans isomerase A (PPIA) and heterogeneous nuclear ribonucleoprotein H3, were also upregulated in MCF-7/PTX cells. Notably, knockdown of the stress-response chaperone PPIA using small interfering RNA in MCF-7/PTX cells restored their sensitivity to paclitaxel. These findings indicated that PPIA may have an important role in paclitaxel resistance in MCF-7/PTX cells. PMID:28123557

  2. 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.

  3. A cyclophilin A CPR1 overexpression enhances stress acquisition in Saccharomyces cerevisiae.

    PubMed

    Kim, Il-Sup; Kim, Hyun-Young; Shin, Sun-Young; Kim, Young-Saeng; Lee, Dong Hee; Park, Kyung Moc; Yoon, Ho-Sung

    2010-06-01

    Cyclophilins are conserved cis-trans peptidyl-prolyl isomerase that are implicated in protein folding and function as molecular chaperones. We found the expression of cyclophilin A, Cpr1, changes in response to exposure to yeast Saccharomyces cerevisiae to abiotic stress conditions. The effect of Cpr1 overexpression in stress responses was therefore examined. The CPR1 gene was cloned to the yeast expression vector pVTU260 under regulation of an endogenous alcohol dehydrogenase (ADH) promoter. The overexpression of Cpr1 drastically increased cell viability of yeast in the presence of stress inducers, such as cadmium, cobalt, copper, hydrogen peroxide, tert-butyl hydroperoxide (t-BOOH), and sodium dodecyl sulfate (SDS). The Cpr1 expression also enhanced the cell rescue program resulting in a variety of antioxidant enzymes including thioredoxin system (particularly, thioredoxin peroxidase), metabolic enzymes (glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase), and molecular chaperones (Hsp104, Hsp90, Hsp60 and Hsp42). Thus, our study illustrates the importance of Cpr1 as a molecular chaperone that improves cellular stress responses through collaborative relationships with other proteins when yeast cells are exposed to adverse conditions, and it also premises the improvement of yeast strains.

  4. Stability of Pin1 as revealed by thermal and spectroscopic studies

    NASA Astrophysics Data System (ADS)

    Wang, Jing-Zhang; Lin, Tao; Zhu, Guo-Fei; Du, Lin-Fang

    2010-06-01

    Pin1 is a two-domain enzyme which has peptidyl-prolyl cis/trans isomerase activity. Pin1 recognizes phospho-Ser/Thr-Pro motifs in cell-signaling proteins, and is both a cancer and an Alzheimer's disease target. The thermal stability of Pin1 was studied intensively by SDS-PAGE, enzymatic activity assay, intrinsic fluorescence spectroscopy and circular dichroism spectroscopy. The activity of Pin1 gradually decreased above 40 °C, and the Tm was 57.6 ± 1.0 °C. Fluorescence experiments indicated that heat treatment induced changes in the substructures in Pin1, resulting in that the polarity in the microenvironments of the tryptophan residues increased. It is assumed that the thermal denaturation of Pin1 involved a three-state transition. The intermediate state of Pin1 at about 60 °C was confirmed by fluorescence emission spectra, the synchronous fluorescence spectra and CD measurements. Decreases in α-helix and β-sheet appeared above 40 °C, which was balanced by an enhancement in unordered coil. The Tm values calculated from α-helix transition and β-sheet transition were 54.6 ± 0.6 °C and 70.7 ± 3.3 °C, respectively. Our results illustrated that Pin1 had a relatively high thermal stability and the WW domain had a higher stability than the PPIase domain.

  5. Discovering Conformational Sub-States Relevant to Protein Function

    PubMed Central

    Ramanathan, Arvind; Savol, Andrej J.; Langmead, Christopher J.; Agarwal, Pratul K.; Chennubhotla, Chakra S.

