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Sample records for peptidyl prolyl cis-trans

  1. Peptidyl prolyl cis-trans-isomerase activity associated with the lumen of the endoplasmic reticulum.

    PubMed Central

    Bose, S; Freedman, R B

    1994-01-01

    Peptidyl prolyl cis-trans-isomerase (PPI) activity was detected in microsomal fractions from bovine and rat liver. Extensive washing, proteinase and sonication treatments indicated that although some of this activity was due to adsorbed cytosolic enzymes, there was also an active but latent microsomal PPI activity. Density-gradient subfractionation indicated that activity was associated with vesicles derived from both the rough and the smooth endoplasmic reticulum (ER), suggesting that the activity was located within the ER lumen. The luminal PPI activity was inhibited by cyclosporin A and was active towards an unfolded protein substrate as well as towards the standard peptide substrate. PMID:8010971

  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. Vaccine potential of bacterial macrophage infectivity potentiator (MIP)-like peptidyl prolyl cis/trans isomerase (PPIase) proteins.

    PubMed

    Humbert, María Victoria; Almonacid Mendoza, Hannia L; Jackson, Alexandra C; Hung, Miao-Chiu; Bielecka, Magdalena K; Heckels, John E; Christodoulides, Myron

    2015-01-01

    Peptidyl prolyl cis/trans isomerases (PPIases) are a superfamily of proteins ubiquitously distributed among living organisms, which function primarily to assist the folding and structuring of unfolded and partially folded polypeptide chains and proteins. In this review, we focus specifically on the Macrophage Infectivity Potentiator (MIP)-like PPIases, which are members of the immunophilin family of FK506-binding proteins (FKBP). MIP-like PPIases have accessory roles in virulence and are candidates for inclusion in vaccines protective against both animal and human bacterial pathogens. A structural vaccinology approach obviates any issues over molecular mimicry and potential cross-reactivity with human FKBP proteins and studies with a representative antigen, the Neisseria meningitidis-MIP, support this strategy. Moreover, a dual approach of vaccination and drug targeting could be considered for controlling bacterial infectious diseases of humans and animals.

  4. Determining the roles of a conserved tyrosine residue in a Mip-like peptidyl-prolyl cis-trans isomerase.

    PubMed

    Polley, Soumitra; Chakravarty, Devlina; Chakrabarti, Gopal; Sau, Subrata

    2016-06-01

    The FKBP22 and the related peptidyl-prolyl cis-trans isomerases dimerize using their N-terminal domains. Conversely, their C-terminal domains possess both the substrate and inhibitor binding sites. To delineate the roles of a conserved Tyr residue at their N-terminal domains, we have studied a FKBP22 mutant that carries an Ala in place of the conserved Tyr at position 15. We have demonstrated that the Tyr 15 of FKBP22 is indispensable for preserving its dimerization ability, catalytic activity, and structure. The residue, however, little contributed to its inhibitor binding ability and stability. The mode of action of Tyr 15 has been discussed at length.

  5. 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

  6. Metamorphic protein IscU changes conformation by cis-trans isomerizations of two peptidyl-prolyl peptide bonds.

    PubMed

    Dai, Ziqi; Tonelli, Marco; Markley, John L

    2012-12-01

    IscU from Escherichia coli, the scaffold protein for iron-sulfur cluster biosynthesis and transfer, populates two conformational states with similar free energies and with lifetimes on the order of 1 s that interconvert in an apparent two-state reaction. One state (S) is structured, and the other (D) is largely disordered; however, both play essential functional roles. We report here nuclear magnetic resonance studies demonstrating that all four prolyl residues of apo-IscU (P14, P35, P100, and P101) are trans in the S state but that two absolutely conserved residues (P14 and P101) become cis in the D state. The peptidyl-prolyl peptide bond configurations were determined by analyzing assigned chemical shifts and were confirmed by measurements of nuclear Overhauser effects. We conclude that the S ⇄ D interconversion involves concerted trans-cis isomerization of the N13-P14 and P100-P101 peptide bonds. Although the D state is largely disordered, we show that it contains an ordered domain that accounts for the stabilization of two high-energy cis peptide bonds. Thus, IscU may be classified as a metamorphic protein.

  7. 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. PMID:26341203

  8. 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.

  9. 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

  10. Solution structure of Escherichia coli Par10: The prototypic member of the Parvulin family of peptidyl-prolyl cis/trans isomerases

    PubMed Central

    Kühlewein, Angelika; Voll, Georg; Hernandez Alvarez, Birte; Kessler, Horst; Fischer, Gunter; Rahfeld, Jens-Ulrich; Gemmecker, Gerd

    2004-01-01

    E. coli Par10 is a peptidyl-prolyl cis/trans isomerase (PPIase) from Escherichia coli catalyzing the isomerization of Xaa-Pro bonds in oligopeptides with a broad substrate specificity. The structure of E. coli Par10 has been determined by multidimensional solution-state NMR spectroscopy based on 1207 conformational constraints (1067 NOE-derived distances, 42 vicinal coupling-constant restraints, 30 hydrogen-bond restraints, and 68 φ/ψ restraints derived from the Chemical Shift Index). Simulated-annealing calculations with the program ARIA and subsequent refinement with XPLOR yielded a set of 18 convergent structures with an average backbone RMSD from mean atomic coordinates of 0.50 Å within the well-defined secondary structure elements. E. coli Par10 is the smallest known PPIase so far, with a high catalytic efficiency comparable to that of FKBPs and cyclophilins. The secondary structure of E. coli Par10 consists of four helical regions and a four-stranded antiparallel β-sheet. The N terminus forms a β-strand, followed by a large stretch comprising three α-helices. A loop region containing a short β-strand separates these helices from a fourth α-helix. The C terminus consists of two more β-strands completing the four-stranded anti-parallel β-sheet with strand order 2143. Interestingly, the third β-strand includes a Gly-Pro cis peptide bond. The curved β-strand forms a hydrophobic binding pocket together with α-helix 4, which also contains a number of highly conserved residues. The three-dimensional structure of Par10 closely resembles that of the human proteins hPin1 and hPar14 and the plant protein Pin1At, belonging to the same family of highly homologous proteins. PMID:15322281

  11. The Cellular Peptidyl-Prolyl cis/trans Isomerase Pin1 Regulates Reactivation of Kaposi's Sarcoma-Associated Herpesvirus from Latency

    PubMed Central

    Guito, Jonathan; Gavina, Aileen; Palmeri, Diana

    2014-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma and primary effusion lymphoma. KSHV-infected cells are predominantly latent, with a subset undergoing lytic reactivation. Rta is the essential lytic switch protein that reactivates virus by forming transactivation-competent complexes with the Notch effector protein RBP-Jk and promoter DNA. Strikingly, Rta homolog analysis reveals that prolines constitute 17% of conserved residues. Rta is also highly phosphorylated in vivo. We previously demonstrated that proline content determines Rta homotetramerization and function. We hypothesize that proline-directed modifications regulate Rta function by controlling binding to peptidyl-prolyl cis/trans isomerases (PPIases). Cellular PPIase Pin1 binds specifically to phosphoserine- or phosphothreonine-proline (pS/T-P) motifs in target proteins. Pin1 dysregulation is implicated in myriad human cancers and can be subverted by viruses. Our data show that KSHV Rta protein contains potential pS/T-P motifs and binds directly to Pin1. Rta transactivation is enhanced by Pin1 at two delayed early viral promoters in uninfected cells. Pin1's effect, however, suggests a rheostat-like influence on Rta function. We show that in infected cells, endogenous Pin1 is active during reactivation and enhances Rta-dependent early protein expression induced by multiple signals, as well as DNA replication. Surprisingly, ablation of Pin1 activity by the chemical juglone or dominant-negative Pin1 enhanced late gene expression and production of infectious virus, while ectopic Pin1 showed inhibitory effects. Our data thus suggest that Pin1 is a unique, dose-dependent molecular timer that enhances Rta protein function, but inhibits late gene synthesis and virion production, during KSHV lytic reactivation. PMID:24173213

  12. 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

  13. Separation of cis/trans isomers of a prolyl peptide bond by capillary zone electrophoresis.

    PubMed

    Meyer, S; Jabs, A; Schutkowski, M; Fischer, G

    1994-01-01

    On capillary electrophoresis of the chemically pure thioxo peptide Ala-Phe-psi[CS-N]-Pro-Phe-4-nitroanilide a peak splitting was observed at a capillary temperature of 25 degrees C. By contrast, the oxo peptide analogue exhibits a single, sharp peak under these conditions. Both peaks of the thioxo compound coincided gradually when the temperature was increased to 60 degrees C. Peak fusion was reverted by cooling down the heated sample. This behavior could be attributed to the electrophoresis-mediated separation of the cis/trans prolyl bond isomers of the thioxo peptide, allowing data of this conformational equilibrium to be determined. Derived from computational data about molecular volume and the hydration energy of low-energy cis and trans isomeric structures, the more rapid migration of the cis form in comparison to trans may be explained by structural parameters.

  14. Mycobacterium tuberculosis Peptidyl-Prolyl Isomerases Also Exhibit Chaperone like Activity In-Vitro and In-Vivo

    PubMed Central

    Pandey, Saurabh; Sharma, Ashish; Tripathi, Deeksha; Kumar, Ashutosh; Khubaib, Mohd; Bhuwan, Manish; Chaudhuri, Tapan Kumar; Hasnain, Seyed Ehtesham; Ehtesham, Nasreen Zafar

    2016-01-01

    Peptidyl-prolyl cis-trans isomerases (Ppiases), also known as cyclophilins, are ubiquitously expressed enzymes that assist in protein folding by isomerization of peptide bonds preceding prolyl residues. Mycobacterium tuberculosis (M.tb) is known to possess two Ppiases, PpiA and PpiB. However, our understanding about the biological significance of mycobacterial Ppiases with respect to their pleiotropic roles in responding to stress conditions inside the macrophages is restricted. This study describes chaperone-like activity of mycobacterial Ppiases. We show that recombinant rPpiA and rPpiB can bind to non-native proteins in vitro and can prevent their aggregation. Purified rPpiA and rPpiB exist in oligomeric form as evident from gel filtration chromatography.E. coli cells overexpressing PpiA and PpiB of M.tb could survive thermal stress as compared to plasmid vector control. HEK293T cells transiently expressing M.tb PpiA and PpiB proteins show increased survival as compared to control cells in response to oxidative stress and hypoxic conditions generated after treatment with H2O2 and CoCl2 thereby pointing to their likely role in adaption under host generated oxidative stress and conditions of hypoxia. The chaperone-like function of these M.tuberculosis cyclophilins may possibly function as a stress responder and consequently contribute to virulence. PMID:26981873

  15. 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.

  16. 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

  17. Interaction of FkpA, a peptidyl-prolyl cis/trans isomerase with EspP autotransporter protein

    PubMed Central

    Henderson, Ian R; Nataro, James P

    2010-01-01

    The serine protease autotransporters of Enterobacteriaceae (SPATEs) represent a large class of proteases with contributions to virulence. They are synthesized with a C-terminal domain that forms a β-barrel pore in the outer membrane implicated in translocation of the N-terminal ‘passenger’ domain across the outer membrane. The most recent model for autotransporter secretion comprises entry to the periplasm via the Sec apparatus, followed by the insertion of the C-terminus into the outer membrane as a β-barrel protein and accompanied by translocation of the passenger domain to the bacterial cell surface, all of this with the assistance of the Bam complex insertase/foldase and periplasmic chaperone proteins. We have recently observed direct involvement of periplasmic chaperones in the biogenesis of EspP, a prototypical autotransporter protein produced by Escherichia coli O157:H7. Using molecular and biophysical approaches we demonstrated for the first time, direct protein-protein interactions between the periplasmic SurA and DegP chaperones and either the EspP-β or EspP passenger domains. Such chaperone interactions took place on conserved aromatic residues on the SPATE family. In this report, we now demonstrate direct binding of the periplasmic chaperone FkpA to the EspP passanger domain in Surface Plasmon Resonance experiments with relatively high affinity. We also provide evidence of interaction between the SurA and Skp chaperones with the Bam. These findings in conjunction with newly published data support the role of chaperones in preventing misfolding of AT passenger domains before translocation throughout the Bam complex. PMID:21327044

  18. Kinetic Isotope Effects Support the Twisted Amide Mechanism of Pin1 Peptidyl-Prolyl Isomerase

    PubMed Central

    Mercedes-Camacho, Ana Y.; Mullins, Ashley B.; Mason, Matthew D.; Xu, Guoyan G.; Mahoney, Brendan J.; Wang, Xingsheng; Peng, Jeffrey W.; Etzkorn, Felicia A.

    2013-01-01

    The Pin1 peptidyl-prolyl isomerase (PPIase) catalyzes isomerization of pSer/pThr-Pro motifs in regulating the cell cycle. Peptide substrates, Ac–Phe–Phe–phosphoSer–Pro–Arg–p-nitroaniline, were synthesized in unlabeled form, and with deuterium labeled Ser-d3 and Pro-d7 amino acids. Kinetic data was collected as a function of Pin1 concentration to measure kinetic isotope effects (KIE) on catalytic efficiency (kcat/Km). The normal secondary (2°) KIE value measured for the Ser-d3 substrate (kH/kD = 1.6 ± 0.2) indicates that the serine carbonyl does not rehybridize from sp2 to sp3 in the rate-determining step, ruling out a nucleophilic addition mechanism. The normal 2° KIE can be explained by hyperconjugation between Ser α-C–H/D and C=O, and release of steric strain upon rotation of the amide bond from cis to syn-exo. The inverse 2° KIE value (kH/kD = 0.86 ± 0.08) measured for the Pro-d7 substrate indicates rehybridization of the prolyl nitrogen from sp2 to sp3 during the rate-limiting step of isomerization. No solvent kinetic isotope was measured by NMR exchange spectroscopy (EXSY) (kH2O/kD2O = 0.92 ± 0.12), indicating little or no involvement of exchangeable protons in the mechanism. These results support the formation of a simple twisted-amide transition state as the mechanism for peptidyl prolyl isomerization catalyzed by Pin1. A model of the reaction mechanism is presented using crystal structures of Pin1 with ground state analogues and an inhibitor that resembles a twisted amide transition state. PMID:24116866

  19. 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.

  20. 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. PMID:25998391

  1. Calcineurin Undergoes a Conformational Switch Evoked via Peptidyl-Prolyl Isomerization

    PubMed Central

    Guasch, Alicia; Aranguren-Ibáñez, Álvaro; Pérez-Luque, Rosa; Aparicio, David; Martínez-Høyer, Sergio; Mulero, M. Carmen; Serrano-Candelas, Eva

    2015-01-01

    A limited repertoire of PPP family of serine/threonine phosphatases with a highly conserved catalytic domain acts on thousands of protein targets to orchestrate myriad central biological roles. A major structural reorganization of human calcineurin, a ubiquitous Ser/Thr PPP regulated by calcium and calmodulin and targeted by immunosuppressant drugs cyclosporin A and FK506, is unveiled here. The new conformation involves trans- to cis- isomerization of proline in the SAPNY sequence, highly conserved across PPPs, and remodels the main regulatory site where NFATc transcription factors bind. Transitions between cis- and trans- conformations may involve peptidyl prolyl isomerases such as cyclophilin A and FKBP12, which are known to physically interact with and modulate calcineurin even in the absence of immunosuppressant drugs. Alternative conformations in PPPs provide a new perspective on interactions with substrates and other protein partners and may foster development of more specific inhibitors as drug candidates. PMID:26248042

  2. 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

  3. Nerve Growth Factor Stimulates Interaction of Cayman Ataxia Protein BNIP-H/Caytaxin with Peptidyl-Prolyl Isomerase Pin1 in Differentiating Neurons

    PubMed Central

    Buschdorf, Jan Paul; Chew, Li Li; Soh, Unice Jim Kim; Liou, Yih-Cherng; Low, Boon Chuan

    2008-01-01

    Mutations in ATCAY that encodes the brain-specific protein BNIP-H (or Caytaxin) lead to Cayman cerebellar ataxia. BNIP-H binds to glutaminase, a neurotransmitter-producing enzyme, and affects its activity and intracellular localization. Here we describe the identification and characterization of the binding between BNIP-H and Pin1, a peptidyl-prolyl cis/trans isomerase. BNIP-H interacted with Pin1 after nerve growth factor-stimulation and they co-localized in the neurites and cytosol of differentiating pheochromocytoma PC12 cells and the embryonic carcinoma P19 cells. Deletional mutagenesis revealed two cryptic binding sites within the C-terminus of BNIP-H such that single point mutants affecting the WW domain of Pin1 completely abolished their binding. Although these two sites do not contain any of the canonical Pin1-binding motifs they showed differential binding profiles to Pin1 WW domain mutants S16E, S16A and W34A, and the catalytically inert C113A of its isomerase domain. Furthermore, their direct interaction would occur only upon disrupting the ability of BNIP-H to form an intramolecular interaction by two similar regions. Furthermore, expression of Pin1 disrupted the BNIP-H/glutaminase complex formation in PC12 cells under nerve growth factor-stimulation. These results indicate that nerve growth factor may stimulate the interaction of BNIP-H with Pin1 by releasing its intramolecular inhibition. Such a mechanism could provide a post-translational regulation on the cellular activity of BNIP-H during neuronal differentiation. (213 words) PMID:18628984

  4. Local control of cis-peptidyl-prolyl bonds mediated by CH···π interactions: the Xaa-Pro-Tyr motif.

    PubMed

    Ganguly, Himal K; Kaur, Hundeep; Basu, Gautam

    2013-09-17

    Compared to generic peptide bonds, the peptidyl-prolyl bond shows a strong propensity for the cis conformer. The presence of a sequence-contiguous aromatic (Aro) residue can further stabilize the cis conformer, as observed for the Aro-Pro motif. The cis propensity of the reverse sequence motif, Pro-Aro, is not so well understood, especially the effect of N-capping the Pro-Aro motif with different amino acid residues. From a comparative nuclear magnetic resonance study of two peptide series with the general sequences Ac-Xaa-Pro-Tyr-NH2 and Ac-Xaa-Pro-Ala-NH2, we present a relative thermodynamic scale that reflects how the nature of the Xaa side chain influences the cis propensity of the Xaa-Pro-Tyr motif, with Gly, Pro, and Ala at position Xaa giving the greatest enhancement of the cis-peptidyl-prolyl population. We also show that CH···π interaction between Xaa and Tyr is responsible for the enhanced cis population. However, the mere presence of the CH···π interaction does not guarantee that the peptidyl-prolyl bond will have a higher cis content in Xaa-Pro-Tyr than in Xaa-Pro-Ala. Xaa-dependent intramolecular interactions present in Xaa-trans-Pro-Tyr can nullify favorable CH···π interactions in Xaa-cis-Pro-Tyr. The relative cis-peptidyl-prolyl stabilizing propensities of Xaa (Xaa-Pro-Tyr) in proteins and in our peptide series show strong linear correlation except when Xaa is aromatic. We also explore the Xaa-Pro-Gly-Tyr sequence motif and show that mediated by a Pro-Tyr CH···π interaction, the cis-peptidyl-prolyl bond in the motif is stabilized when Xaa is Pro.

  5. 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.

  6. 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

  7. Inhibition of the FKBP family of peptidyl prolyl isomerases induces abortive translocation and degradation of the cellular prion protein.

    PubMed

    Stocki, Pawel; Sawicki, Maxime; Mays, Charles E; Hong, Seo Jung; Chapman, Daniel C; Westaway, David; Williams, David B

    2016-03-01

    Prion diseases are fatal neurodegenerative disorders for which there is no effective treatment. Because the cellular prion protein (PrP(C)) is required for propagation of the infectious scrapie form of the protein, one therapeutic strategy is to reduce PrP(C) expression. Recently FK506, an inhibitor of the FKBP family of peptidyl prolyl isomerases, was shown to increase survival in animal models of prion disease, with proposed mechanisms including calcineurin inhibition, induction of autophagy, and reduced PrP(C) expression. We show that FK506 treatment results in a profound reduction in PrP(C) expression due to a defect in the translocation of PrP(C) into the endoplasmic reticulum with subsequent degradation by the proteasome. These phenotypes could be bypassed by replacing the PrP(C) signal sequence with that of prolactin or osteopontin. In mouse cells, depletion of ER luminal FKBP10 was almost as potent as FK506 in attenuating expression of PrP(C). However, this occurred at a later stage, after translocation of PrP(C) into the ER. Both FK506 treatment and FKBP10 depletion were effective in reducing PrP(Sc) propagation in cell models. These findings show the involvement of FKBP proteins at different stages of PrP(C) biogenesis and identify FKBP10 as a potential therapeutic target for the treatment of prion diseases. PMID:26764098

  8. Inhibition of the FKBP family of peptidyl prolyl isomerases induces abortive translocation and degradation of the cellular prion protein

    PubMed Central

    Stocki, Pawel; Sawicki, Maxime; Mays, Charles E.; Hong, Seo Jung; Chapman, Daniel C.; Westaway, David; Williams, David B.