    2011-01-01

    Background Internal motions enable proteins to explore a range of conformations, even in the vicinity of native state. The role of conformational fluctuations in the designated function of a protein is widely debated. Emerging evidence suggests that sub-groups within the range of conformations (or sub-states) contain properties that may be functionally relevant. However, low populations in these sub-states and the transient nature of conformational transitions between these sub-states present significant challenges for their identification and characterization. Methods and Findings To overcome these challenges we have developed a new computational technique, quasi-anharmonic analysis (QAA). QAA utilizes higher-order statistics of protein motions to identify sub-states in the conformational landscape. Further, the focus on anharmonicity allows identification of conformational fluctuations that enable transitions between sub-states. QAA applied to equilibrium simulations of human ubiquitin and T4 lysozyme reveals functionally relevant sub-states and protein motions involved in molecular recognition. In combination with a reaction pathway sampling method, QAA characterizes conformational sub-states associated with cis/trans peptidyl-prolyl isomerization catalyzed by the enzyme cyclophilin A. In these three proteins, QAA allows identification of conformational sub-states, with critical structural and dynamical features relevant to protein function. Conclusions Overall, QAA provides a novel framework to intuitively understand the biophysical basis of conformational diversity and its relevance to protein function. PMID:21297978

  6. Epigallocatechin-gallate Suppresses Tumorigenesis by Directly Targeting Pin1

    SciTech Connect

    Urusova, Darya V.; Shim, Jung-Hyun; Kim, Dong Joon; Jung, Sung Keun; Zykova, Tatyana A.; Carper, Andria; Bode, Ann M.; Dong, Zigang

    2011-09-01

    The most active anticancer component in green tea is epigallocatechin-3-gallate (EGCG). The human peptidyl prolyl cis/trans isomerase (Pin1) plays a critical role in oncogenic signaling. Herein, we report the X-ray crystal structure of the Pin1/EGCG complex resolved at 1.9 Å resolution. Notably, the structure revealed the presence of EGCG in both the WW and PPIase domains of Pin1. The direct binding of EGCG with Pin1 was confirmed and the interaction inhibited Pin1 PPIase activity. In addition, proliferation of cells expressing Pin1 was inhibited and tumor growth in a xenograft mouse model was suppressed. The binding of EGCG with Arg17 in the WW domain prevented the binding of c-Jun, a well-known Pin1 substrate. EGCG treatment corresponded with a decreased abundance of cyclin D1 and diminution of 12-O-tetradecanoylphorbol-l3-acetate–induced AP-1 or NF-κB promoter activity in cells expressing Pin1. Overall, these results showed that EGCG directly suppresses the tumor-promoting effect of Pin1.

  7. Dipentamethylene thiuram monosulfide is a novel inhibitor of Pin1

    SciTech Connect

    Tatara, Yota; Lin, Yi-Chin; Bamba, Yoshimasa; Mori, Tadashi; Uchida, Takafumi

    2009-07-03

    Pin1 is involved in eukaryotic cell proliferation by changing the structure and function of phosphorylated proteins. PiB, the Pin1 specific inhibitor, blocks cancer cell proliferation. However, low solubility of PiB in DMSO has limited studies of its effectiveness. We screened for additional Pin1 inhibitors and identified the DMSO-soluble compound dipentamethylene thiuram monosulfide (DTM) that inhibits Pin1 activity with an EC50 value of 4.1 {mu}M. Molecular modeling and enzyme kinetic analysis indicated that DTM competitively inhibits Pin1 activity, with a K{sub i} value of 0.05 {mu}M. The K{sub D} value of DTM with Pin1 was determined to be 0.06 {mu}M by SPR technology. Moreover, DTM specifically inhibited peptidyl-prolyl cis/trans isomerase activity in HeLa cells. FACS analysis showed that DTM induced G0 arrest of the HCT116 cells. Our results suggest that DTM has the potential to guide the development of novel antifungal and/or anticancer drugs.

  8. Acute Heat Stress and Reduced Nutrient Intake Alter Intestinal Proteomic Profile and Gene Expression in Pigs

    PubMed Central

    Pearce, Sarah C.; Lonergan, Steven M.; Huff-Lonergan, Elisabeth; Baumgard, Lance H.; Gabler, Nicholas K.