    2016-01-01

    Prion diseases are fatal neurodegenerative disorders for which there is no effective treatment. Because the cellular prion protein (PrPC) is required for propagation of the infectious scrapie form of the protein, one therapeutic strategy is to reduce PrPC expression. Recently FK506, an inhibitor of the FKBP family of peptidyl prolyl isomerases, was shown to increase survival in animal models of prion disease, with proposed mechanisms including calcineurin inhibition, induction of autophagy, and reduced PrPC expression. We show that FK506 treatment results in a profound reduction in PrPC expression due to a defect in the translocation of PrPC into the endoplasmic reticulum with subsequent degradation by the proteasome. These phenotypes could be bypassed by replacing the PrPC signal sequence with that of prolactin or osteopontin. In mouse cells, depletion of ER luminal FKBP10 was almost as potent as FK506 in attenuating expression of PrPC. However, this occurred at a later stage, after translocation of PrPC into the ER. Both FK506 treatment and FKBP10 depletion were effective in reducing PrPSc propagation in cell models. These findings show the involvement of FKBP proteins at different stages of PrPC biogenesis and identify FKBP10 as a potential therapeutic target for the treatment of prion diseases. PMID:26764098

  9. 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.

  10. 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.

  11. Cis-trans photoisomerization of fluorescent-protein chromophores.

    PubMed

    Voliani, Valerio; Bizzarri, Ranieri; Nifosì, Riccardo; Abbruzzetti, Stefania; Grandi, Elena; Viappiani, Cristiano; Beltram, Fabio

    2008-08-28

    Photochromic variants of fluorescent proteins are opening the way to a number of opportunities for high-sensitivity regioselective studies in the cellular environment and may even lead to applications in information and communication technology. Yet, the detailed photophysical processes at the basis of photoswitching have not been fully clarified. In this paper, we used synthetic FP chromophores to clarify the photophysical processes associated with the photochromic behavior. In particular, we investigated the spectral modification of synthetic chromophore analogues of wild-type green fluorescent protein (GFP), Y66F GFP (BFPF), and Y66W GFP (CFP) upon irradiation in solutions of different polarities. We found that the cis-trans photoisomerization mechanism can be induced in all the chromophores. The structural assignments were carried out both by NMR measurements and DFT calculations. Remarkably, we determined for the first time the spectra of neutral trans isomers in different solvents. Finally, we calculated the photoconversion quantum yields by absorption measurements under continuous illumination at different times and by a nanosecond laser-flash photolysis method. Our results indicate that cis-trans photoisomerization is a general mechanism of FP chromophores whose efficiency is modulated by the detailed mutant-specific protein environment. PMID:18671358

  12. Wavelength dependent cis-trans isomerization in vision.

    PubMed

    Kim, J E; Tauber, M J; Mathies, R A

    2001-11-20

    The primary event in vision is the light-driven cis-trans isomerization of the 11-cis-retinal chromophore in the G-protein coupled receptor rhodopsin. Early measurements showed that this photoisomerization has a reaction quantum yield phi of approximately 0.67 [Dartnall (1936) Proc. R. Soc. A 156, 158-170; Dartnall (1968) Vision Res. 8, 339-358] and suggested that the quantum yield was wavelength independent [Schneider (1939) Proc. Natl. Acad. Sci. U.S.A. 170, 102-112]. Here we more accurately determine phi(500) = 0.65 +/- 0.01 and reveal that phi surprisingly depends on the wavelength of the incident light. Although there is no difference in the quantum yield between 450 and 480 nm, the quantum yield falls significantly as the photon energy is reduced below 20 000 cm(-1) (500 nm). At the reddest wavelength measured (570 nm), the quantum yield is reduced by 5 +/- 1% relative to the 500 nm value. These experiments correct the long-held presumption that the quantum yield in vision is wavelength independent, and support the hypothesis that the 200 fs photoisomerization reaction that initiates vision is dictated by nonstationary excited-state vibrational wave packet dynamics. PMID:11705366

  13. Wavelength Dependent Cis-Trans Isomerization in Vision†

    PubMed Central

    Kim, Judy E.; Tauber, Michael J.; Mathies, Richard A.

    2005-01-01

    The primary event in vision is the light-driven cis-trans isomerization of the 11-cis-retinal chromophore in the G-protein coupled receptor rhodopsin. Early measurements showed that this photoisomerization has a reaction quantum yield Φ of ∼0.67 [Dartnall (1936) Proc. R. Soc. A 156, 158-170; Dartnall (1968) Vision Res. 8, 339-358] and suggested that the quantum yield was wavelength independent [Schneider (1939) Proc. Natl. Acad. Sci. U.S.A. 170, 102-112]. Here we more accurately determine Φ 500) = 0.65 ± 0.01 and reveal that Φ surprisingly depends on the wavelength of the incident light. Although there is no difference in the quantum yield between 450 and 480 nm, the quantum yield falls significantly as the photon energy is reduced below 20 000 cm-1 (500 nm). At the reddest wavelength measured (570 nm), the quantum yield is reduced by 5 ± 1% relative to the 500 nm value. These experiments correct the long-held presumption that the quantum yield in vision is wavelength independent, and support the hypothesis that the 200 fs photoisomerization reaction that initiates vision is dictated by nonstationary excited-state vibrational wave packet dynamics. PMID:11705366

  14. A β-Turn Motif in the Steroid Hormone Receptor's Ligand-Binding Domains Interacts with the Peptidyl-prolyl Isomerase (PPIase) Catalytic Site of the Immunophilin FKBP52.

    PubMed

    Byrne, Cillian; Henen, Morkos A; Belnou, Mathilde; Cantrelle, François-Xavier; Kamah, Amina; Qi, Haoling; Giustiniani, Julien; Chambraud, Béatrice; Baulieu, Etienne-Emile; Lippens, Guy; Landrieu, Isabelle; Jacquot, Yves

    2016-09-27

    The immunophilin FKBP52 interacts with nuclear steroid hormone receptors. Studying the crystal structure of human estrogen receptor α (hERα) and using nuclear magnetic resonance, we show here that the short V(364)PGF(367) sequence, which is located within its ligand-binding domain and adopts a type II β-turn conformation in the protein, binds the peptidyl-prolyl isomerase (PPIase or rotamase) FK1 domain of FKBP52. Interestingly, this turn motif displays strong similarities with the FKBP52 FK1 domain-binding moiety of macrolide immunomodulators such as rapamycin and GPI-1046, an immunophilin ligand with neuroprotective characteristics. An increase in the hydrophobicity of the residue preceding the proline and cyclization of the VPGF peptide strengthen its recognition by the FK1 domain of FKBP52. Replacement of the Pro residue with a dimethylproline also enhances this interaction. Our study not only contributes to a better understanding of how the interaction between the FK1 domain of FKBP52 and steroid hormone receptors most likely works but also opens new avenues for the synthesis of FKBP52 FK1 peptide ligands appropriate for the control of hormone-dependent physiological mechanisms or of the functioning of the Tau protein. Indeed, it has been shown that FKBP52 is involved in the intraneuronal dynamics of the Tau protein. PMID:27641460

  15. A β-Turn Motif in the Steroid Hormone Receptor's Ligand-Binding Domains Interacts with the Peptidyl-prolyl Isomerase (PPIase) Catalytic Site of the Immunophilin FKBP52.

    PubMed

    Byrne, Cillian; Henen, Morkos A; Belnou, Mathilde; Cantrelle, François-Xavier; Kamah, Amina; Qi, Haoling; Giustiniani, Julien; Chambraud, Béatrice; Baulieu, Etienne-Emile; Lippens, Guy; Landrieu, Isabelle; Jacquot, Yves

    2016-09-27

    The immunophilin FKBP52 interacts with nuclear steroid hormone receptors. Studying the crystal structure of human estrogen receptor α (hERα) and using nuclear magnetic resonance, we show here that the short V(364)PGF(367) sequence, which is located within its ligand-binding domain and adopts a type II β-turn conformation in the protein, binds the peptidyl-prolyl isomerase (PPIase or rotamase) FK1 domain of FKBP52. Interestingly, this turn motif displays strong similarities with the FKBP52 FK1 domain-binding moiety of macrolide immunomodulators such as rapamycin and GPI-1046, an immunophilin ligand with neuroprotective characteristics. An increase in the hydrophobicity of the residue preceding the proline and cyclization of the VPGF peptide strengthen its recognition by the FK1 domain of FKBP52. Replacement of the Pro residue with a dimethylproline also enhances this interaction. Our study not only contributes to a better understanding of how the interaction between the FK1 domain of FKBP52 and steroid hormone receptors most likely works but also opens new avenues for the synthesis of FKBP52 FK1 peptide ligands appropriate for the control of hormone-dependent physiological mechanisms or of the functioning of the Tau protein. Indeed, it has been shown that FKBP52 is involved in the intraneuronal dynamics of the Tau protein.

  16. Torquoselective Ring Opening of Fused Cyclobutenamides: Evidence for a Cis,Trans-Cyclooctadienone Intermediate

    PubMed Central

    2015-01-01

    Electrocyclic ring opening of 4,6-fused cyclobutenamides 1 under thermal conditions leads to cis,trans-cyclooctadienones 2-E,E as transient intermediates, en route to 5,5-bicyclic products 3. Theoretical calculations predict that 4,5-fused cyclobutenamides should likewise undergo thermal ring opening, giving cis,trans-cycloheptadienones, but in this case conversion to 5,4-bicyclic products is thermodynamically disfavored, and these cyclobutenamides instead rearrange to vinyl cyclopentenones. PMID:24992255

  17. 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.

  18. Prolyl isomerase Pin1 regulates doxorubicin-inducible P-glycoprotein level by reducing Foxo3 stability.

    PubMed

    Shimizu, Taiki; Bamba, Yoshimasa; Kawabe, Yosuke; Fukuda, Tomokazu; Fujimori, Fumihiro; Takahashi, Katsuhiko; Uchida, Chiyoko; Uchida, Takafumi

    2016-03-01

    It has been known that the phosphoSer/Thr-Pro-specific peptidyl prolyl cis/trans isomerase Pin1 regulates a variety of intracellular signaling pathways, including the response to the genotoxic drug doxorubicin. Pin1 binds phosphorylated p53 and stabilizes p53 to cause cell cycle arrest and apoptosis quickly in response to doxorubicin. Here we show another mechanism of Pin1 to maintain cell sensitivity to genotoxic stress, irrespective of whether p53 is present or not. In response to the genotoxic drug, Pin1 binds and decreases levels of the phosphorylated Foxo3, the positive transcription factor of P-glycoprotein (P-gp) gene. Through this mechanism of action, Pin1 decreases the level of P-gp and signals the cell to pump the genotoxic drugs out. This shows that Pin1 is implemented in maintaining the susceptibility to the genotoxic drugs by controlling P-gp level as well as p53-dependent apoptosis and cell cycle signaling pathways.

  19. IRIS Toxicological Review of cis- & trans-1,2-Dichloroethylene (2010 Final)

    EPA Science Inventory

    The final Toxicological Review of cis- & trans-1,2-Dichloroethylene provides scientific support and rationale for the hazard and dose-response assessment pertaining to chronic exposure to cis- and trans-1,2-dichloroethylene. 1,2-Dichloroethylene is used as a solvent for wa...

  20. Prolyl isomerase Pin1 negatively regulates the stability of SUV39H1 to promote tumorigenesis in breast cancer.

    PubMed

    Khanal, Prem; Kim, Garam; Lim, Sung-Chul; Yun, Hyo-Jeong; Lee, Kwang Youl; Choi, Hoo-Kyun; Choi, Hong Seok

    2013-11-01

    Pin1, a conserved eukaryotic peptidyl-prolyl cis/trans isomerase, has profound effects on numerous key-signaling molecules, and its deregulation contributes to disease, particularly cancer. Although Pin1-mediated prolyl isomerization of protein servers as a regulatory switch in signaling pathways, the significance of proline isomerase activity in chromatin modifying complex remains unclear. Here, we identify Pin1 as a key negative regulator for suppressor of variegation 3-9 homologue 1 (SUV39H1) stability, a major methyltransferase responsible for histone H3 trimethylation on Lys9 (H3K9me3). Pin1 interacts with SUV39H1 in a phosphorylation-dependent manner and promotes ubiquitination-mediated degradation of SUV39H1. Consequently, Pin1 reduces SUV39H1 abundance and suppresses SUV39H1 ability to induce H3K9me3. In contrast, depletion of Pin1 in cancer cells leads to elevated SUV39H1 expression, which subsequently increases H3K9me3, inhibiting tumorigenecity of cancer cells. In a xenograft model with 4T1 metastatic mouse breast carcinoma cells, Pin1 overexpression increases tumor growth, whereas SUV39H1 overexpression abrogates it. In human breast cancer patients, immunohistochemical staining shows that Pin1 levels are negatively correlated with SUV39H1 as well as H3K9me3 levels. Thus, Pin1-mediated reduction of SUV39H1 stability contributes to convey oncogenic signals for aggressiveness of human breast cancer, suggesting that Pin1 may be a promising drug target for anticancer therapy. PMID:23934277

  1. 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.

  2. Parallel channels and rate-limiting steps in complex protein folding reactions: prolyl isomerization and the alpha subunit of Trp synthase, a TIM barrel protein.

    PubMed

    Wu, Ying; Matthews, C Robert

    2002-10-18

    A kinetic folding mechanism for the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, involving four parallel channels with multiple native, intermediate and unfolded forms, has recently been proposed. The hypothesis that cis/trans isomerization of several Xaa-Pro peptide bonds is the source of the multiple folding channels was tested by measuring the sensitivity of the three rate-limiting phases (tau(1), tau(2), tau(3)) to catalysis by cyclophilin, a peptidyl-prolyl isomerase. Although the absence of catalysis for the tau(1) (fast) phase leaves its assignment ambiguous, our previous mutational analysis demonstrated its connection to the unique cis peptide bond preceding proline 28. The acceleration of the tau(2) (medium) and tau(3) (slow) refolding phases by cyclophilin demonstrated that cis/trans prolyl isomerization is also the source of these phases. A collection of proline mutants, which covered all of the remaining 18 trans proline residues of alphaTS, was constructed to obtain specific assignments for these phases. Almost all of the mutant proteins retained the complex equilibrium and kinetic folding properties of wild-type alphaTS; only the P217A, P217G and P261A mutations caused significant changes in the equilibrium free energy surface. Both the P78A and P96A mutations selectively eliminated the tau(1) folding phase, while the P217M and P261A mutations eliminated the tau(2) and tau(3) folding phases, respectively. The redundant assignment of the tau(1) phase to Pro28, Pro78 and Pro96 may reflect their mutual interactions in non-random structure in the unfolded state. The non-native cis isomers for Pro217 and Pro261 may destabilize an autonomous C-terminal folding unit, thereby giving rise to kinetically distinct unfolded forms. The nature of the preceding amino acid, the solvent exposure, or the participation in specific elements of secondary structure in the native state, in general, are not determinative of the proline residues whose

  3. Discrimination of cis-trans sex pheromone components in two sympatric Lepidopteran species.

    PubMed

    Zhang, Sufang; Kong, Xiangbo; Ze, Sangzi; Wang, Hongbin; Lin, Aizhu; Liu, Fu; Zhang, Zhen

    2016-06-01

    Pheromone-binding proteins (PBPs) play an important role in the recognition of pheromones by insects. However, the abilities of these PBPs to discriminate pheromone components and recognize the isomers are unclear. Dendrolimus houi and Dendrolimus kikuchii are two sympatric coniferous pests whose pheromones have cis-trans isomers. We used these insect species to detect the precise recognition abilities of PBPs. The four PBPs examined showed male-biased antenna-intensive expression patterns, whereas PBP1 showed higher expression than PBP2 in the antenna. DhouPBP1 only bound to a minor interspecific pheromone component, whereas DhouPBP2 bound to all three intraspecific components and another minor interspecific component. DkikPBP1 and DkikPBP2 could recognize all three intraspecific components with affinities negatively correlated with their ratios, and they bound to interspecific pheromones with affinity that was positively correlated with the ratios. The four PBPs have different cis-trans isomer discrimination abilities, i.e., DhouPBP1 and DkikPBP1 could not discriminate the two cis-trans isomer pairs of pheromones from the two species, whereas DhouPBP2 could discriminate between both pairs, and DkikPBP2 could only discriminate one pair. Overall, PBPs from D. houi and D. kikuchii use different strategies to help the moths to discriminate the intra- and interspecific pheromone components. Our work will contribute to better understanding of the sex pheromone recognition mechanism in these two sister species of moths and provide insights into more effective management practices of these pest species. PMID:27107681

  4. Discrimination of cis-trans sex pheromone components in two sympatric Lepidopteran species.

    PubMed

    Zhang, Sufang; Kong, Xiangbo; Ze, Sangzi; Wang, Hongbin; Lin, Aizhu; Liu, Fu; Zhang, Zhen

    2016-06-01

    Pheromone-binding proteins (PBPs) play an important role in the recognition of pheromones by insects. However, the abilities of these PBPs to discriminate pheromone components and recognize the isomers are unclear. Dendrolimus houi and Dendrolimus kikuchii are two sympatric coniferous pests whose pheromones have cis-trans isomers. We used these insect species to detect the precise recognition abilities of PBPs. The four PBPs examined showed male-biased antenna-intensive expression patterns, whereas PBP1 showed higher expression than PBP2 in the antenna. DhouPBP1 only bound to a minor interspecific pheromone component, whereas DhouPBP2 bound to all three intraspecific components and another minor interspecific component. DkikPBP1 and DkikPBP2 could recognize all three intraspecific components with affinities negatively correlated with their ratios, and they bound to interspecific pheromones with affinity that was positively correlated with the ratios. The four PBPs have different cis-trans isomer discrimination abilities, i.e., DhouPBP1 and DkikPBP1 could not discriminate the two cis-trans isomer pairs of pheromones from the two species, whereas DhouPBP2 could discriminate between both pairs, and DkikPBP2 could only discriminate one pair. Overall, PBPs from D. houi and D. kikuchii use different strategies to help the moths to discriminate the intra- and interspecific pheromone components. Our work will contribute to better understanding of the sex pheromone recognition mechanism in these two sister species of moths and provide insights into more effective management practices of these pest species.

  5. Structural characterization of the HIV-1 Vpr N terminus: evidence of cis/trans-proline isomerism.

    PubMed

    Bruns, Karsten; Fossen, Torgils; Wray, Victor; Henklein, Peter; Tessmer, Uwe; Schubert, Ulrich

    2003-10-31

    The 96-residue human immunodeficiency virus (HIV) accessory protein Vpr serves manifold functions in the retroviral life cycle including augmentation of viral replication in non-dividing host cells, induction of G2 cell cycle arrest, and modulation of HIV-induced apoptosis. Using a combination of dynamic light scattering, circular dichroism, and NMR spectroscopy the N terminus of Vpr is shown to be a unique domain of the molecule that behaves differently from the C-terminal domain in terms of self-association and secondary structure folding. Interestingly, the four highly conserved proline residues in the N terminus are predicted to have a high propensity for cis/trans isomerism. Thus the high resolution structure and folding of a synthetic N-terminal peptide (Vpr1-40) and smaller fragments thereof have been investigated. 1H NMR data indicate Vpr1-40 possesses helical structure between residues 17-32, and for the first time, this helix, which is bound by proline residues, was observed even in aqueous solution devoid of any detergent supplements. In addition, NMR data revealed that all of the proline residues undergo a cis/ trans isomerism to such an extent that approximately 40% of all Vpr molecules possess at least one proline in a cis conformation. This phenomenon of cis/trans isomerism, which is unprecedented for HIV-1 Vpr, not only provides an explanation for the molecular heterogeneity observed in the full-length molecule but also indicates that in vivo the folding and function of Vpr should depend on a cis/trans-proline isomerase activity, particularly as two of the proline residues in positions 14 and 35 show considerable amounts of cis isomers. This prediction correlates well with our recent observation (Zander, K., Sherman, M. P., Tessmer, U., Bruns, K., Wray, V., Prechtel, A. T., Schubert, E., Henklein, P., Luban, J., Neidleman, J., Greene, W. C., and Schubert, U. (2003) J. Biol. Chem. 278, 43170-43181) of a functional interaction between the major

  6. 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.