    2015-01-01

    Heat stress and reduced feed intake negatively affect intestinal integrity and barrier function. Our objective was to compare ileum protein profiles of pigs subjected to 12 hours of HS, thermal neutral ad libitum feed intake, or pair-fed to heat stress feed intake under thermal neutral conditions (pair-fed thermal neutral). 2D-Differential In Gel Electrophoresis and gene expression were performed. Relative abundance of 281 and 138 spots differed due to heat stress, compared to thermal neutral and pair-fed thermal neutral pigs, respectively. However, only 20 proteins were different due to feed intake (thermal neutral versus pair-fed thermal neutral). Heat stress increased mRNA expression of heat shock proteins and protein abundance of heat shock proteins 27, 70, 90-α and β were also increased. Heat stress reduced ileum abundance of several metabolic enzymes, many of which are involved in the glycolytic or TCA pathways, indicating a change in metabolic priorities. Stress response enzymes peroxiredoxin-1 and peptidyl-prolyl cis-trans isomerase A were decreased in pair-fed thermal neutral and thermal neutral pigs compared to heat stress. Heat stress increased mRNA abundance markers of ileum hypoxia. Altogether, these data show that heat stress directly alters intestinal protein and mRNA profiles largely independent of reduced feed intake. These changes may be related to the reduced intestinal integrity associated with heat stress. PMID:26575181

  9. Hydroxytyrosol rich extract from olive leaves modulates cell cycle progression in MCF-7 human breast cancer cells.

    PubMed

    Bouallagui, Zouhaier; Han, Junkuy; Isoda, Hiroko; Sayadi, Sami

    2011-01-01

    Throughout the history, olive (Olea europea L.) leaves have been heavily exploited for the prevention or the treatment of hypertension, carcinogenesis, diabetes, atherosclerosis and so many other traditional therapeutic uses. These activities are thought to be the output of olive micronutrients especially polyphenols. Hydroxytyrosol and oleuropein are considered as major polyphenolic compounds in olive leaf. In this work, a hydroxytyrosol rich olive leaves extract was investigated for potential anti-tumoral activities. In vitro cytotoxic effects against MCF-7 breast cancer cells were examined using MTT and neutral red tests. The anti-tumor activities were further investigated by flow cytometry and western blotting. Cytotoxicity assays resulted in a dose dependent growth inhibition of MCF-7 cells. This inhibition was due to the cell cycle arrest in the G0/G1 phase. The understanding of the molecular mechanism by which olive leaves extract arrested cell growth showed a down-expression of the peptidyl-prolyl cis-trans isomerase Pin1 which in turn decreased the level of a G1 key protein; Cyclin D1. Additionally, olive leaves extract treatment up-regulated the AP1 transcription factor member, c-jun. Therefore, olive leaves extract will necessitate further deep investigation for a probable use as a cancer preventive food additive.

  10. Cyclophilin-40 has a cellular role in the aryl hydrocarbon receptor signaling.

    PubMed

    Luu, Tony C; Bhattacharya, Pompeya; Chan, William K

    2008-09-22

    Cyclophilin-40 (CyP40) promotes the formation of the gel shift complex that contains the aryl hydrocarbon receptor (AhR), AhR nuclear translocator (Arnt) and dioxin response element (DRE) using baculovirus expressed proteins. Here we reported that CyP40 plays a role in the AhR signaling. When the CyP40 content in MCF-7 cells is reduced, up-regulation of cyp1a1 and cyp1b1 by 3-methylchloranthrene (3MC) is also reduced, suggesting that CyP40 is essential for maximal AhR function. The CyP40 region containing amino acids 186-215, but not the peptidyl-prolyl cis-trans isomerase and tetratricopeptide repeat domains, is essential for forming the AhR/Arnt/DRE complex. CyP40 is found in the cell nucleus after 3MC treatment and appears to promote the DRE binding form of the AhR/Arnt heterodimer.