  7. Ensemble of Transition State Structures for the Cis-Trans Isomerization of N-Methylacetamide

    SciTech Connect

    Mantz, Yves A.; Branduardi, Davide; Bussi, Giovanni; Parrinello, Michele

    2009-09-17

    The cis-trans isomerization of N-methylacetamide (NMA), a model peptidic fragment, is studied theoretically in vacuo and in explicit water solvent at 300 K using the metadynamics technique. The computed cis-trans free energy difference is very similar for NMA(g) and NMA(aq), in agreement with experimental measurements of population ratios and theoretical studies at 0 K. By exploiting the flexibility in the definition of a pair of recently introduced collective variables (Branduardi, D.; Gervasio, F. L.; Parrinello, M. J. Chem. Phys. 2007, 126, 054103), an ensemble of transition state structures is generated at finite temperature for both NMA(g) and NMA(aq), as verified by computing committor distribution functions. Ensemble members of NMA(g) are shown to have correlated values of the backbone dihedral angle and a second dihedral angle involving the amide hydrogen atom. The dynamical character of these structures is preserved in the presence of solvent, whose influence on the committor functions can be modeled using effective friction/noise terms.

  8. Ensemble of transition state structures for the cis-trans isomerization of N-methylacetamide.

    PubMed

    Mantz, Yves A; Branduardi, Davide; Bussi, Giovanni; Parrinello, Michele

    2009-09-17

    The cis-trans isomerization of N-methylacetamide (NMA), a model peptidic fragment, is studied theoretically in vacuo and in explicit water solvent at 300 K using the metadynamics technique. The computed cis-trans free energy difference is very similar for NMA(g) and NMA(aq), in agreement with experimental measurements of population ratios and theoretical studies at 0 K. By exploiting the flexibility in the definition of a pair of recently introduced collective variables (Branduardi, D.; Gervasio, F. L.; Parrinello, M. J. Chem. Phys. 2007, 126, 054103), an ensemble of transition state structures is generated at finite temperature for both NMA(g) and NMA(aq), as verified by computing committor distribution functions. Ensemble members of NMA(g) are shown to have correlated values of the backbone dihedral angle and a second dihedral angle involving the amide hydrogen atom. The dynamical character of these structures is preserved in the presence of solvent, whose influence on the committor functions can be modeled using effective friction/noise terms. PMID:19694484

  9. AlCl₃-Catalyzed Ring Expansion Cascades of Bicyclic Cyclobutenamides Involving Highly Strained Cis,Trans-Cycloheptadienone Intermediates.

    PubMed

    Wang, Xiao-Na; Krenske, Elizabeth H; Johnston, Ryne C; Houk, K N; Hsung, Richard P

    2015-04-29

    We report the first experimental evidence for the generation of highly strained cis,trans-cycloheptadienones by electrocyclic ring opening of 4,5-fused cyclobutenamides. In the presence of AlCl3, the cyclobutenamides rearrange to [2.2.1]-bicyclic ketones; DFT calculations provide evidence for a mechanism involving torquoselective 4π-electrocyclic ring opening to a cis,trans-cycloheptadienone followed by a Nazarov-like recyclization and a 1,2-alkyl shift. Similarly, 4,6-fused cyclobutenamides undergo AlCl3-catalyzed rearrangements to [3.2.1]-bicyclic ketones through cis,trans-cyclooctadienone intermediates. The products can be further elaborated via facile cascade reactions to give complex tri- and tetracyclic molecules. PMID:25895058

  10. AlCl3-Catalyzed Ring-Expansion Cascades of Bicyclic Cyclobutenamides Involving Highly Strained Cis,Trans-Cycloheptadienone Intermediates

    PubMed Central

    Wang, Xiao-Na; Krenske, Elizabeth H.; Johnston, Ryne C.; Houk, K. N.; Hsung, Richard P.

    2015-01-01

    We report the first experimental evidence for the generation of highly strained cis,trans-cycloheptadienones by electrocyclic ring opening of 4,5-fused cyclobutenamides. In the presence of AlCl3, the cyclobutenamides rearrange to [2.2.1]-bicyclic ketones; DFT calculations provide evidence for a mechanism involving torquoselective 4π-electrocyclic ring opening to a cis,trans-cycloheptadienone followed by a Nazarov-like recyclization and a 1,2-alkyl shift. Similarly, 4,6-fused cyclobutenamides undergo AlCl3-catalyzed rearrangements to [3.2.1]-bicyclic ketones through cis,trans-cyclooctadienone intermediates. The products can be further elaborated via facile cascade reactions to give complex tri- and tetracyclic molecules. PMID:25895058

  11. Cis-trans isomerisation of azobenzenes studied by laser-coupled NMR spectroscopy and DFT calculations.

    PubMed

    Wazzan, Nuha A; Richardson, Patricia R; Jones, Anita C

    2010-07-30

    In a combined experimental and computational study of a group of para-substituted azobenzenes, the effects of substituents and solvent on the kinetics of thermal cis-to-trans isomerisation have been examined and the success of DFT calculations in predicting kinetic parameters assessed. Mono-substituted species are predicted to isomerise by inversion in both non-polar and polar solvent, whereas for push-pull azobenzenes the mechanism is predicted to change from inversion to rotation on going from non-polar to polar solvent. Computed free energies of activation qualitatively reproduce experimental trends but do not quantitatively predict the kinetics of cis-trans isomerisation. The polarisable continuum model of solvation fails to predict the experimentally observed influence of solvent on the entropy of activation.

  12. 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. PMID:27130626

  13. 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.

  14. cis-trans isomerization of carbon double bonds in monounsaturated triacylglycerols via generation of free radicals.

    PubMed

    Tsuzuki, Wakako

    2010-09-01

    We investigated the heat-induced cis/trans isomerization of double bonds in monounsaturated lipids. When triolein (9-cis, 18:1) was heated around 180 degrees C, small amounts of isomerization products were obtained depending on the heating period. The heat-induced isomerization of triolein was considerably suppressed by the addition of different antioxidants or under nitrogen stream, and these additives simultaneously inhibited the thermal oxidation of double bonds in triolein. Therefore, an intermediate of the thermal oxidation reaction might be responsible for the heat-induced isomerization of the double bonds in triolein. The thermodynamics of the heat-induced isomerization of triolein (9-cis, 18:1) and trielaidin (9-trans, 18:1) were investigated using Arrhenius plot. The Arrhenius activation energies of cis double bonds in triolein and trans double bonds in trielaidin were 106 kJ/mol and 137 kJ/mol, respectively. The calculated internal rotational barrier heights of these double bonds were similar to those of the double bond of 2-butene radical and significantly lower than those of non-radicalized double bonds in 2-butene. These results suggest that heat-induced cis/trans isomerization of triolein and trielaidin occurs mainly through the formation of radical species, which are the intermediates produced during thermal oxidation. The activation energy difference between the two forms suggests that trans trielaidin radicals are more stable than cis triolein radicals. The high thermodynamic stability of the trans double bonds in lipid radicals would influence the population of cis and trans isomers in edible oils and contribute to slight accumulation of trans-18:1 isomers during heating or industrial processing.

  15. 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

    2016-08-01

    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. PMID:27434776

  16. Control of carotenoid biosynthesis through a heme-based cis-trans isomerase.

    PubMed

    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

    2015-08-01

    Plants synthesize carotenoids, which are essential for plant development and survival. These metabolites also serve as essential nutrients for human health. The biosynthetic pathway for all plant carotenoids occurs in chloroplasts and other plastids and requires 15-cis-ζ-carotene isomerase (Z-ISO). It was not known whether Z-ISO catalyzes isomerization 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-trans isomerization of the 15-15' carbon-carbon double bond in 9,15,9'-cis-ζ-carotene to produce the substrate required by the subsequent 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 was previously undescribed. As an isomerase, Z-ISO represents a new prototype for heme b proteins and potentially uses a new chemical mechanism.

  17. Conformational Preferences in Small Peptide Models: The Relevance of cis/trans-Conformations.

    PubMed

    Jangra, Harish; Haindl, Michael H; Achrainer, Florian; Hioe, Johnny; Gschwind, Ruth M; Zipse, Hendrik

    2016-09-01

    The accurate description of cis/trans peptide structures is of fundamental relevance for the field of protein modeling and protein structure determination. A comprehensive conformational analysis of dipeptide model Ace-Gly-NMe (1) has been carried out by using a combination of theoretical calculations and experimental ((1) H and (13) C NMR and NOESY) spectroscopic measurements to assess the relevance of cis-peptide conformers. NMR measurements in dimethyl sulfoxide (DMSO) solution and calculations employing a continuum solvation model both point to the extended trans,trans conformer C5_tt as the global minimum. The cis-peptide structures C5_ct and C5_tc, with the N- or C-terminal amide group in cis-conformation, are observed separately and located 13.0±2 kJ mol(-1) higher in energy. This is in close agreement with the theoretical prediction of around 12 kJ mol(-1) in DMSO. The ability of common protein force fields to reproduce the energies of the cis-amide conformers C5_ct and C5_tc in 1 is limited, making these methods unsuitable for the description of cis-peptide structures in protein simulations. PMID:27535479

  18. Analysis of oxidation sensitivity of maleate cis-trans isomerase from Serratia marcescens.

    PubMed

    Hatakeyama, K; Goto, M; Kobayashi, M; Terasawa, M; Yukawa, H

    2000-07-01

    The maleate cis-trans isomerase gene (maiA) from Serratia marcescens IFO3736 was cloned and sequenced. Serratia MaiA has 62.4% amino acid identity with Alcaligenes faecalis IFO13111 MaiA and 64.9% with Bacillus stearothermophilus MI-102 MaiA. All known ten amino acid sequences of MaiA had significant conserved regions containing cysteine residues, which were previously suggested to be involved in an active site of the enzyme. The maiA gene was expressed in Escherichia coli, and expressed products MaiA was purified and characterized. The purified enzyme of strain IFO3736 showed high activity at room temperature and high heat stability. It also showed higher activity in the presence of high concentration of aspartic acid than the enzyme of A. faecalis IFO13111, but it was also sensitive to chemical oxidation. By amino acid composition analysis, cysteine, methionine, and tyrosine residues were suggested to be oxidized to inactivate the enzyme by chemical oxidation. To investigate the mechanism of chemical oxidation of the enzyme, six methionine residues in the conserved regions of S. marcescens MaiA were replaced with cysteine residues by site-directed mutagenesis. The analysis of the constructed mutants suggested that the Met201 residue near the Cys198 residue is involved in the sensitivity of the enzyme to chemical oxidation.

  19. Red- and blue-shifted hydrogen bonds in the cis-trans noncyclic formic acid dimer.

    PubMed

    Zhou, Pan-Pan; Qiu, Wen-Yuan

    2009-08-01

    The cis-trans noncyclic formic acid dimer was studied by means of MP2 method with 6-31G(d,p), 6-31+G(d,p) and 6-311+G(d,p) basis sets. It exhibits simultaneously red-shifted O-H...O and blue-shifted C-H...O hydrogen bonds. AIM and NBO analyses are performed at the MP2/6-31+G(d,p) level to explore their properties and origins. AIM analysis provides the evidence that the O-H bond becomes weaker and the C-H bond becomes stronger upon the hydrogen bond formations. Intermolecular and intramolecular hyperconjugations have important influence on the electron densities in the X-H (X = O, C) sigma bonding orbital and its sigma* antibonding orbital. The electron densities in the two orbitals are closely connected with the X-H (X = O, C) bond length, and they are used to quantitatively estimate the bond length variation. The larger amount of charge transfer in the red-shifted O-H...O hydrogen bond is due to its favorable H...O electron channel, whereas the H...O electron channel in the blue-shifted C-H...O hydrogen bond is weaker. Structural reorganization effects shorten the C-H bond by approximately 30% when compared to the C-H bond contraction upon the dimerization. Strikingly, it leads to a small elongation and a slight red shift of the O-H bond. Both rehybridization and repolarization result in the X-H (X = O, C) bond contraction, but their effects on the O-H bond do not hold a dominant position. The hydrogen-bonding processes go through the electrostatic attractions, van der Waals interactions, charge-transfer interactions, hydrogen-bonding interactions and electrostatic repulsions. Electrostatic attractions are of great importance on the origin of the red-shifted O-H...O hydrogen bond, especially the strong H(delta+)...O(delta-) attraction. For the blue-shifted C-H...O hydrogen bond, the considerable nucleus-nucleus repulsion between H and O atoms caused by the strong electrostatic attraction between C and O atoms is a possible reason for the C-H bond contraction and

  20. 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

  1. A mechanistic hypothesis for the cytochrome P450-catalyzed cis-trans isomerization of 4-hydroxytamoxifen: an unusual redox reaction.

    PubMed

    Gao, Li; Tu, Yaoquan; Wegman, Pia; Wingren, Sten; Eriksson, Leif A

    2011-09-26

    We provide a detailed description of the cis-trans isomerization of 4-hydroxytamoxifen/endoxifen catalyzed by several isoforms from the cytochrome P450 (CYP) superfamily, including CYP1B1, CYP2B6, and CYP2C19. We show that the reactions mainly involve redox processes catalyzed by CYP. DFT calculation results strongly suggest that the isomerization occurs via a cationic intermediate. The cationic cis-isomer is more than 3 kcal/mol more stable than the trans form, resulting in an easier conversion from trans-to-cis than cis-to-trans. The cis-trans isomerization is a rarely reported CYP reaction and is ascribed to the lack of a second abstractable proton on the ethenyl group of the triarylvinyl class of substrates. The cationic intermediates thus formed instead of the stable dehydrogenation products allow for isomerization to occur. As a comparison, the reactions for the tamoxifen derivatives are compared to those of other substrates, 4-hydroxyacetanilide and raloxifene, for which the stable dehydrogenation products are formed. PMID:21870861

  2. Studies on the s-cis-trans isomerism for some furan derivatives through IR and NMR spectroscopies and theoretical calculations.

    PubMed

    Rittner, Roberto; Ducati, Lucas C; Tormena, Cláudio F; Cormanich, Rodrigo A; Fiorin, Barbara C; Braga, Carolyne B; Abraham, Raymond J

    2013-02-15

    The s-cis-trans isomerism of two furan derivatives [2-acetyl- (AF) and 2-acetyl-5-methylfuran, (AMF)] was analyzed, using data from the deconvolution of their carbonyl absorption band in two solvents (CH(2)Cl(2) and CH(3)CN). These infrared data showed that the O,O-trans conformers predominate in the less polar solvent (CH(2)Cl(2)), but these equilibria change in a more polar solvent (CH(3)CN) leading to a slight predominance of the O,O-cis conformers, in agreement with the theoretical calculations. The later results were obtained using B3LYP-IEFPCM/6-31++g(3df,3p) level of theory, which taking into account the solvent effects at IEFPCM (Integral Equation Formalism Polarizable Continuum Model). Low temperature (13)CNMR spectra in CD(2)Cl(2) (ca. -75 °C) showed pairs of signals for each carbon, due to the known high energy barrier for the cis-trans interconversion leading to a large predominance of the trans isomers, which decreases in acetone-d(6). This was confirmed by their (1)HNMR spectra at the same temperatures. Moreover, despite the larger hyperconjugative interactions for the O,O-cis isomers, obtained from NBO data, these isomers are destabilized by the their Lewis energy.

  3. cis/trans isomerization of unsaturated fatty acids as possible control mechanism of membrane fluidity in Pseudomonas putida P8.

    PubMed

    Loffeld, B; Keweloh, H

    1996-08-01

    Exponentially growing cells of Pseudomonas putida had an increased ratio of saturated to unsaturated fatty acids in response to increased growth temperatures. Resting cells in which fatty acid biosynthesis was stopped reacted to a thermal increase by converting cis-monounsaturated fatty acids to trans isomers. cis/trans Isomerization of up to 60% of the unsaturated fatty acids was also activated by alcohols of different chain length. Their effective concentrations apparently depended on the lipophilic character of the alcohols. Also, a salt shock caused by the addition of NaCl resulted in the production of trans fatty acids. However, cells that were adapted to growth media of high osmolarity synthesized cyclopropane fatty acids instead of trans fatty acids. Activity of cis/trans-isomerase was dependent on the growth phase and was significantly higher during logarithmic growth than during the stationary phase. The results of this study agree with the hypothesis that the isomerization of cis into trans unsaturated fatty acids is an emergency action of cells of P. putida to adapt membrane fluidity to drastic changes of environmental conditions.

  4. Solvent-Triggered Cis/Trans Isomerism in Cobalt Dioxolene Chemistry: Distinguishing Effects of Packing on Valence Tautomerism.

    PubMed

    Panja, Anangamohan; Jana, Narayan Ch; Bauzá, Antonio; Frontera, Antonio; Mathonière, Corine

    2016-09-01

    In this article, the synthesis and X-ray crystal structures of two cis/trans isomers of valence tautomeric (VT) cobalt dioxolene compounds are reported. The cis isomer (1) was isolated from the polar protic methanol solvent as a kinetic product, whereas the less polar nonprotic solvent acetone yielded the trans isomer (2). It should be noted that, although some coordination polymers involving cobalt bis(dioxolene) with the cis disposition are known for bridging ancillary ligands, such an arrangement is unprecedented for mononuclear compounds. A careful study of intermocular interactions revealed that the methanol solvent does not have much influence on the crystal growth in 1, whereas acetone forms strong halogen-bonding interactions that are crucial in the solid-state architecture of 2. This behavior can likely be used in crystal engineering to design new organic-inorganic hybrid materials. The energy difference between the two isomers was examined using DFT calculations, confirming that the trans form is in the thermodynamic state whereas the cis isomer is a kinetic product that can be converted into the trans isomer with time. Finally, both isomers exhibit solvent loss at elevated temperatures that is accompanied by a change in magnetic properties, associated with an irreversible valence tautomerism. Our results highlight the crucial role of the solvents for the isolation of cis/trans isomers in cobalt dioxolene chemistry, as well as the distinguishing effects of intermolecular forces and the solid-state packing on VT behavior. PMID:27557848

  5. Solvent-Triggered Cis/Trans Isomerism in Cobalt Dioxolene Chemistry: Distinguishing Effects of Packing on Valence Tautomerism.

    PubMed

    Panja, Anangamohan; Jana, Narayan Ch; Bauzá, Antonio; Frontera, Antonio; Mathonière, Corine

    2016-09-01

    In this article, the synthesis and X-ray crystal structures of two cis/trans isomers of valence tautomeric (VT) cobalt dioxolene compounds are reported. The cis isomer (1) was isolated from the polar protic methanol solvent as a kinetic product, whereas the less polar nonprotic solvent acetone yielded the trans isomer (2). It should be noted that, although some coordination polymers involving cobalt bis(dioxolene) with the cis disposition are known for bridging ancillary ligands, such an arrangement is unprecedented for mononuclear compounds. A careful study of intermocular interactions revealed that the methanol solvent does not have much influence on the crystal growth in 1, whereas acetone forms strong halogen-bonding interactions that are crucial in the solid-state architecture of 2. This behavior can likely be used in crystal engineering to design new organic-inorganic hybrid materials. The energy difference between the two isomers was examined using DFT calculations, confirming that the trans form is in the thermodynamic state whereas the cis isomer is a kinetic product that can be converted into the trans isomer with time. Finally, both isomers exhibit solvent loss at elevated temperatures that is accompanied by a change in magnetic properties, associated with an irreversible valence tautomerism. Our results highlight the crucial role of the solvents for the isolation of cis/trans isomers in cobalt dioxolene chemistry, as well as the distinguishing effects of intermolecular forces and the solid-state packing on VT behavior.

  6. Peptidylprolyl cis/trans isomerase activity and molecular evolution of vertebrate Cyclophilin A.

    PubMed

    Liqian, Ren; Wei, Liu; Wenbo, Li; Wenjun, Liu; Lei, Sun

    2016-08-01

    Peptidylprolyl isomerases (PPIase) cyclophilin A (CypA, encoded by PPIA) is a typical member of the Cyclophilin family and is involved in protein folding/translocation, signal transduction, inflammation, immune system regulation, apoptosis and virus replication. In the present study, we investigated the PPIase activity and genetic variation of vertebrate CypA. According to the GenBank reference sequences, vertebrate PPIA genes were cloned, among which the bat (Myotis davidi) and duck (Anas platyrhynchos) PPIA genes were reported for the first time. Then PPIA genes were sub-cloned into the expression vector pGEX-6p-1 and expressed in Escherichia coli. Recombinant CypA proteins were purified by using sepharose 4B affinity chromatography and the GST tag was cleaved, followed by gel filtration. The PPIase activity assay indicated that there was no significant difference in the catalytic activity of prolyl peptide bond isomerization among 12 different vertebrate CypA proteins. In addition, the genetic variation and molecular evolution analysis showed that these vertebrate CypA proteins had the same CsA binding site and the PPIase active sites. Furthermore, the predicted structure and gene localization were remarkable conserved. Our data suggested that the important residues of CypA were highly conserved, which is crucial for its PPIase activity and cellular functions. PMID:27531612

  7. CIS-TRANS ISOMERS OF VITAMIN A AND RETINENE IN THE RHODOPSIN SYSTEM

    PubMed Central

    Hubbard, Ruth; Wald, George

    1952-01-01

    Vitamin A and retinene, the carotenoid precursors of rhodopsin, occur in a variety of molecular shapes, cis-trans isomers of one another. For the synthesis of rhodopsin a specific cis isomer of vitamin A is needed. Ordinary crystalline vitamin A, as also the commercial synthetic product, both primarily all-trans, are ineffective. The main site of isomer specificity is the coupling of retinene with opsin. It is this reaction that requires a specific cis isomer of retinene. The oxidation of vitamin A to retinene by the alcohol dehydrogenase-cozymase system displays only a low degree of isomer specificity. Five isomers of retinene have been isolated in crystalline condition: all-trans; three apparently mono-cis forms, neoretinenes a and b and isoretinene a; and one apparently di-cis isomer, isoretinene b. Neoretinenes a and b were first isolated in our laboratory, and isoretinenes a and b in the Organic Research Laboratory of Distillation Products Industries. Each of these substances is converted to an equilibrium mixture of stereoisomers on simple exposure to light. For this reaction, light is required which retinene can absorb; i.e., blue, violet, or ultraviolet light. Yellow, orange, or red light has little effect. The single geometrical isomers of retinene must therefore be protected from low wave length radiation if their isomerization is to be avoided. By incubation with opsin in the dark, the capacity of each of the retinene isomers to synthesize rhodopsin was examined. All-trans retinene and neoretinene a are inactive. Neoretinene b yields rhodopsin indistinguishable from that extracted from the dark-adapted retina (λmax· 500 mµ). Isoretinene a yields a similar light-sensitive pigment, isorhodopsin, the absorption spectrum of which is displaced toward shorter wave lengths (λmax· 487 mµ). Isoretinene b appears to be inactive, but isomerizes preferentially to isoretinene a, which in the presence of opsin is removed to form isorhodopsin before the

  8. 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

    2013-06-01

    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

  9. 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

  10. 1H-13C HSQC NMR spectroscopy for estimating procyanidin/prodelphinidin and cis/trans flavan-3-ol ratios of condensed tannin samples: correlation with thiolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  11. 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…

  12. Automated determination of fatty acid methyl ester and cis/trans methyl ester composition of fats and oils.

    PubMed

    de Koning, S; van der Meer, B; Alkema, G; Janssen, H G; Brinkman, U A

    2001-07-13

    The determination of the fatty acid composition (as methyl esters, FAMEs) of fats and oils and their cis/trans (CTME) distribution requires a simple, but manual and time-consuming sample preparation. The so-called BF3 method is often the preferred procedure. Because FAME/CTME analyses are encountered very frequently in the food industry, an automated, robot-based alternative is proposed which uses the sodium methylate procedure. After sample weighing and the (manual) addition of heptane (2 min), a XYZ robotic autosampler is used for all remaining work, which includes reagent addition, agitation, sample settling and the final injection into the gas chromatograph (10 min). The performance of the sodium methylate and BF3 methods are compared by analysing some 30 oil and fat samples. The novel procedure is much faster (less than 15 min versus ca. 1 h) and manual sample handling is drastically decreased. The experimental results obtained with the two methods frequently are the same, while small differences can be explained by (known) differences of the two methods in the conversion of minor oil/fat constituents, such as free fatty acids, wax esters and sterol esters. In case of FAME analyses, a hot injection is to be preferred over a cold injection. The RSDs of the peak areas were 1.5% for the major fatty acids to 11% for peaks that were just above the noise level. The detection limit were approximately 0.03%.

  13. Thermal cis-trans isomerization of cis,cis-3,7-decadiene - A model for cis-1,4-polybutadiene

    NASA Technical Reports Server (NTRS)

    Golub, M. A.; Lee, W. M.

    1983-01-01

    The thermal cis-trans isomerization of cis,cis-3,7-decadiene (DD), a model compound for cis-PBD, is reported. It is demonstrated that the rather low E for the polyalkenamer isomerizations compared with that for the 2-olefins is not an artifact of the solid polymer structures, but rather is characteristic of both small and large molecules possessing pairs of nonconjugated vinylene double bonds in a suitable arrangement.

  14. 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.

  15. Differential Effects of Collagen Prolyl 3-Hydroxylation on Skeletal Tissues

    PubMed Central

    Homan, Erica P.; Lietman, Caressa; Grafe, Ingo; Lennington, Jennifer; Morello, Roy; Napierala, Dobrawa; Jiang, Ming-Ming; Munivez, Elda M.; Dawson, Brian; Bertin, Terry K.; Chen, Yuqing; Lua, Rhonald; Lichtarge, Olivier; Hicks, John; Weis, Mary Ann; Eyre, David; Lee, Brendan H. L.

    2014-01-01

    Mutations in the genes encoding cartilage associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1 encoded by LEPRE1) were the first identified causes of recessive Osteogenesis Imperfecta (OI). These proteins, together with cyclophilin B (encoded by PPIB), form a complex that 3-hydroxylates a single proline residue on the α1(I) chain (Pro986) and has cis/trans isomerase (PPIase) activity essential for proper collagen folding. Recent data suggest that prolyl 3-hydroxylation of Pro986 is not required for the structural stability of collagen; however, the absence of this post-translational modification may disrupt protein-protein interactions integral for proper collagen folding and lead to collagen over-modification. P3H1 and CRTAP stabilize each other and absence of one results in degradation of the other. Hence, hypomorphic or loss of function mutations of either gene cause loss of the whole complex and its associated functions. The relative contribution of losing this complex's 3-hydroxylation versus PPIase and collagen chaperone activities to the phenotype of recessive OI is unknown. To distinguish between these functions, we generated knock-in mice carrying a single amino acid substitution in the catalytic site of P3h1 (Lepre1H662A). This substitution abolished P3h1 activity but retained ability to form a complex with Crtap and thus the collagen chaperone function. Knock-in mice showed absence of prolyl 3-hydroxylation at Pro986 of the α1(I) and α1(II) collagen chains but no significant over-modification at other collagen residues. They were normal in appearance, had no growth defects and normal cartilage growth plate histology but showed decreased trabecular bone mass. This new mouse model recapitulates elements of the bone phenotype of OI but not the cartilage and growth phenotypes caused by loss of the prolyl 3-hydroxylation complex. Our observations suggest differential tissue consequences due to selective inactivation of P3H1 hydroxylase activity

  16. 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.

  17. Secreted Cyclophilin A, a Peptidylprolyl cis-trans Isomerase, Mediates Matrix Assembly of Hensin, a Protein Implicated in Epithelial Differentiation*S⃞

    PubMed Central

    Peng, Hu; Vijayakumar, Soundarapandian; Schiene-Fischer, Cordelia; Li, Hui; Purkerson, Jeffrey M.; Malesevic, Miroslav; Liebscher, Jürgen; Al-Awqati, Qais; Schwartz, George J.

    2009-01-01

    Hensin is a rabbit ortholog of DMBT1, a multifunctional, multidomain protein implicated in the regulation of epithelial differentiation, innate immunity, and tumorigenesis. Hensin in the extracellular matrix (ECM) induced morphological changes characteristic of terminal differentiation in a clonal cell line (clone C) of rabbit kidney intercalated cells. Although hensin is secreted in monomeric and various oligomeric forms, only the polymerized ECM form is able to induce these phenotypic changes. Here we report that hensin secretion and matrix assembly were inhibited by the peptidylprolyl cis-trans isomerase (PPIase) inhibitors cyclosporin A (CsA) and a derivative of cyclosporin A with modifications in the d-Ser side chain (Cs9) but not by the calcineurin pathway inhibitor FK506. PPIase inhibition led to failure of hensin polymerization in the medium and ECM, plus the loss of apical cytoskeleton, apical microvilli, and the columnar epithelial shape of clone C cells. Cyclophilin A was produced and secreted into the media to a much greater extent than cyclophilins B and C. Our results also identified the direct CsA-sensitive interaction of cyclophilin A with hensin, suggesting that cyclophilin A is the PPIase that mediates the polymerization and matrix assembly of hensin. These results are significant because this is the first time a direct role of peptidylprolyl cis-trans isomerase activity has been implicated in the process of epithelial differentiation. PMID:19112104

  18. Details of the ultrafast DNA photo-cross-linking reaction of 3-cyanovinylcarbazole nucleoside: cis-trans isomeric effect and the application for SNP-based genotyping.

    PubMed

    Fujimoto, Kenzo; Yamada, Asuka; Yoshimura, Yoshinaga; Tsukaguchi, Tadashi; Sakamoto, Takashi

    2013-10-30

    To clarify the cis-trans isomeric effect on the ultrafast DNA photo-cross-linking reaction of 3-cyanovinylcarbazole nucleoside ((CNV)K) in the DNA duplex, which gives a single photodimer on the reversed-phase HPLC chromatogram, the kinetics of the cis-trans photoisomerization of (CNV)K in double-stranded DNA was evaluated. Since the photoisomerization rate constant for cis to trans isomerization in double-stranded DNA was significantly larger than that for trans to cis isomerization, and the thermodynamic stability of the trans isomer was higher than that of the cis isomer, it was strongly suggested that the trans isomer of (CNV)K is a reactive species of the photo-cross-linking reaction. (1)H-(1)H NOESY analysis of the photoadduct consisting of (CNV)K and T also supported the trans-mediated photo-cross-linking reaction of (CNV)K. By using this ultrafast photo-cross-linking reaction for the molecular beacon-based SNPs typing, four individual Japanese rice strains were clearly distinguishable with simple photoirradiation and fluorescence imaging using double-stranded target DNAs.

  19. 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

  20. Prolyl Isomerization as a Molecular Memory in the Allosteric Regulation of the Signal Adapter Protein c-CrkII

    PubMed Central

    Schmidpeter, Philipp A. M.; Schmid, Franz X.

    2015-01-01

    c-CrkII is a central signal adapter protein. A domain opening/closing reaction between its N- and C-terminal Src homology 3 domains (SH3N and SH3C, respectively) controls signal propagation from upstream tyrosine kinases to downstream targets. In chicken but not in human c-CrkII, opening/closing is coupled with cis/trans isomerization at Pro-238 in SH3C. Here, we used advanced double-mixing experiments and kinetic simulations to uncover dynamic domain interactions in c-CrkII and to elucidate how they are linked with cis/trans isomerization and how this regulates substrate binding to SH3N. Pro-238 trans → cis isomerization is not a simple on/off switch but converts chicken c-CrkII from a high affinity to a low affinity form. We present a double-box model that describes c-CrkII as an allosteric system consisting of an open, high affinity R state and a closed, low affinity T state. Coupling of the T-R transition with an intrinsically slow prolyl isomerization provides c-CrkII with a kinetic memory and possibly functions as a molecular attenuator during signal transduction. PMID:25488658

  1. Conformations of Prolyl-Peptide Bonds in the Bradykinin 1-5 Fragment in Solution and in the Gas Phase.

    PubMed

    Voronina, Liudmila; Masson, Antoine; Kamrath, Michael; Schubert, Franziska; Clemmer, David; Baldauf, Carsten; Rizzo, Thomas

    2016-07-27

    The dynamic nature of intrinsically disordered peptides makes them a challenge to characterize by solution-phase techniques. In order to gain insight into the relation between the disordered state and the environment, we explore the conformational space of the N-terminal 1-5 fragment of bradykinin (BK[1-5](2+)) in the gas phase by combining drift tube ion mobility, cold-ion spectroscopy, and first-principles simulations. The ion-mobility distribution of BK[1-5](2+) consists of two well-separated peaks. We demonstrate that the conformations within the peak with larger cross-section are kinetically trapped, while the more compact peak contains low-energy structures. This is a result of cis-trans isomerization of the two prolyl-peptide bonds in BK[1-5](2+). Density-functional theory calculations reveal that the compact structures have two very different geometries with cis-trans and trans-cis backbone conformations. Using the experimental CCSs to guide the conformational search, we find that the kinetically trapped species have a trans-trans configuration. This is consistent with NMR measurements performed in a solution, which show that 82% of the molecules adopt a trans-trans configuration and behave as a random coil. PMID:27366919

  2. Different shapes of spherical vaterite by photo-induced cis?trans isomerization of an azobenzene-containing polymer in a mixture of dimethyl sulfoxide and water

    NASA Astrophysics Data System (ADS)

    Keum, Dong-Ki; Na, Hai-Sub; Naka, Kensuke; Chujo, Yoshiki

    2004-10-01

    We studied the crystallization of CaCO3 by the photoisomerization of azobenzene groups in poly[1-[4-[3-carboxy-4-hydroxyphenylazobenzenesulfonamido]-1,2-ethanediyl, sodium salt] (PAZO) in a mixture of dimethyl sulfoxide and water at 30 °C. The products were characterized by scanning electron microscopy (SEM), FT-IR, and powder X-ray diffraction (XRD) analysis. We observed that the different shapes of spherical vaterite particles were produced by the changes of configuration and polarity of the azobenzene groups in the polymer which resulted from photo-induced isomerization. The results indicate that the nucleation of primary particles of CaCO3 was inhibited by in situ photo-induced cis-trans isomerization of PAZO. Therefore, we suggest that the shapes of the spherical vaterite can be effectively modified by photoisomerization of the azobenzene groups in the polymer at the initial stage of CaCO3 crystallization.

  3. 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

  4. Positron annihilation and relaxation dynamics from dielectric spectroscopy and nuclear magnetic resonance: Cis-trans-1,4-poly(butadiene)

    NASA Astrophysics Data System (ADS)

    Bartoš, J.; Šauša, O.; Schwartz, G. A.; Alegría, A.; Alberdi, J. M.; Arbe, A.; Krištiak, J.; Colmenero, J.

    2011-04-01

    We report a joint analysis of positron annihilation lifetime spectroscopy (PALS), dielectric spectroscopy (BDS), and nuclear magnetic resonance (NMR) on cis-trans-1,4-poly(butadiene) (c-t-1,4-PBD). Phenomenological analysis of the orthopositronium lifetime τ3 - T dependence by linear fitting reveals four characteristic PALS temperatures: T_{b1} ^G = 0{.63}T_g^{PALS}, T_g^{PALS}, T_{b1} ^L = 1.22T_g^{PALS}, and T_{b2} ^L = 1.52T_g^{PALS}. Slight bend effects in the glassy and supercooled liquid states are related to the fast or slow secondary β process, from neutron scattering, respectively, the latter being connected with the trans-isomers. In addition, the first bend effect in the supercooled liquid coincides with a deviation of the slow effective secondary βeff relaxation related to the cis-isomers from low-T Arrhenius behavior to non-Arrhenius one and correlates with the onset of the primary α process from BDS. The second plateau effect in the liquid state occurs when τ3 becomes commensurable with the structural relaxation time τα(Tb2). It is also approximately related to its crossover from non-Arrhenius to Arrhenius regime in the combined BDS and NMR data. Finally, the combined BDS and NMR structural relaxation data, when analyzed in terms of the two-order parameter (TOP) model, suggest the influence of solidlike domains on both the annihilation behavior and the local and segmental chain mobility in the supercooled liquid. All these findings indicate the influence of the dynamic heterogeneity in both the primary and secondary relaxations due to the cis-trans isomerism in c-t-1,4-PBD and their impact into the PALS response.

  5. 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.

  6. 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.

  7. Preparative separation and purification of four cis-trans isomers of coumaroylspermidine analogs from safflower by high-speed counter-current chromatography.

    PubMed

    Li, Wen-Cong; Wang, Xiao-Yan; Lin, Peng-Cheng; Hu, Na; Zhang, Qiu-Long; Suo, You-Rui; Ding, Chen-Xu

    2013-11-01

    High-speed counter-current chromatography (HSCCC) was successfully applied for the first time to isolate and purify four cis-trans isomers of coumaroylspermidine analogs from Safflower. HSCCC separation was achieved with a two-phase solvent system composed of chloroform-methanol-water (1:1:1, v/v/v) with the upper phase as the mobile phase. In a single run, a total of 1.3mg of N(1), N(5), N(10)-(E)-tri-p-coumaroylspermidine (EEE), 4.4mg of N(1)(E)-N(5)-(Z)-N(10)-(E)-tri-p-coumaroylspermidine (EZE), 7.2mg of N(1)(Z)-N(5)-(Z)-N(10)-(E)-tri-p-coumaroylspermidine (ZZE), and 11.5mg of N(1),N(5),N(10)-(Z)-tri-p-coumaroylspermidine (ZZZ) were obtained from 100mg of crude sample. High Performance Liquid Chromatography (HPLC) analysis showed that the purities of these four components are 95.5%, 98.1%, 97.5% and 96.2%, respectively. The chemical structures were identified by ESI-MS, (1)H NMR and (13)C NMR.

  8. 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. PMID:24990165

  9. 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.

  10. Preparative separation and purification of four cis-trans isomers of coumaroylspermidine analogs from safflower by high-speed counter-current chromatography.

    PubMed

    Li, Wen-Cong; Wang, Xiao-Yan; Lin, Peng-Cheng; Hu, Na; Zhang, Qiu-Long; Suo, You-Rui; Ding, Chen-Xu

    2013-11-01

    High-speed counter-current chromatography (HSCCC) was successfully applied for the first time to isolate and purify four cis-trans isomers of coumaroylspermidine analogs from Safflower. HSCCC separation was achieved with a two-phase solvent system composed of chloroform-methanol-water (1:1:1, v/v/v) with the upper phase as the mobile phase. In a single run, a total of 1.3mg of N(1), N(5), N(10)-(E)-tri-p-coumaroylspermidine (EEE), 4.4mg of N(1)(E)-N(5)-(Z)-N(10)-(E)-tri-p-coumaroylspermidine (EZE), 7.2mg of N(1)(Z)-N(5)-(Z)-N(10)-(E)-tri-p-coumaroylspermidine (ZZE), and 11.5mg of N(1),N(5),N(10)-(Z)-tri-p-coumaroylspermidine (ZZZ) were obtained from 100mg of crude sample. High Performance Liquid Chromatography (HPLC) analysis showed that the purities of these four components are 95.5%, 98.1%, 97.5% and 96.2%, respectively. The chemical structures were identified by ESI-MS, (1)H NMR and (13)C NMR. PMID:24055753

  11. High-performance liquid chromatography separation of cis-trans anthocyanin isomers from wild Lycium ruthenicum Murr. employing a mixed-mode reversed-phase/strong anion-exchange stationary phase.

    PubMed

    Jin, Hongli; Liu, Yanfang; Guo, Zhimou; Yang, Fan; Wang, Jixia; Li, Xiaolong; Peng, Xiaojun; Liang, Xinmiao

    2015-01-21

    The cis-trans isomerism is a common phenomenon for acylated anthocyanins. Nevertheless, few studies reported effective methods for the preparation of isomeric anthocyanins from natural products. In this work, a high-performance liquid chromatography (HPLC) method was developed to efficiently purify anthocyanin isomers from Lycium ruthenicum Murr. based on a mixed-mode reversed-phase/strong anion-exchange column (named XCharge C8SAX). Four commercially available columns were evaluated with a pair of isomeric anthocyanins, and the results demonstrated that the XCharge C8SAX column exhibited improved selectivity and column efficiency for the isomers. The chromatographic parameters, including pH, organic content, and ionic strength, were investigated. Optimal separation quality for the anthocyanin isomers was achieved on the XCharge C8SAX column. Six pure anthocyanins, including two pairs of cis-trans isomeric anthocyanins with one new anthocyanin, were purified from L. ruthenicum and identified. All of the results indicated that this method is an effective way to separate anthocyanins, especially for cis-trans isomers. PMID:25539032

  12. High-performance liquid chromatography separation of cis-trans anthocyanin isomers from wild Lycium ruthenicum Murr. employing a mixed-mode reversed-phase/strong anion-exchange stationary phase.