  11. Target cell cyclophilins facilitate human papillomavirus type 16 infection.

    PubMed

    Bienkowska-Haba, Malgorzata; Patel, Hetalkumar D; Sapp, Martin

    2009-07-01

    Following attachment to primary receptor heparan sulfate proteoglycans (HSPG), human papillomavirus type 16 (HPV16) particles undergo conformational changes affecting the major and minor capsid proteins, L1 and L2, respectively. This results in exposure of the L2 N-terminus, transfer to uptake receptors, and infectious internalization. Here, we report that target cell cyclophilins, peptidyl-prolyl cis/trans isomerases, are required for efficient HPV16 infection. Cell surface cyclophilin B (CyPB) facilitates conformational changes in capsid proteins, resulting in exposure of the L2 N-terminus. Inhibition of CyPB blocked HPV16 infection by inducing noninfectious internalization. Mutation of a putative CyP binding site present in HPV16 L2 yielded exposed L2 N-terminus in the absence of active CyP and bypassed the need for cell surface CyPB. However, this mutant was still sensitive to CyP inhibition and required CyP for completion of infection, probably after internalization. Taken together, these data suggest that CyP is required during two distinct steps of HPV16 infection. Identification of cell surface CyPB will facilitate the study of the complex events preceding internalization and adds a putative drug target for prevention of HPV-induced diseases.

  12. Proteomic changes in female rat hippocampus following exposure to a terrified sound stress.

    PubMed

    Yang, Juan; Hu, Lili; Song, Tusheng; Liu, Yong; Wu, Qiuhua; Zhao, Lingyu; Liu, Liying; Zhao, Xiaoge; Zhang, Dianzeng; Huang, Chen

    2014-06-01

    Stress plays a profound role in the onset of affective disorders, including an elevation in risk factors for depression and anxiety. Women are twice as vulnerable to stress as men because of greater sensitivity to a substance produced during times of anxiety. To better define the abnormal proteins implicated in cognitive deficits and other stress-induced dysfunction, female rats were exposed to terrified sound stress, and two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) were utilized to determine the differential protein expression in the hippocampus in sound-stressed female rats compared with controls. Quantitative differences were found in 44 protein spots which were differentially expressed between the stressed and control groups (fold change of >2; p < 0.01). Eighteen protein spots were downregulated, and 26 protein spots were upregulated in the stressed group. The seven most differentially expressed proteins were identified and validated as follows: dihydropyrimidinase-related protein 2 (DRP-2), creatine kinase B type, dynamin-1 protein, alpha-internexin, glial fibrillary acidic protein beta, gamma-enolase, and peptidyl-prolyl cis-trans isomerase A. Changes in protein levels were detected in the hippocampus of female rats subjected to terrified sound stress. The findings herein may open new opportunities for further investigations on the modulation induced in the hippocampus by stress at the molecular level, especially with respect to females stress.

  13. 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.

  14. The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae

    PubMed Central

    Kessler, Daniel; Papatheodorou, Panagiotis; Stratmann, Tina; Dian, Elke Andrea; Hartmann-Fatu, Cristina; Rassow, Joachim; Bayer, Peter; Mueller, Jonathan Wolf

    2007-01-01

    Background The parvulin-type peptidyl prolyl cis/trans isomerase Par14 is highly conserved in all metazoans. The recently identified parvulin Par17 contains an additional N-terminal domain whose occurrence and function was the focus of the present study. Results Based on the observation that the human genome encodes Par17, but bovine and rodent genomes do not, Par17 exon sequences from 10 different primate species were cloned and sequenced. Par17 is encoded in the genomes of Hominidae species including humans, but is absent from other mammalian species. In contrast to Par14, endogenous Par17 was found in mitochondrial and membrane fractions of human cell lysates. Fluorescence of EGFP fusions of Par17, but not Par14, co-localized with mitochondrial staining. Par14 and Par17 associated with isolated human, rat and yeast mitochondria at low salt concentrations, but only the Par17 mitochondrial association was resistant to higher salt concentrations. Par17 was imported into mitochondria in a time and membrane potential-dependent manner, where it reached the mitochondrial matrix. Moreover, Par17 was shown to bind to double-stranded DNA under physiological salt conditions. Conclusion Taken together, the DNA binding parvulin Par17 is targeted to the mitochondrial matrix by the most recently evolved mitochondrial prepeptide known to date, thus adding a novel protein constituent to the mitochondrial proteome of Hominidae. PMID:17875217