    PubMed

    Jin, Hongli; Liu, Yanfang; Guo, Zhimou; Yang, Fan; Wang, Jixia; Li, Xiaolong; Peng, Xiaojun; Liang, Xinmiao

    2015-01-21

    The cis-trans isomerism is a common phenomenon for acylated anthocyanins. Nevertheless, few studies reported effective methods for the preparation of isomeric anthocyanins from natural products. In this work, a high-performance liquid chromatography (HPLC) method was developed to efficiently purify anthocyanin isomers from Lycium ruthenicum Murr. based on a mixed-mode reversed-phase/strong anion-exchange column (named XCharge C8SAX). Four commercially available columns were evaluated with a pair of isomeric anthocyanins, and the results demonstrated that the XCharge C8SAX column exhibited improved selectivity and column efficiency for the isomers. The chromatographic parameters, including pH, organic content, and ionic strength, were investigated. Optimal separation quality for the anthocyanin isomers was achieved on the XCharge C8SAX column. Six pure anthocyanins, including two pairs of cis-trans isomeric anthocyanins with one new anthocyanin, were purified from L. ruthenicum and identified. All of the results indicated that this method is an effective way to separate anthocyanins, especially for cis-trans isomers.

  13. 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. PMID:27327214

  14. 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

    2015-09-01

    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

  15. 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.

  16. Biochemical characterization and selective inhibition of β-carotene cis-trans isomerase D27 and carotenoid cleavage dioxygenase CCD8 on the strigolactone biosynthetic pathway.

    PubMed

    Harrison, Peter J; Newgas, Sophie A; Descombes, Flora; Shepherd, Sarah A; Thompson, Andrew J; Bugg, Timothy D H

    2015-10-01

    The first three enzymatic steps of the strigolactone biosynthetic pathway catalysed by β-carotene cis-trans isomerase Dwarf27 (D27) from Oryza sativa and carotenoid cleavage dioxygenases CCD7 and CCD8 from Arabidopsis thaliana have been reconstituted in vitro, and kinetic assays have been developed for each enzyme, in order to develop selective enzyme inhibitors. Recombinant OsD27 shows a UV-visible λmax at 422 nm and is inactivated by silver(I) acetate, consistent with the presence of an iron-sulfur cluster that is used in catalysis. OsD27 and AtCCD7 are not inhibited by hydroxamic acids that cause shoot branching in planta, but OsD27 is partially inhibited by terpene-like hydroxamic acids. The reaction catalysed by AtCCD8 is shown to be a two-step kinetic mechanism using pre-steady-state kinetic analysis. Kinetic evidence is presented for acid-base catalysis in the CCD8 catalytic cycle and the existence of an essential cysteine residue in the CCD8 active site. AtCCD8 is inhibited in a time-dependent fashion by hydroxamic acids D2, D4, D5 and D6 (> 95% inhibition at 100 μm) that cause a shoot branching phenotype in A. thaliana, and selective inhibition of CCD8 is observed using hydroxamic acids D13H and D15 (82%, 71% inhibition at 10 μm). The enzyme inhibition data imply that the biochemical basis of the shoot branching phenotype is due to inhibition of CCD8.

  17. Plant carotene cis-trans isomerase CRTISO: a new member of the FAD(RED)-dependent flavoproteins catalyzing non-redox reactions.

    PubMed

    Yu, Qiuju; Ghisla, Sandro; Hirschberg, Joseph; Mann, Varda; Beyer, Peter

    2011-03-11

    The carotene cis-trans isomerase CRTISO is a constituent of the carotene desaturation pathway as evolved in cyanobacteria and prevailing in plants, in which a tetra-cis-lycopene species, termed prolycopene, is formed. CRTISO, an evolutionary descendant of the bacterial carotene desaturase CRTI, catalyzes the cis-to-trans isomerization reactions leading to all-trans-lycopene, the substrate for the subsequent lycopene cyclization to form all-trans-α/β-carotene. CRTISO and CRTI share a dinucleotide binding motif at the N terminus. Here we report that this site is occupied by FAD in CRTISO. The reduced form of this cofactor catalyzes a reaction not involving net redox changes. Results obtained with C(1)- and C(5)-deaza-FAD suggest mechanistic similarities with type II isopentenyl diphosphate: dimethylallyl diphosphate isomerase (IDI-2). CRTISO, together with lycopene cyclase CRTY and IDI-2, thus represents the third enzyme in isoprenoid metabolism belonging to the class of non-redox enzymes depending on reduced flavin for activity. The regional specificity and the kinetics of the isomerization reaction were investigated in vitro using purified enzyme and biphasic liposome-based systems carrying specific cis-configured lycopene species as substrates. The reaction proceeded from cis to trans, recognizing half-sides of the symmetrical prolycopene and was accompanied by one trans-to-cis isomerization step specific for the C(5)-C(6) double bond. Rice lycopene β-cyclase (OsLCY-b), when additionally introduced into the biphasic in vitro system used, was found to be stereospecific for all-trans-lycopene and allowed the CRTISO reaction to proceed toward completion by modifying the thermodynamics of the overall reaction. PMID:21209101

  18. Biochemical characterization and selective inhibition of β-carotene cis-trans isomerase D27 and carotenoid cleavage dioxygenase CCD8 on the strigolactone biosynthetic pathway.

    PubMed

    Harrison, Peter J; Newgas, Sophie A; Descombes, Flora; Shepherd, Sarah A; Thompson, Andrew J; Bugg, Timothy D H

    2015-10-01

    The first three enzymatic steps of the strigolactone biosynthetic pathway catalysed by β-carotene cis-trans isomerase Dwarf27 (D27) from Oryza sativa and carotenoid cleavage dioxygenases CCD7 and CCD8 from Arabidopsis thaliana have been reconstituted in vitro, and kinetic assays have been developed for each enzyme, in order to develop selective enzyme inhibitors. Recombinant OsD27 shows a UV-visible λmax at 422 nm and is inactivated by silver(I) acetate, consistent with the presence of an iron-sulfur cluster that is used in catalysis. OsD27 and AtCCD7 are not inhibited by hydroxamic acids that cause shoot branching in planta, but OsD27 is partially inhibited by terpene-like hydroxamic acids. The reaction catalysed by AtCCD8 is shown to be a two-step kinetic mechanism using pre-steady-state kinetic analysis. Kinetic evidence is presented for acid-base catalysis in the CCD8 catalytic cycle and the existence of an essential cysteine residue in the CCD8 active site. AtCCD8 is inhibited in a time-dependent fashion by hydroxamic acids D2, D4, D5 and D6 (> 95% inhibition at 100 μm) that cause a shoot branching phenotype in A. thaliana, and selective inhibition of CCD8 is observed using hydroxamic acids D13H and D15 (82%, 71% inhibition at 10 μm). The enzyme inhibition data imply that the biochemical basis of the shoot branching phenotype is due to inhibition of CCD8. PMID:26257333

  19. Investigation of the binding of cis/trans-[MCl4(1H-indazole)(NO)](-) (M = Ru, Os) complexes to human serum albumin.

    PubMed

    Dömötör, Orsolya; Rathgeb, Anna; Kuhn, Paul-Steffen; Popović-Bijelić, Ana; Bačić, Goran; Enyedy, Eva Anna; Arion, Vladimir B

    2016-06-01

    Overall binding affinity of sodium or indazolium cis/trans-[MCl4(1H-indazole)(NO)] (M = Ru, Os) complexes towards human serum albumin (HSA) and high molecular mass components of the blood serum was monitored by ultrafiltration. HSA was found to be mainly responsible for the binding of the studied ruthenium and osmium complexes. In other words, this protein can provide a depot for the compounds and can affect their biodistribution and transport processes. In order to elucidate the HSA binding sites tryptophan fluorescence quenching studies and displacement reactions with the established site markers warfarin and dansylglycine were performed. Conditional stability constants for the binding to sites I and II on HSA were computed showing that the studied ruthenium and osmium complexes are able to bind into both sites with moderately strong affinity (logK' = 4.4-5.1). Site I is slightly more favored over site II for all complexes. No significant differences in the HSA binding properties were found for these metal complexes demonstrating negligible influence of the type of counterion (sodium vs indazolium), the metal ion center identity (Ru vs. Os) or the position of the nitrosyl group on the binding event. Electron paramagnetic resonance spin labeling of HSA revealed that indazolium trans-[RuCl4(1H-indazole)(NO)] and long-chain fatty acids show competitive binding to HSA. Moreover, this complex has a higher affinity for site I, but when present in excess, it is able to bind to site II as well, and displace fatty acids. PMID:26908285

  20. NMR analysis of cleaved Escherichia coli thioredoxin (1-73/74-108) and its P76A variant: cis/trans peptide isomerization.

    PubMed Central

    Yu, W. F.; Tung, C. S.; Wang, H.; Tasayco, M. L.

    2000-01-01

    Inspection of high resolution three-dimensional (3D) structures from the protein database reveals an increasing number of cis-Xaa-Pro and cis-Xaa-Yaa peptide bonds. However, we are still far from being able to predict whether these bonds will remain cis upon single-site substitution of Pro or Yaa and/or cleavage of a peptide bond close to it in the sequence. We have chosen oxidized Escherichia coli thioredoxin (Trx), a member of the Trx superfamily with a single alpha/beta domain and cis P76 to determine the effect of single-site substitution and/or cleavage on this isomer. Standard two-dimensional (2D) NMR analysis were performed on cleaved Trx (1-73/74-108) and its P76A variant. Analysis of the NOE connectivities indicates remarkable similarity between the secondary and supersecondary structure of the noncovalent complexes and Trx. Analysis of the 2D version of the HCCH-TOCSY and HMQC-NOESY-HMQC and 13C-filtered HMQC-NOESY spectra of cleaved Trx with uniformly 13C-labeled 175 and P76 shows surprising conservation of both cis P76 and packing of 175 against W31. A similar NMR analysis of its P76A variant provides no evidence for cis A76 and shows only subtle local changes in both the packing of 175 and the interstrand connectivities between its most protected hydrophobic strands (beta2 and beta4). Indeed, a molecular simulation model for the trans P76A variant of Trx shows only subtle local changes around the substitution site. In conclusion, cleavage of R73 is insufficient to provoke cis/trans isomerization of P76, but cleavage and single-site substitution (P76A) favors the trans isomer. PMID:10739243

  1. 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

  2. Transesterification and amide cis-trans isomerization in Zn and Cd complexes of the chelating amino acid ligand Boc-Asp(Dpa)-OBzl.

    PubMed

    Niklas, Nicole; Zahl, Achim; Alsfasser, Ralf

    2007-01-01

    The amino acid derivative Boc-Asp-OBzl (Boc=N-butyloxycarbonyl; Asp=aspartic acid; Bzl=benzyl) was functionalized by coupling its carboxylate side chain to dipicolylamine. This yielded the tridentate nitrogen donor ligand Boc-Asp(Dpa)-OBzl (-OBzl). The compound -OBzl contains three different carbonyl groups: a tertiary amide linkage between Asp and Dpa, a C-terminal benzyl ester function, and an N-terminal urethane protecting group. NMR spectra were used to compare the reactivity of these moieties. The Boc protecting group gives rise to two isomers, (E, 9%) and (Z, 91%). Coordination of Cd(NO3)2 and Zn(NO3)2 yielded the complexes and. These compounds have significantly reduced barriers to rotation about the tertiary amide C-N bond compared with the free ligand (-OBzl:18.5 kcal mol-1 in CDBr3;: 12.9 kcal mol-1 in (CD3)2CO;: 13.8 kcal mol-1 in (CD3)2CO). Both complexes readily undergo transesterification in methanol or CD3OD. Experimental pseudo-first order rate constants were determined in CD3OD and (CD3)2CO:CD3OD (3:1;). It was found that the zinc complex (k=(2.28+/-0.02)x10(-4) s-1) is significantly more reactive than the cadmium complex (k=(1.41+/-0.03)x10(-6) s-1). In order to study their tertiary amide cis-trans isomerization, the cadmium complex [(-OCH3)Cd(NO3)2] was synthesized, and the zinc complex [(-OCD3)Zn(NO3)2] was generated in situ in (CD3)2CO:CD3OD (3:1). The barriers to rotation were determined (:14.1 kcal mol-1 in CD3OD;: 13.4 kcal mol-1 in (CD3)2CO:CD3OD (3:1)). Our results show that the stronger Lewis-acid zinc(II) is significantly more active than cadmium(II) in the acceleration of the transesterification. This is in marked contrast to the tertiary amide bond rotation which is comparably fast with both metal ions. PMID:17160185

  3. 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

  4. 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.

  5. 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. PMID:26320351

  6. 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.

  7. Slow tight-binding inhibition of prolyl endopeptidase by benzyloxycarbonyl-prolyl-prolinal.

    PubMed Central

    Bakker, A V; Jung, S; Spencer, R W; Vinick, F J; Faraci, W S

    1990-01-01

    Prolyl endopeptidase is a serine proteinase that specifically cleaves peptides on the carboxy side of proline residues. Wilk & Orlowski [(1983) J. Neurochem. 41, 69-75] have shown that benzyloxycarbonyl-prolyl-prolinal (Z-prolyl-prolinal) is a potent inhibitor of prolyl endopeptidase. We show that Z-prolyl-prolinal is a slow-binding inhibitor of mouse brain prolyl endopeptidase with Ki 0.35 +/- 0.05 nM. Kinetic analysis indicates that the mechanism is a simple, but slow, reversible equilibrium between free and bound enzyme (E + I in equilibrium EI) with rate constants for association (kon) and dissociation (koff) of 1.6 X 10(5) M-1.s-1 and approx. 4 X 10(-5) s-1 respectively. Slow-binding inhibition is dependent on the presence of the aldehyde group since the alcohol (Z-prolyl-prolinol) is a rapid and 50,000-fold poorer inhibitor (Ki 19 microM). Prolyl endopeptidase from human brain is also inhibited by Z-prolyl-prolinal with kinetics similar to those of the mouse brain enzyme. PMID:2241932

  8. Dissociability of free and peptidyl-tRNA bound ribosomes.

    PubMed

    Surguchov, A P; Fominykch, E S; Lyzlova, L V

    1978-06-16

    The influence of peptidyl-tRNA on the dissociation of yeast 80 S ribosomes into subunits was studied. For this purpose temperature-sensitive (ts) suppressor strain of yeast Saccharomyces cervisiae carrying a defect in peptide chain termination was used. It was found that peptidyl-tRNA did not influence the dissociation of ribosomes either at high salt concentration or in the presence of dissociation factor (DF) from yeast. After dissociation of yeast ribosomes in 0.5 M KCl, peptidyl-tRNA remains bound to the 60 S subunit. Some characteristics of the termination process and release of nascent polypeptides from yeast ribosomes are discussed. PMID:355860

  9. Spontaneous Formation of RNA Strands, Peptidyl RNA, and Cofactors.

    PubMed

    Jauker, Mario; Griesser, Helmut; Richert, Clemens

    2015-11-23

    How the biochemical machinery evolved from simple precursors is an open question. Here we show that ribonucleotides and amino acids condense to peptidyl RNAs in the absence of enzymes under conditions established for genetic copying. Untemplated formation of RNA strands that can encode genetic information, formation of peptidyl chains linked to RNA, and formation of the cofactors NAD(+), FAD, and ATP all occur under the same conditions. In the peptidyl RNAs, the peptide chains are phosphoramidate-linked to a ribonucleotide. Peptidyl RNAs with long peptide chains were selected from an initial pool when a lipophilic phase simulating the interior of membranes was offered, and free peptides were released upon acidification. Our results show that key molecules of genetics, catalysis, and metabolism can emerge under the same conditions, without a mineral surface, without an enzyme, and without the need for chemical pre-activation.

  10. Spontaneous Formation of RNA Strands, Peptidyl RNA, and Cofactors

    PubMed Central

    Jauker, Mario; Griesser, Helmut; Richert, Clemens

    2015-01-01

    How the biochemical machinery evolved from simple precursors is an open question. Here we show that ribonucleotides and amino acids condense to peptidyl RNAs in the absence of enzymes under conditions established for genetic copying. Untemplated formation of RNA strands that can encode genetic information, formation of peptidyl chains linked to RNA, and formation of the cofactors NAD+, FAD, and ATP all occur under the same conditions. In the peptidyl RNAs, the peptide chains are phosphoramidate-linked to a ribonucleotide. Peptidyl RNAs with long peptide chains were selected from an initial pool when a lipophilic phase simulating the interior of membranes was offered, and free peptides were released upon acidification. Our results show that key molecules of genetics, catalysis, and metabolism can emerge under the same conditions, without a mineral surface, without an enzyme, and without the need for chemical pre-activation. PMID:26435376

  11. 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.

  12. 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

  13. Topographic regulation of neuronal intermediate filaments by phosphorylation, role of peptidyl-prolyl isomerase 1: significance in neurodegeneration.

    PubMed

    Binukumar, B K; Shukla, Varsha; Amin, Niranjana D; Reddy, Preethi; Skuntz, Suzanne; Grant, Philip; Pant, Harish C

    2013-07-01

    The neuronal cytoskeleton is tightly regulated by phosphorylation and dephosphorylation reactions mediated by numerous associated kinases, phosphatases and their regulators. Defects in the relative kinase and phosphatase activities and/or deregulation of compartment-specific phosphorylation result in neurodegenerative disorders. The largest family of cytoskeletal proteins in mammalian cells is the superfamily of intermediate filaments (IFs). The neurofilament (NF) proteins are the major IFs. Aggregated forms of hyperphosphorylated tau and phosphorylated NFs are found in pathological cell body accumulations in the central nervous system of patients suffering from Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis. The precise mechanisms for this compartment-specific phosphorylation of cytoskeletal proteins are not completely understood. In this review, we focus on the mechanisms of neurofilament phosphorylation in normal physiology and neurodegenerative diseases. We also address the recent breakthroughs in our understanding the role of different kinases and phosphatases involved in regulating the phosphorylation status of the NFs. In addition, special emphasis has been given to describe the role of phosphatases and Pin1 in phosphorylation of NFs.

  14. Escherichia coli cyclophilin B binds a highly distorted form of trans-prolyl peptide isomer.

    PubMed

    Konno, Michiko; Sano, Yumi; Okudaira, Kayoko; Kawaguchi, Yoko; Yamagishi-Ohmori, Yoko; Fushinobu, Shinya; Matsuzawa, Hiroshi

    2004-09-01

    Cyclophilins facilitate the peptidyl-prolyl isomerization of a trans-isomer to a cis-isomer in the refolding process of unfolded proteins to recover the natural folding state with cis-proline conformation. To date, only short peptides with a cis-form proline have been observed in complexes of human and Escherichia coli proteins of cyclophilin A, which is present in cytoplasm. The crystal structures analyzed in this study show two complexes in which peptides having a trans-form proline, i.e. succinyl-Ala-trans-Pro-Ala-p-nitroanilide and acetyl-Ala-Ala-trans-Pro-Ala-amidomethylcoumarin, are bound on a K163T mutant of Escherichia coli cyclophilin B, the preprotein of which has a signal sequence. Comparison with cis-form peptides bound to cyclophilin A reveals that in any case the proline ring is inserted into the hydrophobic pocket and a hydrogen bond between CO of Pro and Neta2 of Arg is formed to fix the peptide. On the other hand, in the cis-isomer, the formation of two hydrogen bonds of NH and CO of Ala preceding Pro with the protein fixes the peptide, whereas in the trans-isomer formation of a hydrogen bond between CO preceding Ala-Pro and His47 Nepsilon2 via a mediating water molecule allows the large distortion in the orientation of Ala of Ala-Pro. Although loss of double bond character of the amide bond of Ala-Pro is essential to the isomerization pathway occurring by rotating around its bond, these peptides have forms impossible to undergo proton transfer from the guanidyl group of Arg to the prolyl N atom, which induces loss of double bond character.

  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. 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

  17. 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.

  18. 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.

  19. Oligopeptide cyclophilin inhibitors: a reassessment.

    PubMed

    Schumann, Michael; Jahreis, Günther; Kahlert, Viktoria; Lücke, Christian; Fischer, Gunter

    2011-11-01

    Potent cyclophilin A (CypA) inhibitors such as non-immunosuppressive cyclosporin A (CsA) derivatives have been already used in clinical trials in patients with viral infections. CypA is a peptidyl prolyl cis/trans isomerase (PPIase) that catalyzes slow prolyl bond cis/trans interconversions of the backbone of substrate peptides and proteins. In this study we investigate whether the notoriously low affinity inhibitory interaction of linear proline-containing peptides with the active site of CypA can be increased through a combination of a high cis/trans ratio and a negatively charged C-terminus as has been recently reported for Trp-Gly-Pro. Surprisingly, isothermal titration calorimetry did not reveal formation of an inhibitory CypA/Trp-Gly-Pro complex previously described within a complex stability range similar to CsA, a nanomolar CypA inhibitor. Moreover, despite of cis content of 41% at pH 7.5 Trp-Gly-Pro cannot inhibit CypA-catalyzed standard substrate isomerization up to high micromolar concentrations. However, in the context of the CsA framework a net charge of -7 clustered at the amino acid side chain of position 1 resulted in slightly improved CypA inhibition.