  15. The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae.

    PubMed

    Kessler, Daniel; Papatheodorou, Panagiotis; Stratmann, Tina; Dian, Elke Andrea; Hartmann-Fatu, Cristina; Rassow, Joachim; Bayer, Peter; Mueller, Jonathan Wolf

    2007-09-17

    The parvulin-type peptidyl prolyl cis/trans isomerase Par14 is highly conserved in all metazoans. The recently identified parvulin Par17 contains an additional N-terminal domain whose occurrence and function was the focus of the present study. Based on the observation that the human genome encodes Par17, but bovine and rodent genomes do not, Par17 exon sequences from 10 different primate species were cloned and sequenced. Par17 is encoded in the genomes of Hominidae species including humans, but is absent from other mammalian species. In contrast to Par14, endogenous Par17 was found in mitochondrial and membrane fractions of human cell lysates. Fluorescence of EGFP fusions of Par17, but not Par14, co-localized with mitochondrial staining. Par14 and Par17 associated with isolated human, rat and yeast mitochondria at low salt concentrations, but only the Par17 mitochondrial association was resistant to higher salt concentrations. Par17 was imported into mitochondria in a time and membrane potential-dependent manner, where it reached the mitochondrial matrix. Moreover, Par17 was shown to bind to double-stranded DNA under physiological salt conditions. Taken together, the DNA binding parvulin Par17 is targeted to the mitochondrial matrix by the most recently evolved mitochondrial prepeptide known to date, thus adding a novel protein constituent to the mitochondrial proteome of Hominidae.

  16. 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

  17. Differential composition of culture supernatants from wild-type Brucella abortus and its isogenic virB mutants.

    PubMed

    Delpino, M Victoria; Comerci, Diego J; Wagner, Mary Ann; Eschenbrenner, Michel; Mujer, Cesar V; Ugalde, Rodolfo A; Fossati, Carlos A; Baldi, Pablo C; Delvecchio, Vito G

    2009-07-01

    The virB genes coding type IV secretion system are necessary for the intracellular survival and replication of Brucella spp. In this study, extracellular proteins from B. abortus 2308 (wild type, WT) and its isogenic virB10 polar mutant were compared. Culture supernatants harvested in the early stationary phase were concentrated and subjected to 2D electrophoresis. Spots present in the WT strain but absent in the virB10 mutant (differential spots) were considered extracellular proteins released in a virB-related manner, and were identified by MALDI-TOF analysis and matching with Brucella genomes. Among the 11 differential proteins identified, DnaK chaperone (Hsp70), choloylglycine hydrolase (CGH) and a peptidyl-prolyl cis-trans isomerase (PPIase) were chosen for further investigation because of their homology with extracellular and/or virulence factors from other bacteria. The three proteins were obtained in recombinant form and specific monoclonal antibodies (mAbs) were prepared. By Western blot with these mAbs, the three proteins were detected in supernatants from the WT but not in those from the virB10 polar mutant or from strains carrying non-polar mutations in virB10 or virB11 genes. These results suggest that the expression of virB genes affects the extracellular release of DnaK, PPIase and CGH, and possibly other proteins from B. abortus.

  18. Scalable Text Mining Assisted Curation of Post-Translationally Modified Proteoforms in the Protein Ontology.

    PubMed

    Ross, Karen E; Natale, Darren A; Arighi, Cecilia; Chen, Sheng-Chih; Huang, Hongzhan; Li, Gang; Ren, Jia; Wang, Michael; Vijay-Shanker, K; Wu, Cathy H

    2016-08-01

    The Protein Ontology (PRO) defines protein classes and their interrelationships from the family to the protein form (proteoform) level within and across species. One of the unique contributions of PRO is its representation of post-translationally modified (PTM) proteoforms. However, progress in adding PTM proteoform classes to PRO has been relatively slow due to the extensive manual curation effort required. Here we report an automated pipeline for creation of PTM proteoform classes that leverages two phosphorylation-focused text mining tools (RLIMS-P, which detects mentions of kinases, substrates, and phosphorylation sites, and eFIP, which detects phosphorylation-dependent protein-protein interactions (PPIs)) and our integrated PTM database, iPTMnet. By applying this pipeline, we obtained a set of ~820 substrate-site pairs that are suitable for automated PRO term generation with literature-based evidence attribution. Inclusion of these terms in PRO will increase PRO coverage of species-specific PTM proteoforms by 50%. Many of these new proteoforms also have associated kinase and/or PPI information. Finally, we show a phosphorylation network for the human and mouse peptidyl-prolyl cis-trans isomerase (PIN1/Pin1) derived from our dataset that demonstrates the biological complexity of the information we have extracted. Our approach addresses scalability in PRO curation and will be further expanded to advance PRO representation of phosphorylated proteoforms.