  20. The natural product berberine is a human prolyl oligopeptidase inhibitor.

    PubMed

    Tarrago, Teresa; Kichik, Nessim; Seguí, Josep; Giralt, Ernest

    2007-03-01

    Prolyl oligopeptidase is a cytosolic serine peptidase that hydrolyzes proline-containing peptides at the carboxy terminus. This peptidase has been associated with schizophrenia, bipolar affective disorder, and related neuropsychiatric disorders, and therefore may have important clinical implications. Among the strategies used to find novel prolyl oligopeptidase inhibitors, traditional Chinese medicinal plants provide a rich source of unexplored compounds. We used (19)F NMR spectroscopy to search for new prolyl oligopeptidase inhibitors in a library of traditional Chinese medicine plant extracts. Several extracts were identified as powerful inhibitors of this peptidase. The alkaloid berberine was the prolyl oligopeptidase inhibitory molecule isolated from Rhizoma coptidis extract. Berberine inhibited prolyl oligopeptidase in a dose-dependent manner. As berberine is a natural compound that has been safely administered to humans, it opens up new perspectives for the treatment of neuropsychiatric diseases. The results described herein suggest that the initiation of clinical trials in patients with schizophrenia, bipolar affective disorder, or related diseases in which cognitive capabilities are affected should be undertaken with either the extract or pure BBR.

  1. 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

  2. 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

  3. The Dipeptidyl Peptidase Family, Prolyl Oligopeptidase, and Prolyl Carboxypeptidase in the Immune System and Inflammatory Disease, Including Atherosclerosis

    PubMed Central

    Waumans, Yannick; Baerts, Lesley; Kehoe, Kaat; Lambeir, Anne-Marie; De Meester, Ingrid

    2015-01-01

    Research from over the past 20 years has implicated dipeptidyl peptidase (DPP) IV and its family members in many processes and different pathologies of the immune system. Most research has been focused on either DPPIV or just a few of its family members. It is, however, essential to consider the entire DPP family when discussing any one of its members. There is a substantial overlap between family members in their substrate specificity, inhibitors, and functions. In this review, we provide a comprehensive discussion on the role of prolyl-specific peptidases DPPIV, FAP, DPP8, DPP9, dipeptidyl peptidase II, prolyl carboxypeptidase, and prolyl oligopeptidase in the immune system and its diseases. We highlight possible therapeutic targets for the prevention and treatment of atherosclerosis, a condition that lies at the frontier between inflammation and cardiovascular disease. PMID:26300881

  4. Recycling of Peptidyl-tRNAs by Peptidyl-tRNA Hydrolase Counteracts Azithromycin-Mediated Effects on Pseudomonas aeruginosa

    PubMed Central

    Gödeke, Julia; Pustelny, Christian

    2013-01-01

    Acute and chronic infections caused by the opportunistic pathogen Pseudomonas aeruginosa pose a serious threat to human health worldwide, and its increasing resistance to antibiotics requires alternative treatments that are more effective than available strategies. Clinical studies have clearly demonstrated that cystic fibrosis (CF) patients with chronic P. aeruginosa infections benefit from long-term low-dose azithromycin (AZM) treatment. Immunomodulating activity, the impact of AZM on the expression of quorum-sensing-dependent virulence factors, type three secretion, and motility in P. aeruginosa seem to contribute to the therapeutic response. However, to date, the molecular mechanisms underlying these AZM effects have remained elusive. Our data indicate that the AZM-mediated phenotype is caused by a depletion of the intracellular pools of tRNAs available for protein synthesis. Overexpression of the P. aeruginosa peptidyl-tRNA hydrolase, which recycles the tRNA from peptidyl-tRNA drop-off during translation, counteracted the effects of AZM on stationary-phase cell killing, cytotoxicity, and the production of rhamnolipids and partially restored swarming motility. Intriguingly, the exchange of a rare for a frequent codon in rhlR also explicitly diminished the AZM-mediated decreased production of rhamnolipids. These results indicate that depletion of the tRNA pools by AZM seems to affect the translation of genes that use rare aminoacyl-tRNA isoacceptors to a great extent and might explain the selective activity of AZM on the P. aeruginosa proteome and possibly also on the protein expression profiles of other bacterial pathogens. PMID:23318806

  5. A digestive prolyl carboxypeptidase in Tenebrio molitor larvae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  6. 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.

  7. Structure and Mechanism of a Viral Collagen Prolyl Hydroxylase

    PubMed Central

    2015-01-01

    The Fe(II)- and 2-oxoglutarate (2-OG)-dependent dioxygenases comprise a large and diverse enzyme superfamily the members of which have multiple physiological roles. Despite this diversity, these enzymes share a common chemical mechanism and a core structural fold, a double-stranded β-helix (DSBH), as well as conserved active site residues. The prolyl hydroxylases are members of this large superfamily. Prolyl hydroxylases are involved in collagen biosynthesis and oxygen sensing in mammalian cells. Structural–mechanistic studies with prolyl hydroxylases have broader implications for understanding mechanisms in the Fe(II)- and 2-OG-dependent dioxygenase superfamily. Here, we describe crystal structures of an N-terminally truncated viral collagen prolyl hydroxylase (vCPH). The crystal structure shows that vCPH contains the conserved DSBH motif and iron binding active site residues of 2-OG oxygenases. Molecular dynamics simulations are used to delineate structural changes in vCPH upon binding its substrate. Kinetic investigations are used to report on reaction cycle intermediates and compare them to the closest homologues of vCPH. The study highlights the utility of vCPH as a model enzyme for broader mechanistic analysis of Fe(II)- and 2-OG-dependent dioxygenases, including those of biomedical interest. PMID:26368022

  8. 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.

  9. Uranium-Carbene-Imido Metalla-Allenes: Ancillary-Ligand-Controlled cis-/trans-Isomerisation and Assessment of trans Influence in the R2 C=U(IV) =NR' Unit (R=Ph2 PNSiMe3 ; R'=CPh3 ).

    PubMed

    Lu, Erli; Cooper, Oliver J; Tuna, Floriana; Wooles, Ashley J; Kaltsoyannis, Nikolas; Liddle, Stephen T

    2016-08-01

    Uranium(IV)-carbene-imido complexes [U(BIPM(TMS) )(NCPh3 )(κ(2) -N,N'-BIPY)] (2; BIPM(TMS) =C(PPh2 NSiMe3 )2 ; BIPY=2,2-bipyridine) and [U(BIPM(TMS) )(NCPh3 )(DMAP)2 ] (3; DMAP=4-dimethylamino-pyridine) that contain unprecedented, discrete R2 C=U=NR' units are reported. These complexes complete the family of E=U=E (E=CR2 , NR, O) metalla-allenes with feasible first-row hetero-element combinations. Intriguingly, 2 and 3 contain cis- and trans-C=U=N units, respectively, representing rare examples of controllable cis/trans isomerisation in f-block chemistry. This work reveals a clear-cut example of the trans influence in a mid-valent uranium system, and thus a strong preference for the cis isomer, which is computed in a co-ligand-free truncated model-to isolate the electronic trans influence from steric contributions-to be more stable than the trans isomer by approximately 12 kJ mol(-1) with an isomerisation barrier of approximately 14 kJ mol(-1) . PMID:27405793

  10. The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center

    PubMed Central

    Englander, Michael T.; Avins, Joshua L.; Fleisher, Rachel C.; Liu, Bo; Effraim, Philip R.; Wang, Jiangning; Schulten, Klaus; Leyh, Thomas S.; Gonzalez, Ruben L.; Cornish, Virginia W.

    2015-01-01

    The cellular translational machinery (TM) synthesizes proteins using exclusively L- or achiral aminoacyl-tRNAs (aa-tRNAs), despite the presence of D-amino acids in nature and their ability to be aminoacylated onto tRNAs by aa-tRNA synthetases. The ubiquity of L-amino acids in proteins has led to the hypothesis that D-amino acids are not substrates for the TM. Supporting this view, protein engineering efforts to incorporate D-amino acids into proteins using the TM have thus far been unsuccessful. Nonetheless, a mechanistic understanding of why D-aa-tRNAs are poor substrates for the TM is lacking. To address this deficiency, we have systematically tested the translation activity of D-aa-tRNAs using a series of biochemical assays. We find that the TM can effectively, albeit slowly, accept D-aa-tRNAs into the ribosomal aa-tRNA binding (A) site, use the A-site D-aa-tRNA as a peptidyl-transfer acceptor, and translocate the resulting peptidyl-D-aa-tRNA into the ribosomal peptidyl-tRNA binding (P) site. During the next round of continuous translation, however, we find that ribosomes carrying a P-site peptidyl-D-aa-tRNA partition into subpopulations that are either translationally arrested or that can continue translating. Consistent with its ability to arrest translation, chemical protection experiments and molecular dynamics simulations show that P site-bound peptidyl-D-aa-tRNA can trap the ribosomal peptidyl-transferase center in a conformation in which peptidyl transfer is impaired. Our results reveal a novel mechanism through which D-aa-tRNAs interfere with translation, provide insight into how the TM might be engineered to use D-aa-tRNAs, and increase our understanding of the physiological role of a widely distributed enzyme that clears D-aa-tRNAs from cells. PMID:25918365

  11. The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center.

    PubMed

    Englander, Michael T; Avins, Joshua L; Fleisher, Rachel C; Liu, Bo; Effraim, Philip R; Wang, Jiangning; Schulten, Klaus; Leyh, Thomas S; Gonzalez, Ruben L; Cornish, Virginia W

    2015-05-12

    The cellular translational machinery (TM) synthesizes proteins using exclusively L- or achiral aminoacyl-tRNAs (aa-tRNAs), despite the presence of D-amino acids in nature and their ability to be aminoacylated onto tRNAs by aa-tRNA synthetases. The ubiquity of L-amino acids in proteins has led to the hypothesis that D-amino acids are not substrates for the TM. Supporting this view, protein engineering efforts to incorporate D-amino acids into proteins using the TM have thus far been unsuccessful. Nonetheless, a mechanistic understanding of why D-aa-tRNAs are poor substrates for the TM is lacking. To address this deficiency, we have systematically tested the translation activity of D-aa-tRNAs using a series of biochemical assays. We find that the TM can effectively, albeit slowly, accept D-aa-tRNAs into the ribosomal aa-tRNA binding (A) site, use the A-site D-aa-tRNA as a peptidyl-transfer acceptor, and translocate the resulting peptidyl-D-aa-tRNA into the ribosomal peptidyl-tRNA binding (P) site. During the next round of continuous translation, however, we find that ribosomes carrying a P-site peptidyl-D-aa-tRNA partition into subpopulations that are either translationally arrested or that can continue translating. Consistent with its ability to arrest translation, chemical protection experiments and molecular dynamics simulations show that P site-bound peptidyl-D-aa-tRNA can trap the ribosomal peptidyl-transferase center in a conformation in which peptidyl transfer is impaired. Our results reveal a novel mechanism through which D-aa-tRNAs interfere with translation, provide insight into how the TM might be engineered to use D-aa-tRNAs, and increase our understanding of the physiological role of a widely distributed enzyme that clears D-aa-tRNAs from cells.

  12. Peptidyl transferase inhibition by the nascent leader peptide of an inducible cat gene.

    PubMed Central

    Gu, Z; Rogers, E J; Lovett, P S

    1993-01-01

    The site of ribosome stalling in the leader of cat transcripts is critical to induction of downstream translation. Site-specific stalling requires translation of the first five leader codons and the presence of chloramphenicol, a sequence-independent inhibitor of ribosome elongation. We demonstrate in this report that a synthetic peptide (the 5-mer) corresponding to the N-terminal five codons of the cat-86 leader inhibits peptidyl transferase in vitro. The N-terminal 2-, 3-, and 4-mers and the reverse 5-mer (reverse amino acid sequence of the 5-mer) are virtually without effect on peptidyl transferase. A missense mutation in the cat-86 leader that abolishes induction in vivo corresponds to an amino acid replacement in the 5-mer that completely relieves peptidyl transferase inhibition. In contrast, a missense mutation that does not interfere with in vivo induction corresponds to an amino acid replacement in the 5-mer that does not significantly alter peptidyl transferase inhibition. Our results suggest that peptidyl transferase inhibition by the nascent cat-86 5-mer peptide may be the primary determinant of the site of ribosome stalling in the leader. A model based on this concept can explain the site specificity of ribosome stalling as well as the response of induction to very low levels of the antibiotic inducer. Images PMID:7690023

  13. 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.

  14. 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. PMID:25104352

  15. Peptidyl cyclopropenones: Reversible inhibitors, irreversible inhibitors, or substrates of cysteine proteases?

    PubMed Central

    Cohen, Meital; Bretler, Uriel; Albeck, Amnon

    2013-01-01

    Peptidyl cyclopropenones were previously introduced as selective cysteine protease reversible inhibitors. In the present study we synthesized one such peptidyl cyclopropenone and investigated its interaction with papain, a prototype cysteine protease. A set of kinetics, biochemical, HPLC, MS, and 13C-NMR experiments revealed that the peptidyl cyclopropenone was an irreversible inhibitor of the enzyme, alkylating the catalytic cysteine. In parallel, this cyclopropenone also behaved as an alternative substrate of the enzyme, providing a product that was tentatively suggested to be either a spiroepoxy cyclopropanone or a gamma-lactone. Thus, a single family of compounds exhibits an unusual variety of activities, being reversible inhibitors, irreversible inhibitors and alternative substrates towards enzymes of the same family. PMID:23553793

  16. Cell adhesion promoting peptide GVKGDKGNPGWPGAP from the collagen type IV triple helix: Cis/trans proline-induced multiple sup 1 H NMR conformations and evidence for a KG/PG multiple turn repeat motif in the all-trans proline state

    SciTech Connect

    Mayo, K.H.; Parra-Diaz, D. ); McCarthy, J.B.; Chelberg, M. )

    1991-08-20

    Peptide GVKGDKGNPGWPGAPY (called peptide IV-H1), derived from the protein sequence of human collagen type IV, triple-helix domain residues 1263-1277, represents an RGD-independent, cell-specific, adhesion, spreading, and motility promoting domain in type IV collagen. In this study, peptide IV-H1 has been investigated by {sup 1}H NMR (500 MHz) spectroscopy. Cis-trans proline isomerization at each of the three proline residues gives rise to a number of slowly exchanging (500-MHz NMR time scale) conformation states. The presence of more than two sets of resonances for residues sequentially proximal to a proline, e.g., A14-cis-P15 K3, indicates long range conformation interactions and the presence of preferred structure in this short linear peptide. Many resonances belonging to these multiple species have been assigned by using mono-proline-substituted analogues. Conformational (isomer) state-specific 2D {sup 1}H NMR assignments for the combination of cis and trans proline states have been made via analysis of COSY-type, HOHAHA, and NOESY spectra. The NMR data indicate significant {beta}-turn populations centered at K3-G4, K5-G6, P9-G10, and P12-G13, and a C-terminal {gamma}-turn within the A14-P15-Y16 sequence. These NMR data are supported by circular dichroic studies which indicate the presence of 52% {beta}-turn, 10% helix, and 38% random coil structural populations. Since equally spaced KG and PG residues are found on both sides of peptide IV-H1 in the native collagen type IV sequence, this multiple turn repeat motif may continue through a longer segment of the protein.

  17. Erasable holographic medium using cis-trans isomerization

    NASA Technical Reports Server (NTRS)

    Adelman, A. H.; Grotta, H. M.

    1972-01-01

    Photochemical process has been developed for recording of erasable holograms by utilizing reversible transformation of two isomers of molecule upon exposure to light. Hologram system records, reads, and erases in response to changes in refractive index of mixture of isomers.

  18. 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.

  19. 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. PMID:26809121

  20. Anti-peptidyl transferase leader peptides of attenuation-regulated chloramphenicol-resistance genes.

    PubMed Central

    Gu, Z; Harrod, R; Rogers, E J; Lovett, P S

    1994-01-01

    The chloramphenicol (Cm)-inducible cmlA gene of Tn1696 specifies nonenzymatic resistance to Cm and is regulated by attenuation. The first eight codons of the leader specify a peptide that inhibits peptidyl transferase in vitro. Functionally similar, but less inhibitory, peptides are encoded by the leaders of Cm-inducible cat genes. However, the cat and cmlA coding sequences are unrelated and specify proteins of unrelated function. The inhibition of peptidyl transferase by the leader peptides is additive with that of Cm. Erythromycin competes with the inhibitory action of the peptides, and erythromycin and the peptides footprint to overlapping sites at the peptidyl transferase center of 23S rRNA. It is proposed that translation of the cmlA and cat leaders transiently pauses upon synthesis of the inhibitor peptides. The predicted site of pausing is identical to the leader site where long-term occupancy by a ribosome (ribosome stalling) will activate downstream gene expression. We therefore propose the inducer, Cm, converts a peptide-paused ribosome to the stalled state. We discuss the idea that cooperativity between leader peptide and inducer is necessary for ribosome stalling and may link the activation of a specific drug-resistance gene with a particular antibiotic. Images PMID:7515506

  1. Context-specific inhibition of translation by ribosomal antibiotics targeting the peptidyl transferase center

    PubMed Central

    Marks, James; Kannan, Krishna; Roncase, Emily J.; Klepacki, Dorota; Kefi, Amira; Orelle, Cédric; Vázquez-Laslop, Nora; Mankin, Alexander S.

    2016-01-01

    The first broad-spectrum antibiotic chloramphenicol and one of the newest clinically important antibacterials, linezolid, inhibit protein synthesis by targeting the peptidyl transferase center of the bacterial ribosome. Because antibiotic binding should prevent the placement of aminoacyl-tRNA in the catalytic site, it is commonly assumed that these drugs are universal inhibitors of peptidyl transfer and should readily block the formation of every peptide bond. However, our in vitro experiments showed that chloramphenicol and linezolid stall ribosomes at specific mRNA locations. Treatment of bacterial cells with high concentrations of these antibiotics leads to preferential arrest of translation at defined sites, resulting in redistribution of the ribosomes on mRNA. Antibiotic-mediated inhibition of protein synthesis is most efficient when the nascent peptide in the ribosome carries an alanine residue and, to a lesser extent, serine or threonine in its penultimate position. In contrast, the inhibitory action of the drugs is counteracted by glycine when it is either at the nascent-chain C terminus or at the incoming aminoacyl-tRNA. The context-specific action of chloramphenicol illuminates the operation of the mechanism of inducible resistance that relies on programmed drug-induced translation arrest. In addition, our findings expose the functional interplay between the nascent chain and the peptidyl transferase center. PMID:27791002

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

    PubMed Central

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

    2009-01-01

    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 in order to successfully eradicate all secondary oxidizing species and prevent uncontrolled oxidation of sulfur-containing residues. PMID:19278868

  3. 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. PMID:26808442

  4. 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

  5. Further delineation of FKBP14-related Ehlers-Danlos syndrome: A patient with early vascular complications and non-progressive kyphoscoliosis, and literature review.

    PubMed

    Dordoni, Chiara; Ciaccio, Claudia; Venturini, Marina; Calzavara-Pinton, Piergiacomo; Ritelli, Marco; Colombi, Marina

    2016-08-01

    FKBP14-related Ehlers-Danlos syndrome (EDS) is an extremely rare recessive connective tissue disorder described for the first time in 2012 by Baumann and coworkers. The causal gene, FKBP14, encodes a member of the F506-binding family of peptidyl-prolyl cis-trans isomerases. The paucity of patients described so far makes this disorder poorly defined at clinical level. Here, we report an additional pediatric patient, who is compound heterozygous for a recurrent and a novel FKBP14 mutation, and compare his phenotype with those available in literature. This evaluation confirms that kyphoscoliosis (either progressive or non-progressive), myopathy, joint hypermobility, and congenital hearing loss (sensorineural, conductive, or mixed) are the typical features of the syndrome. Since the patient showed a severe cardiovascular event in childhood and atlantoaxial instability, this report expands the phenotype of the disorder and the allelic repertoire of FKBP14. © 2016 Wiley Periodicals, Inc.

  6. 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

  7. 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. PMID:11690648

  8. 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

  9. Further delineation of FKBP14-related Ehlers-Danlos syndrome: A patient with early vascular complications and non-progressive kyphoscoliosis, and literature review.

    PubMed

    Dordoni, Chiara; Ciaccio, Claudia; Venturini, Marina; Calzavara-Pinton, Piergiacomo; Ritelli, Marco; Colombi, Marina

    2016-08-01

    FKBP14-related Ehlers-Danlos syndrome (EDS) is an extremely rare recessive connective tissue disorder described for the first time in 2012 by Baumann and coworkers. The causal gene, FKBP14, encodes a member of the F506-binding family of peptidyl-prolyl cis-trans isomerases. The paucity of patients described so far makes this disorder poorly defined at clinical level. Here, we report an additional pediatric patient, who is compound heterozygous for a recurrent and a novel FKBP14 mutation, and compare his phenotype with those available in literature. This evaluation confirms that kyphoscoliosis (either progressive or non-progressive), myopathy, joint hypermobility, and congenital hearing loss (sensorineural, conductive, or mixed) are the typical features of the syndrome. Since the patient showed a severe cardiovascular event in childhood and atlantoaxial instability, this report expands the phenotype of the disorder and the allelic repertoire of FKBP14. © 2016 Wiley Periodicals, Inc. PMID:27149304

  10. 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

  11. Parasite cyclophilins and antiparasite activity of cyclosporin A.

    PubMed

    Page, A P; Kumar, S; Carlow, C K

    1995-10-01

    Cyclosporin A (CsA) was initially developed as an immunosuppressive drug. In the past several years, it has been shown to possess antiparasite activity independent of the immune system. It is not known how the drug exerts these antiparasite effects, or why it is stage and/or species specific. The answers may lie in the enzymatic function of cyclophilins. The cyclophilins are a growing family of proteins that exhibit peptidyl-prolyl cis-trans isomerase (PPiase) activity and bid CsA to varying degrees. PPiases have been shown to play a role in the folding of many essential proteins. Antony Page, Sanjay Kumar and Clotilde Carlow here review parasite cyclophilins and their association with CsA. The possible biological function of parasite cyclophilins and their potential role in future drug discovery are also discussed.

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

    PubMed

    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

  13. 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

  14. Green tea gets molecular.

    PubMed

    Rouzer, Carol A; Marnett, Lawrence J

    2011-09-01

    Green tea and its major polyphenolic flavonoid, epigallocatechin gallate (EGCG), have been credited with cancer chemopreventive activity for many years; the mechanism for this activity, however, has remained obscure. Now, as reported in this issue of the journal (beginning on page 1366), Urusova and colleagues showed direct binding of EGCG to the peptidyl prolyl cis/trans isomerase Pin1, which inhibited Pin1 enzymatic activity. They showed that Pin1 expression is required for EGCG effects on cell growth, c-Jun activation, and transcription regulation mediated by NF-κB and activator protein-1. The data provide a glimpse of the mechanism of action of EGCG and set a new bar for the future study of natural products with chemopreventive activity. PMID:21893494

  15. 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

  16. 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.

  17. 23S rRNA nucleotides in the peptidyl transferase center are essential for tryptophanase operon induction.

    PubMed

    Yang, Rui; Cruz-Vera, Luis R; Yanofsky, Charles

    2009-06-01

    Distinct features of the ribosomal peptide exit tunnel are known to be essential for recognition of specific amino acids of a nascent peptidyl-tRNA. Thus, a tryptophan residue at position 12 of the peptidyl-tRNA TnaC-tRNA(Pro) leads to the creation of a free tryptophan binding site within the ribosome at which bound tryptophan inhibits normal ribosome functions. The ribosomal processes that are inhibited are hydrolysis of TnaC-tRNA(Pro) by release factor 2 and peptidyl transfer of TnaC of TnaC-tRNA(Pro) to puromycin. These events are normally performed in the ribosomal peptidyl transferase center. In the present study, changes of 23S rRNA nucleotides in the 2585 region of the peptidyl transferase center, G2583A and U2584C, were observed to reduce maximum induction of tna operon expression by tryptophan in vivo without affecting the concentration of tryptophan necessary to obtain 50% induction. The growth rate of strains with ribosomes with either of these changes was not altered appreciably. In vitro analyses with mutant ribosomes with these changes showed that tryptophan was not as efficient in protecting TnaC-tRNA(Pro) from puromycin action as wild-type ribosomes. However, added tryptophan did prevent sparsomycin action as it normally does with wild-type ribosomes. These findings suggest that these two mutational changes act by reducing the ability of ribosome-bound tryptophan to inhibit peptidyl transferase activity rather than by reducing the ability of the ribosome to bind tryptophan. Thus, the present study identifies specific nucleotides within the ribosomal peptidyl transferase center that appear to be essential for effective tryptophan induction of tna operon expression. PMID:19329641

  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. An efficient solid-phase synthesis of peptidyl-N-acetylguanidines for use in native chemical ligation.

    PubMed

    Okamoto, Ryo; Isoe, Madoka; Izumi, Masayuki; Kajihara, Yasuhiro

    2016-05-01

    In the modern protocols of chemical protein syntheses, peptide-α-thioesters have been used as key components for the assembly of full-length polypeptides through chemoselective peptide coupling reactions. A variety of thioester precursors have been developed for the synthesis of the peptide-α-thioesters by Fmoc solid phase peptide synthesis (Fmoc-SPPS). Recently our group found a peptidyl-N-acetylguanidine as a new peptide-α-thioester precursor. This peptide derivative can be converted into a corresponding peptide-α-thioester only by treatment with an excess amount of a thiol in aqueous buffers at around neutral pH. This unique property allowed us to envision the practical use of the peptidyl-N-acetylguanidines for the chemical syntheses of proteins; however, an efficient synthetic method has been lacking. Herein, we report an efficient solid-phase synthesis of peptidyl-N-acetylguanidines. This new synthetic method employing selective activation and cleavage of a peptide bond successfully provided peptidyl-N-acetylguanidines from the on-resin protected peptides prepared by standard Fmoc-SPPS. We also evaluated the reactivity of a peptidyl-N-acetylguanidine in native chemical ligation through the synthesis of glucose-dependent insulinotropic polypeptide analogue. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27005965

  20. Activities of the peptidyl transferase center of ribosomes lacking protein L27

    PubMed Central

    Maracci, Cristina; Wohlgemuth, Ingo; Rodnina, Marina V.

    2015-01-01

    The ribosome is the molecular machine responsible for protein synthesis in all living organisms. Its catalytic core, the peptidyl transferase center (PTC), is built of rRNA, although several proteins reach close to the inner rRNA shell. In the Escherichia coli ribosome, the flexible N-terminal tail of the ribosomal protein L27 contacts the A- and P-site tRNA. Based on computer simulations of the PTC and on previous biochemical evidence, the N-terminal α-amino group of L27 was suggested to take part in the peptidyl-transfer reaction. However, the contribution of this group to catalysis has not been tested experimentally. Here we investigate the role of L27 in peptide-bond formation using fast kinetics approaches. We show that the rate of peptide-bond formation at physiological pH, both with aminoacyl-tRNA or with the substrate analog puromycin, is independent of the presence of L27; furthermore, translation of natural mRNAs is only marginally affected in the absence of L27. The pH dependence of the puromycin reaction is unaltered in the absence of L27, indicating that the N-terminal α-amine is not the ionizing group taking part in catalysis. Likewise, L27 is not required for the peptidyl-tRNA hydrolysis during termination. Thus, apart from the known effect on subunit association, which most likely explains the phenotype of the deletion strains, L27 does not appear to be a key player in the core mechanism of peptide-bond formation on the ribosome. PMID:26475831

  1. Activities of the peptidyl transferase center of ribosomes lacking protein L27.

    PubMed

    Maracci, Cristina; Wohlgemuth, Ingo; Rodnina, Marina V

    2015-12-01

    The ribosome is the molecular machine responsible for protein synthesis in all living organisms. Its catalytic core, the peptidyl transferase center (PTC), is built of rRNA, although several proteins reach close to the inner rRNA shell. In the Escherichia coli ribosome, the flexible N-terminal tail of the ribosomal protein L27 contacts the A- and P-site tRNA. Based on computer simulations of the PTC and on previous biochemical evidence, the N-terminal α-amino group of L27 was suggested to take part in the peptidyl-transfer reaction. However, the contribution of this group to catalysis has not been tested experimentally. Here we investigate the role of L27 in peptide-bond formation using fast kinetics approaches. We show that the rate of peptide-bond formation at physiological pH, both with aminoacyl-tRNA or with the substrate analog puromycin, is independent of the presence of L27; furthermore, translation of natural mRNAs is only marginally affected in the absence of L27. The pH dependence of the puromycin reaction is unaltered in the absence of L27, indicating that the N-terminal α-amine is not the ionizing group taking part in catalysis. Likewise, L27 is not required for the peptidyl-tRNA hydrolysis during termination. Thus, apart from the known effect on subunit association, which most likely explains the phenotype of the deletion strains, L27 does not appear to be a key player in the core mechanism of peptide-bond formation on the ribosome.

  2. Serum immunoreactive prolyl hydroxylase in inflammatory rheumatic diseases.

    PubMed Central

    Kuutti-Savolainen, E R; Kivirikko, K I; Laitinen, O

    1980-01-01

    Serum immunoreactive prolyl hydroxylase protein (S-IRPH) was measured in 56 patients with inflammatory rheumatic diseases, and the values were compared with those in 32 control subjects. S-IRPH was above the 95% confidence limit of the controls in about 70% of the patients with active systemic lupus erythematosus, rheumatoid arthritis, scleroderma, Reiter's syndrome, Sjögren's syndrome, polyarteritis nodosa, or polymyositis. Raised values were observed in about half of the patients with an erythrocyte sedimentation rate (ESR) of 21-50 and in about 90% of those with ESR of over 50, whereas only about 10% of the patients with an inactive disease had an S-IRPH concentration exceeding this limit. Only 1 out of 8 patients with active ankylosing spondylitis had a raised S-IRPH value. The results support previous data indicating that significant changes in collagen metabolism occur in active connective tissue diseases. Assays of S-IRPH might be of some value in assessing the activity of these diseases and in monitoring the treatment provided. PMID:6251755

  3. Regulation of Cardiac Hypertrophic Signaling by Prolyl Isomerase Pin1

    PubMed Central

    Toko, Haruhiro; Konstandin, Mathias H.; Doroudgar, Shirin; Ormachea, Lucia; Joyo, Eri; Joyo, Anya Y.; Din, Shabana; Gude, Natalie A.; Collins, Brett; Völkers, Mirko; Thuerauf, Donna J.; Glembotski, Christopher C.; Chen, Chun-Hau; Lu, Kun Ping; Müller, Oliver J.; Uchida, Takafumi; Sussman, Mark A.

    2013-01-01

    Rationale Cardiac hypertrophy results from the complex interplay of differentially regulated cascades based upon the phosphorylation status of involved signaling molecules. While numerous critical regulatory kinases and phosphatases have been identified in the myocardium, the intracellular mechanism for temporal regulation of signaling duration and intensity remains obscure. In the non-myocyte context, control of folding, activity, and stability of proteins is mediated by the prolyl isomerase Pin1, but the role of Pin1 in the heart is unknown. Objective To establish the role of Pin1 in the heart. Methods and Results Here we show that either genetic deletion or cardiac over-expression of Pin1 blunts hypertrophic responses induced by transaortic constriction and consequent cardiac failure in vivo. Mechanistically, we find that Pin1 directly binds to Akt, MEK and Raf-1 in cultured cardiomyocytes following hypertrophic stimulation. Furthermore, loss of Pin1 leads to diminished hypertrophic signaling of Akt and MEK, while over-expression of Pin1 increases Raf-1 phosphorylation on the auto-inhibitory site Ser259 leading to reduced MEK activation. Conclusions Collectively, these data support a role for Pin1 as a central modulator of the intensity and duration of two major hypertrophic signaling pathways, thereby providing a novel target for regulation and control of cardiac hypertrophy. PMID:23487407

  4. 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.

  5. Developmental Stage-dependent Regulation of Prolyl 3-Hydroxylation in Tendon Type I Collagen.

    PubMed

    Taga, Yuki; Kusubata, Masashi; Ogawa-Goto, Kiyoko; Hattori, Shunji

    2016-01-01

    3-Hydroxyproline (3-Hyp), which is unique to collagen, is a fairly rare post-translational modification. Recent studies have suggested a function of prolyl 3-hydroxylation in fibril assembly and its relationships with certain disorders, including recessive osteogenesis imperfecta and high myopia. However, no direct evidence for the physiological and pathological roles of 3-Hyp has been presented. In this study, we first estimated the overall alterations in prolyl hydroxylation in collagens purified from skin, bone, and tail tendon of 0.5-18-month-old rats by LC-MS analysis with stable isotope-labeled collagen, which was recently developed as an internal standard for highly accurate collagen analyses. 3-Hyp was found to significantly increase in tendon collagen until 3 months after birth and then remain constant, whereas increased prolyl 3-hydroxylation was not observed in skin and bone collagen. Site-specific analysis further revealed that 3-Hyp was increased in tendon type I collagen in a specific sequence region, including a previously known modification site at Pro(707) and newly identified sites at Pro(716) and Pro(719), at the early ages. The site-specific alterations in prolyl 3-hydroxylation with aging were also observed in bovine Achilles tendon. We postulate that significant increases in 3-Hyp at the consecutive modification sites are correlated with tissue development in tendon. The present findings suggest that prolyl 3-hydroxylation incrementally regulates collagen fibril diameter in tendon.

  6. SecM-Stalled Ribosomes Adopt an Altered Geometry at the Peptidyl Transferase Center

    PubMed Central

    Bhushan, Shashi; Hoffmann, Thomas; Seidelt, Birgit; Frauenfeld, Jens; Mielke, Thorsten; Berninghausen, Otto; Wilson, Daniel N.; Beckmann, Roland

    2011-01-01

    As nascent polypeptide chains are synthesized, they pass through a tunnel in the large ribosomal subunit. Interaction between specific nascent chains and the ribosomal tunnel is used to induce translational stalling for the regulation of gene expression. One well-characterized example is the Escherichia coli SecM (secretion monitor) gene product, which induces stalling to up-regulate translation initiation of the downstream secA gene, which is needed for protein export. Although many of the key components of SecM and the ribosomal tunnel have been identified, understanding of the mechanism by which the peptidyl transferase center of the ribosome is inactivated has been lacking. Here we present a cryo-electron microscopy reconstruction of a SecM-stalled ribosome nascent chain complex at 5.6 Å. While no cascade of rRNA conformational changes is evident, this structure reveals the direct interaction between critical residues of SecM and the ribosomal tunnel. Moreover, a shift in the position of the tRNA–nascent peptide linkage of the SecM-tRNA provides a rationale for peptidyl transferase center silencing, conditional on the simultaneous presence of a Pro-tRNAPro in the ribosomal A-site. These results suggest a distinct allosteric mechanism of regulating translational elongation by the SecM stalling peptide. PMID:21267063

  7. 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.

  8. 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.

  9. OsCYP21-4, a novel Golgi-resident cyclophilin, increases oxidative stress tolerance in rice.

    PubMed

    Lee, Sang S; Park, Hyun J; Jung, Won Y; Lee, Areum; Yoon, Dae H; You, Young N; Kim, Hyun-Soon; Kim, Beom-Gi; Ahn, Jun C; Cho, Hye S

    2015-01-01

    OsCYP21-4 is a rice cyclophilin protein that binds to cyclosporine A, an immunosuppressant drug. CYP21-4s in Arabidopsis and rice were previously shown to function as mitochondrial cyclophilins, as determined by TargetP analysis. In the current study, we found that OsCYP21-4-GFP localized to the Golgi, rather than mitochondria, in Nicotiana benthamiana leaves, which was confirmed based on its co-localization with cis Golgi α-ManI-mCherry protein. OsCYP21-4 transcript levels increased in response to treatments with various abiotic stresses and the phytohormone abscisic acid, revealing its stress-responsiveness. CYP21-4 homologs do not possess key peptidyl prolyl cis/trans isomerase (PPIase) activity/cyclosporine A (CsA) binding residues, and recombinant OsCYP21-4 protein did not convert the synthetic substrate Suc-AAPF-pNA via cis- trans- isomerization in vitro. In addition, transgenic plants overexpressing OsCYP21-4 exhibited increased tolerance to salinity and hydrogen peroxide treatment, along with increased peroxidase activity. These results demonstrate that OsCYP21-4 is a novel Golgi-localized cyclophilin that plays a role in oxidative stress tolerance, possibly by regulating peroxidase activity. PMID:26483814

  10. 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.

  11. 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. PMID:25197067

  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. Design and synthesis of a series of novel pyrazolopyridines as HIF-1alpha prolyl hydroxylase inhibitors.

    PubMed

    Warshakoon, Namal C; Wu, Shengde; Boyer, Angelique; Kawamoto, Richard; Renock, Sean; Xu, Kevin; Pokross, Matthew; Evdokimov, Artem G; Zhou, Songtao; Winter, Carol; Walter, Richard; Mekel, Marlene

    2006-11-01

    Recently resolved X-ray crystal structure of HIF-1alpha prolyl hydroxylase was used to design and develop a novel series of pyrazolopyridines as potent HIF-1alpha prolyl hydroxylase inhibitors. The activity of these compounds was determined in a human EGLN-1 assay. Structure-based design aided in optimizing the potency of the initial lead (2, IC(50) of 11 microM) to a potent (11l, 190 nM) EGLN-1 inhibitor. Several of these analogs were potent VEGF inducers in a cell-based assay. These pyrazolopyridines were also effective in stabilizing HIF-1alpha.

  14. [Activity of peptidyl-dipeptidase A and carboxypeptidase N in the serum of patients with Alzheimer disease].

    PubMed

    Solov'ev, V B; Gengin, M T

    2007-01-01

    The activity of peptidyl-dipeptidase A and carboxypeptidase N taking part in peptide metabolism in the serum of patients with Alzheimer disease were studied. The role of these enzymes in the metabolism of neuropeptides and beta-amyloid at the Alzheimer disease was discussed.

  15. Isomerization and Oligomerization of Truncated and Mutated Tau Forms by FKBP52 are Independent Processes.

    PubMed

    Kamah, A; Cantrelle, F X; Huvent, I; Giustiniani, J; Guillemeau, K; Byrne, C; Jacquot, Y; Landrieu, I; Baulieu, E E; Smet, C; Chambraud, B; Lippens, G

    2016-03-27

    The aggregation of the neuronal Tau protein is one molecular hallmark of Alzheimer's disease and other related tauopathies, but the precise molecular mechanisms of the aggregation process remain unclear. The FK506 binding protein FKBP52 is able to induce oligomers in the pathogenic Tau P301L mutant and in a truncated form of the wild-type human Tau protein. Here, we investigate whether FKBP52's capacity to induce Tau oligomers depends on its prolyl cis/trans isomerase activity. We find that FKBP52 indeed can isomerize selected prolyl bonds in the different Tau proteins, and that this activity is carried solely by its first FK506 binding domain. Its capacity to oligomerize Tau is, however, not linked to this peptidyl-prolyl isomerase activity. In addition, we identified a novel molecular interaction implying the PHF6 peptide of Tau and the FK1/FK2 domains of FKBP52 independent of FK506 binding; these data point toward a non-catalytic molecular interaction that might govern the effect of FKBP52 on Tau.

  16. Isomerization and Oligomerization of Truncated and Mutated Tau Forms by FKBP52 are Independent Processes.

    PubMed

    Kamah, A; Cantrelle, F X; Huvent, I; Giustiniani, J; Guillemeau, K; Byrne, C; Jacquot, Y; Landrieu, I; Baulieu, E E; Smet, C; Chambraud, B; Lippens, G

    2016-03-27

    The aggregation of the neuronal Tau protein is one molecular hallmark of Alzheimer's disease and other related tauopathies, but the precise molecular mechanisms of the aggregation process remain unclear. The FK506 binding protein FKBP52 is able to induce oligomers in the pathogenic Tau P301L mutant and in a truncated form of the wild-type human Tau protein. Here, we investigate whether FKBP52's capacity to induce Tau oligomers depends on its prolyl cis/trans isomerase activity. We find that FKBP52 indeed can isomerize selected prolyl bonds in the different Tau proteins, and that this activity is carried solely by its first FK506 binding domain. Its capacity to oligomerize Tau is, however, not linked to this peptidyl-prolyl isomerase activity. In addition, we identified a novel molecular interaction implying the PHF6 peptide of Tau and the FK1/FK2 domains of FKBP52 independent of FK506 binding; these data point toward a non-catalytic molecular interaction that might govern the effect of FKBP52 on Tau. PMID:26903089

  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. 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.