  19. Dipentamethylene thiuram monosulfide is a novel inhibitor of Pin1.

    PubMed

    Tatara, Yota; Lin, Yi-Chin; Bamba, Yoshimasa; Mori, Tadashi; Uchida, Takafumi

    2009-07-03

    Pin1 is involved in eukaryotic cell proliferation by changing the structure and function of phosphorylated proteins. PiB, the Pin1 specific inhibitor, blocks cancer cell proliferation. However, low solubility of PiB in DMSO has limited studies of its effectiveness. We screened for additional Pin1 inhibitors and identified the DMSO-soluble compound dipentamethylene thiuram monosulfide (DTM) that inhibits Pin1 activity with an EC50 value of 4.1 microM. Molecular modeling and enzyme kinetic analysis indicated that DTM competitively inhibits Pin1 activity, with a K(i) value of 0.05 microM. The K(D) value of DTM with Pin1 was determined to be 0.06 microM by SPR technology. Moreover, DTM specifically inhibited peptidyl-prolyl cis/trans isomerase activity in HeLa cells. FACS analysis showed that DTM induced G0 arrest of the HCT116 cells. Our results suggest that DTM has the potential to guide the development of novel antifungal and/or anticancer drugs.

  20. The Rift Valley Fever virus protein NSm and putative cellular protein interactions.

    PubMed

    Engdahl, Cecilia; Näslund, Jonas; Lindgren, Lena; Ahlm, Clas; Bucht, Göran

    2012-07-28

    Rift Valley Fever is an infectious viral disease and an emerging problem in many countries of Africa and on the Arabian Peninsula. The causative virus is predominantly transmitted by mosquitoes and high mortality and abortion rates characterize outbreaks in animals while symptoms ranging from mild to life-threatening encephalitis and hemorrhagic fever are noticed among infected humans. For a better prevention and treatment of the infection, an increased knowledge of the infectious process of the virus is required. The focus of this work was to identify protein-protein interactions between the non-structural protein (NSm), encoded by the M-segment of the virus, and host cell proteins. This study was initiated by screening approximately 26 million cDNA clones of a mouse embryonic cDNA library for interactions with the NSm protein using a yeast two-hybrid system. We have identified nine murine proteins that interact with NSm protein of Rift Valley Fever virus, and the putative protein-protein interactions were confirmed by growth selection procedures and β-gal activity measurements. Our results suggest that the cleavage and polyadenylation specificity factor subunit 2 (Cpsf2), the peptidyl-prolyl cis-trans isomerase (cyclophilin)-like 2 protein (Ppil2), and the synaptosome-associated protein of 25 kDa (SNAP-25) are the most promising targets for the NSm protein of the virus during an infection.

  1. Conformational state of a 25-mer peptide from the cyclophilin-binding loop of the HIV type 1 capsid protein.

    PubMed Central

    Reimer, U; Drewello, M; Jakob, M; Fischer, G; Schutkowski, M

    1997-01-01

    Recently a 25-residue part of Gag polyprotein from HIV type 1 (HIV-1) was reported to bind to the cytosolic 18 kDa cyclophilin (Cyp18) with an IC50 value of 180 microM. This peptide corresponds to the Cyp18-binding domain of HIV-1 Gag. A replacement of Gly with Ala in the cyclophilin-binding loop of HIV-1 Gag polyprotein results in the prevention of the packaging of Cyp18 into virions. We found only two conformers of this peptide among 16 possible expected conformers, owing to cis/trans isomerization of four peptidyl-prolyl bonds. Although this finding implicates the existence of a stabilizing structure, we were not able to detect secondary structure formation by 1H-NMR and CD spectroscopy. We characterized the peptide as a substrate for Cyp18 by two-dimensional exchange 1H-NMR spectroscopy. Surprisingly, we found similar binding characteristics for a peptide corresponding to 25-mer peptide containing the above-mentioned Gly to Ala substitution. PMID:9337866

  2. Human cyclophilin 40 unravels neurotoxic amyloids.

    PubMed

    Baker, Jeremy D; Shelton, Lindsey B; Zheng, Dali; Favretto, Filippo; Nordhues, Bryce A; Darling, April; Sullivan, Leia E; Sun, Zheying; Solanki, Parth K; Martin, Mackenzie D; Suntharalingam, Amirthaa; Sabbagh, Jonathan J; Becker, Stefan; Mandelkow, Eckhard; Uversky, Vladimir N; Zweckstetter, Markus; Dickey, Chad A; Koren, John; Blair, Laura J

    2017-06-01

    The accumulation of amyloidogenic proteins is a pathological hallmark of neurodegenerative disorders. The aberrant accumulation of the microtubule associating protein tau (MAPT, tau) into toxic oligomers and amyloid deposits is a primary pathology in tauopathies, the most common of which is Alzheimer's disease (AD). Intrinsically disordered proteins, like tau, are enriched with proline residues that regulate both secondary structure and aggregation propensity. The orientation of proline residues is regulated by cis/trans peptidyl-prolyl isomerases (PPIases). Here we show that cyclophilin 40 (CyP40), a PPIase, dissolves tau amyloids in vitro. Additionally, CyP40 ameliorated silver-positive and oligomeric tau species in a mouse model of tau accumulation, preserving neuronal health and cognition. Nuclear magnetic resonance (NMR) revealed that CyP40 interacts with tau at sites rich in proline residues. CyP40 was also able to interact with and disaggregate other aggregating proteins that contain prolines. Moreover, CyP40 lacking PPIase activity prevented its capacity for disaggregation in vitro. Finally, we describe a unique structural property of CyP40 that may permit disaggregation to occur in an energy-independent manner. This study identifies a novel human protein disaggregase and, for the first time, demonstrates its capacity to dissolve intracellular amyloids.

  3. Human cyclophilin 40 unravels neurotoxic amyloids

    PubMed Central

    Favretto, Filippo; Nordhues, Bryce A.; Darling, April; Sullivan, Leia E.; Sun, Zheying; Solanki, Parth K.; Martin, Mackenzie D.; Suntharalingam, Amirthaa; Sabbagh, Jonathan J.; Becker, Stefan; Mandelkow, Eckhard; Uversky, Vladimir N.; Zweckstetter, Markus; Dickey, Chad A.; Koren, John; Blair, Laura J.

    2017-01-01

    The accumulation of amyloidogenic proteins is a pathological hallmark of neurodegenerative disorders. The aberrant accumulation of the microtubule associating protein tau (MAPT, tau) into toxic oligomers and amyloid deposits is a primary pathology in tauopathies, the most common of which is Alzheimer’s disease (AD). Intrinsically disordered proteins, like tau, are enriched with proline residues that regulate both secondary structure and aggregation propensity. The orientation of proline residues is regulated by cis/trans peptidyl-prolyl isomerases (PPIases). Here we show that cyclophilin 40 (CyP40), a PPIase, dissolves tau amyloids in vitro. Additionally, CyP40 ameliorated silver-positive and oligomeric tau species in a mouse model of tau accumulation, preserving neuronal health and cognition. Nuclear magnetic resonance (NMR) revealed that CyP40 interacts with tau at sites rich in proline residues. CyP40 was also able to interact with and disaggregate other aggregating proteins that contain prolines. Moreover, CyP40 lacking PPIase activity prevented its capacity for disaggregation in vitro. Finally, we describe a unique structural property of CyP40 that may permit disaggregation to occur in an energy-independent manner. This study identifies a novel human protein disaggregase and, for the first time, demonstrates its capacity to dissolve intracellular amyloids. PMID:28654636