  19. Synthesis and antitumour evaluation of peptidyl-like derivatives containing the 1,3-benzodioxole system.

    PubMed

    Moreira, Diogo Rodrigo de Magalhães; Lima Leite, Ana Cristina; Pinheiro Ferreira, Paulo Michel; da Costa, Patrícia Marçal; Costa Lotufo, Letícia Veras; de Moraes, Manoel Odorico; Brondani, Dalci José; Pessoa, Claudia do O

    2007-03-01

    In the scope of a research program aiming at the synthesis and pharmacological evaluation of novel possible antitumour prototype compounds, we described in this paper the synthesis of peptidyl-like derivatives containing the 1,3-benzodioxole system. The proliferation inhibitors tested against tumour cell lines identified the derivatives tyrosine (4f) and lysine (4 g) as the most active among them, presenting IC(50) values in micromolar range and are more active than Safrole. For the study on the embryonic development, Safrole did not show any selectivity in this latter assay, which indicates that Safrole acts as a 'cell cycle-nonspecific' inhibitory agent. However, compound 4f presented a fair antimitotic effect, mainly on third cleavage and blastulae stages (38% and 1.7% of normal development, at 10 microg/mL), suggesting a time-dependent activity and a 'cell cycle-specific' agent action. Neither derivatives revealed hemolytic action in assay with mouse erythrocytes.

  20. Probing functions of the ribosomal peptidyl transferase center by nucleotide analog interference.

    PubMed

    Erlacher, Matthias D; Polacek, Norbert

    2012-01-01

    The ribosome is a huge ribonucleoprotein complex in charge of protein synthesis in every living cell. The catalytic center of this dynamic molecular machine is entirely built up of 23S ribosomal RNA and therefore the ribosome can be referred to as the largest natural ribozyme known so far. The in vitro reconstitution approach of large ribosomal subunits described herein allows nucleotide analog interference studies to be performed. The approach is based on the site-specific introduction of nonnatural nucleotide analogs into the peptidyl transferase center, the active site located on the interface side of the large ribosomal subunit. This method combined with standard tests of ribosomal functions broadens the biochemical repertoire to investigate the mechanism of diverse aspects of translation considerably and adds another layer of molecular information on top of structural and mutational studies of the ribosome. PMID:22315072

  1. 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

    2013-01-01

    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.

  2. RMF inactivates ribosomes by covering the peptidyl transferase centre and entrance of peptide exit tunnel.

    PubMed

    Yoshida, Hideji; Yamamoto, Hiroshi; Uchiumi, Toshio; Wada, Akira

    2004-04-01

    In gram-negative bacteria such as Escherichia coli, protein synthesis is suppressed by the formation of 100S ribosomes under stress conditions. The 100S ribosome, a dimer of 70S ribosomes, is formed by ribosome modulation factor (RMF) binding to the 70S ribosomes. During the stationary phase, most of the 70S ribosomes turn to 100S ribosomes, which have lost translational activity. This 100S formation is called the hibernation process in the ribosome cycle of the stationary phase. If stationary phase cells are transferred to fresh medium, the 100S ribosomes immediately go back to active 70S ribosomes, showing that inactive 100S <--> active 70S interconversion is a major system regulating translation activity in stationary phase cells. To elucidate the mechanisms of translational inactivation, the binding sites of RMF on 23S rRNA in 100S ribosome of E. coli were examined by a chemical probing method using dimethyl sulphate (DMS). As the results, the nine bases in 23S rRNA were protected from DMS modifications and the modification of one base was enhanced. Interestingly A2451 is included among the protected bases, which is thought to be directly involved in peptidyl transferase activity. We conclude that RMF inactivates ribosomes by covering the peptidyl transferase (PTase) centre and the entrance of peptide exit tunnel. It is surprising that the cell itself produces a protein that seems to inhibit protein synthesis in a similar manner to antibiotics and that it can reversibly bind to and release from the ribosome in response to environmental conditions.

  3. pH-dependent conformational flexibility within the ribosomal peptidyl transferase center.

    PubMed Central

    Muth, G W; Chen, L; Kosek, A B; Strobel, S A

    2001-01-01

    A universally conserved adenosine, A2451, within the ribosomal peptidyl transferase center has been proposed to act as a general acid-base catalyst during peptide bond formation. Evidence in support of this proposal came from pH-dependent dimethylsulfate (DMS) modification within Escherichia coli ribosomes. A2451 displayed reactivity consistent with an apparent acidity constant (pKa) near neutrality, though pH-dependent structural flexibility could not be rigorously excluded as an explanation for the enhanced reactivity at high pH. Here we present three independent lines of evidence in support of the alternative interpretation. First, A2451 in ribosomes from the archaebacteria Haloarcula marismortui displays an inverted pH profile that is inconsistent with proton-mediated base protection. Second, in ribosomes from the yeast Saccharomyces cerevisiae, C2452 rather than A2451 is modified in a pH-dependent manner. Third, within E. coli ribosomes, the position of A2451 modification (N1 or N3 imino group) was analyzed by testing for a Dimroth rearrangement of the N1-methylated base. The data are more consistent with DMS modification of the A2451 N1, a functional group that, according to the 50S ribosomal crystal structure, is solvent inaccessible without structural rearrangement. It therefore appears that pH-dependent DMS modification of A2451 does not provide evidence either for or against a general acid-base mechanism of protein synthesis. Instead the data suggest that there is pH-dependent conformational flexibility within the peptidyl transferase center, the exact nature and physiological relevance of which is not known. PMID:11680845

  4. Tumor suppressive activity of prolyl isomerase Pin1 in renal cell carcinoma

    PubMed Central

    Teng, Brian L.; Hacker, Kathryn E.; Chen, Shufen; Means, Anthony R.; Rathmell, W. Kimryn

    2011-01-01

    Pin1 specifically recognizes and catalyzes the cis-trans isomerization of phosphorylated-Ser/Thr-Pro bonds, which modulate the stability, localization, and function of numerous Pin1 targets involved in tumor progression. However, the role of Pin1 in cancer remains enigmatic as the gene is located on chromosome 19p13.2, which is a region subject to loss of heterozygosity in several tumors. Since Pin1 protein is frequently under-expressed in kidney cancer, we have explored its role in human clear cell renal cell carcinoma (ccRCC). Here we show evidence for PIN1 gene deletion and mRNA under-expression as a mechanism of Pin1 reduction in ccRCC tumors. We demonstrate that restoration of Pin1 in cell lines found to be deficient in Pin1 protein expression can attenuate the growth of ccRCC cells in soft agar and a xenograft tumor model. Moreover, this ability of Pin1 to negatively influence tumor growth in ccRCC cells may be dependent on the presence of functional p53, which is infrequently mutated in ccRCC. These observations suggest Pin1 may have a mild tumor suppressive role in ccRCC. PMID:21764651

  5. Tumor suppressive activity of prolyl isomerase Pin1 in renal cell carcinoma.

    PubMed

    Teng, Brian L; Hacker, Kathryn E; Chen, Shufen; Means, Anthony R; Rathmell, W Kimryn

    2011-10-01

    Pin1 specifically recognizes and catalyzes the cis-trans isomerization of phosphorylated-Ser/Thr-Pro bonds, which modulate the stability, localization, and function of numerous Pin1 targets involved in tumor progression. However, the role of Pin1 in cancer remains enigmatic as the gene is located on chromosome 19p13.2, which is a region subject to loss of heterozygosity in several tumors. Since Pin1 protein is frequently under-expressed in kidney cancer, we have explored its role in human clear cell renal cell carcinoma (ccRCC). Here we show evidence for PIN1 gene deletion and mRNA under-expression as a mechanism of Pin1 reduction in ccRCC tumors. We demonstrate that restoration of Pin1 in cell lines found to be deficient in Pin1 protein expression can attenuate the growth of ccRCC cells in soft agar and a xenograft tumor model. Moreover, this ability of Pin1 to negatively influence tumor growth in ccRCC cells may be dependent on the presence of functional p53, which is infrequently mutated in ccRCC. These observations suggest Pin1 may have a mild tumor suppressive role in ccRCC.

  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. Crystallization and preliminary X-ray characterization of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis

    SciTech Connect

    Nakajima, Yoshitaka; Ito, Kiyoshi Xu, Yue; Yamada, Nozomi; Onohara, Yuko; Ito, Takashi; Yoshimoto, Tadashi

    2005-12-01

    P. gingivalis prolyl tripeptidyl aminopeptidase has been crystallized by the vapour-diffusion method. Diffraction data have been collected and processed to 2.1 Å resolution. A recombinant form of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis has been crystallized by the hanging-drop vapour-diffusion method using potassium sodium tartrate as a precipitating agent. The crystals belong to the hexagonal space group P6{sub 3}22, with unit-cell parameters a = b = 149.4, c = 159.7 Å. The crystals are most likely to contain one subunit of a dimer in the asymmetric unit, with a V{sub M} value of 3.14 Å{sup 3} Da{sup −1}. Diffraction data were collected to 2.1 Å resolution using synchrotron radiation at the BL5 station of the Photon Factory.

  8. Selective inhibition of the hypoxia-inducible factor prolyl hydroxylase PHD3 by Zn(II).

    PubMed

    Na, Yu-Ran; Woo, Dustin J; Choo, Hyunah; Chung, Hak Suk; Yang, Eun Gyeong

    2015-07-01

    We report herein that Zn(II) selectively inhibits the hypoxia-inducible factor prolyl hydroxylase PHD3 over PHD2, and does not compete with Fe(II). Independent of the oligomer formation induced by Zn(II), inhibition of the activity of PHD3 by Zn(II) involves Cys42 and Cys52 residues distantly located from the active site. PMID:26051901

  9. Synthesis of isotopically labeled P-site substrates for the ribosomal peptidyl transferase reaction

    PubMed Central

    Zhong, Minghong

    2010-01-01

    Isotopomers of the ribosomal P-site substrate, the trinucleotide peptide conjugate CCA-pcb,1 have been designed and synthesized in 26–350020steps. These include individual isotopic substitution at the α-proton, carbonyl carbon, and carbonyl oxygen of the amino acid, the O2' and O3' of the adenosine, and a remote label in the N3 and N4 of both cytidines. These isotopomers were synthesized by coupling cytidylyl-(3'5')-cytidine phosphoramidite isotopomers, as the common synthetic intermediates, with isotopically substituted A-Phe-cap-biotin (A-pcb). The isotopic enrichment is higher than 99% for 1-13C (Phe), 2-2H (Phe), and 3,4-15N2 (cytidine), 93% for 2'/3'- 18O (adenosine), and 64% for 1-18O (Phe). A new synthesis of highly enriched [1-18O2] phenylalanine has been developed. The synthesis of [3'-18O] adenosine was improved by Lewis acid aided regioselective ring opening of the epoxide and by an economical SN2-SN2 method with high isotopic enrichment (93%). Such substrates are valuable for studies of the ribosomal peptidyl transferase reaction by complete kinetic isotope effect analysis and of other biological processes catalyzed by nucleic acid related enzymes, including polymerases, reverse transcriptases, ligases, nucleases, and ribozymes. PMID:18081346

  10. 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. PMID:26953800

  11. 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

  12. 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.

  13. 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.

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

    PubMed

    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. Preischemic targeting of HIF prolyl hydroxylation inhibits fibrosis associated with acute kidney injury.

    PubMed

    Kapitsinou, Pinelopi P; Jaffe, Jonathan; Michael, Mark; Swan, Christina E; Duffy, Kevin J; Erickson-Miller, Connie L; Haase, Volker H

    2012-05-01

    Acute kidney injury (AKI) due to ischemia is an important contributor to the progression of chronic kidney disease (CKD). Key mediators of cellular adaptation to hypoxia are oxygen-sensitive hypoxia-inducible factors (HIF), which are regulated by prolyl-4-hydroxylase domain (PHD)-containing dioxygenases. While activation of HIF protects from ischemic cell death, HIF has been shown to promote fibrosis in experimental models of CKD. The impact of HIF activation on AKI-induced fibrosis has not been defined. Here, we investigated the role of pharmacologic HIF activation in AKI-associated fibrosis and inflammation. We found that pharmacologic inhibition of HIF prolyl hydroxylation before AKI ameliorated fibrosis and prevented anemia, while inhibition of HIF prolyl hydroxylation in the early recovery phase of AKI did not affect short- or long-term clinical outcome. Therefore, preischemic targeting of the PHD/HIF pathway represents an effective therapeutic strategy for the prevention of CKD resulting from AKI, and it warrants further investigation in clinical trials. PMID:22262480

  16. Synthesis and evaluation of peptidyl Michael acceptors that inactivate human rhinovirus 3C protease and inhibit virus replication.

    PubMed

    Kong, J S; Venkatraman, S; Furness, K; Nimkar, S; Shepherd, T A; Wang, Q M; Aubé, J; Hanzlik, R P

    1998-07-01

    Human rhinovirus, the chief cause of the common cold, contains a positive-sense strand of RNA which is translated into a large polyprotein in infected cells. Cleavage of the latter to produce the mature viral proteins required for replication is catalyzed in large part by a virally encoded cysteine proteinase (3Cpro) which is highly selective for -Q approximately GP- cleavage sites. We synthesized peptidyl derivatives of vinylogous glutamine or methionine sulfone esters (e.g., Boc-Val-Leu-Phe-vGln-OR: R = Me, 1; R = Et, 2) and evaluated them as inhibitors of HRV-14 3C protease (3Cpro). Compounds 1 and 2 and several related tetra- and pentapeptide analogues rapidly inactivated 3Cpro with submicromolar IC50 values. Electrospray mass spectrometry confirmed the expected 1:1 stoichiometry of 3Cpro inactivation by 1, 2, and several other analogues. Compound 2 also proved to be useful for active site titration of 3Cpro, which has not been possible heretofore because of the lack of a suitable reagent. In contrast to 1, 2, and congeners, peptidyl Michael acceptors lacking a P4 residue have greatly reduced or negligible activity against 3Cpro, consistent with previously established structure-activity relationships for 3Cpro substrates. Hydrolysis of the P1 vinylogous glutamine ester to a carboxylic acid also decreased inhibitory activity considerably, consistent with the decreased reactivity of acrylic acids vs acrylic esters as Michael acceptors. Incorporating a vinylogous methionine sulfone ester in place of the corresponding glutamine derivative in 1 also reduced activity substantially. Compounds 1 and 2 and several of their analogues inhibited HRV replication in cell culture by 50% at low micromolar concentrations while showing little or no evidence of cytotoxicity at 10-fold higher concentrations. Peptidyl Michael acceptors and their analogues may prove useful as therapeutic agents for pathologies involving cysteine proteinase enzymes.

  17. Cloning, Expression, Sequence Analysis and Homology Modeling of the Prolyl Endoprotease from Eurygaster integriceps Puton.

    PubMed

    Yandamuri, Ravi Chandra; Gautam, Ranjeeta; Darkoh, Charles; Dareddy, Vanitha; El-Bouhssini, Mustapha; Clack, Beatrice A

    2014-01-01

    eurygaster integriceps Puton, commonly known as sunn pest, is a major pest of wheat in Northern Africa, the Middle East and Eastern Europe. This insect injects a prolyl endoprotease into the wheat, destroying the gluten. The purpose of this study was to clone the full length cDNA of the sunn pest prolyl endoprotease (spPEP) for expression in E. coli and to compare the amino acid sequence of the enzyme to other known PEPs in both phylogeny and potential tertiary structure. Sequence analysis shows that the 5ꞌ UTR contains several putative transcription factor binding sites for transcription factors known to be expressed in Drosophila that might be useful targets for inhibition of the enzyme. The spPEP was first identified as a prolyl endoprotease by Darkoh et al., 2010. The enzyme is a unique serine protease of the S9A family by way of its substrate recognition of the gluten proteins, which are greater than 30 kD in size. At 51% maximum identity to known PEPs, homology modeling using SWISS-MODEL, the porcine brain PEP (PDB: 2XWD) was selected in the database of known PEP structures, resulting in a predicted tertiary structure 99% identical to the porcine brain PEP structure. A Km for the recombinant spPEP was determined to be 210 ± 53 µM for the zGly-Pro-pNA substrate in 0.025 M ethanolamine, pH 8.5, containing 0.1 M NaCl at 37 °C with a turnover rate of 172 ± 47 µM Gly-Pro-pNA/s/µM of enzyme. PMID:26462938

  18. Evidence for the Slow Reaction of Hypoxia-Inducible Factor Prolyl Hydroxylase 2 with Oxygen

    PubMed Central

    Flashman, Emily; Hoffart, Lee M.; Hamed, Refaat B.; Bollinger, J. Martin; Krebs, Carsten; Schofield, Christopher J.

    2010-01-01

    SUMMARY The response of animals to hypoxia is mediated by the hypoxia-inducible transcription factor (HIF). Human HIF is regulated by four Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases: Prolyl hydroxylase domain enzymes (PHDs or EGLNs) 1–3 catalyse hydroxylation of two prolyl-residues in HIF, triggering its degradation by the proteasome. Factor inhibiting HIF (FIH) catalyses hydroxylation of an asparagine-residue in HIF, inhibiting its transcriptional activity. Collectively, the HIF hydroxylases negatively regulate HIF in response to increasing oxygen concentration. Prolyl hydroxylase domain 2 (PHD2) is the most important oxygen sensor in human cells; however the underlying kinetic basis of the oxygen sensing function of PHD2 is unclear. We report analyses of the reaction of PHD2 with oxygen. Chemical quench/mass spectrometry experiments showed that reaction of a complex of PHD2, Fe(II), 2OG and the C-terminal oxygen-dependent degradation domain of HIF-α (CODD) with oxygen to form hydroxylated CODD and succinate is much slower (~100 fold) than for other similarly studied 2OG oxygenases. Stopped flow/UV-visible spectroscopy experiments showed that the reaction produces a relatively stable species absorbing at 320nm; Mössbauer spectroscopic experiments implied that this species is likely not a Fe(IV)=O intermediate, as observed for other 2OG oxygenases. Overall the results suggest that, at least compared to other studied 2OG oxygenases, PHD2 reacts relatively slowly with oxygen, a property that may be associated with its function as an oxygen sensor. PMID:20840591

  19. Peptidyl-D-amino acid hydrolase from Loligo vulgaris Lam. Purification and characterization.

    PubMed

    D'Aniello, A; Strazzullo, L

    1984-04-10

    An enzyme, tentatively called peptidyl-D-amino acid hydrolase, has been purified from digestive juice from cecum intestine of Loligo vulgaris. The enzyme hydrolyzes peptides that have a low number of D- or L-amino acids. Proteins, polypeptides, and amino acid derivatives are not hydrolyzed. The enzyme acts as a carboxypeptidase with specificity toward small peptides. Polyacrylamide gel electrophoresis, polyacrylamide gel isoelectric focusing, and gel filtration showed the enzyme to be homogeneous. The native enzyme has Mr = 140,000 and consists of two subunits of Mr = 106,000 and 36,000, respectively. The enzyme has an isoelectric point at pH 6.1. The extinction coefficient is 336,000 at 278 nm and the absorption spectrum reveals no chromophoric cofactors. The apparent Km values for Gly-D-Ala, Gly-L-Ala, L-Ala-D-Ala, L-Ala-L-Ala, D-Leu-D-Leu, and L-Leu-L-Leu are 5.2, 7.7, 2.5, 2.8, 5.4, and 8.6 mM, respectively. The enzyme also hydrolyzes Leu-enkephalin, Met-enkephalin, and [D-Ala2] X Met-enkephalin. It has a broad pH optimum from 7.2 to 8.8 with a maximum at pH 8.0. The enzyme activity is not inhibited or increased by Co2+, Mn2+, Mg2+, Zn2+, Cu2+, and Ca2+ at a concentration of 1 mM or by guanidine chloride (50 mM)urea (3 M), and EDTA (50 mM). 50 mM CaCl2, 1 mM CdCl2, and 1 mM Pb(CH3COO)2 inhibited the enzyme activity by 5-10%. Amino acid analysis of the purified enzyme revealed an abundance of aspartic acid, glutamic acid, glycine, and valine. We hypothesize that the enzyme described here serves to hydrolyze D-amino acid peptides, which are probably present in the nervous system of cephalopods.

  20. 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. PMID:25973480

  1. Purification and characterization of human prolyl dipeptidase DPP8 in Sf9 insect cells.

    PubMed

    Chen, Yuan-Shou; Chien, Chia-Hui; Goparaju, Chandra M; Hsu, John Tsu-An; Liang, Po-Huang; Chen, Xin

    2004-05-01

    DPP8 is a new member of the prolyl dipeptidases, many of which have important biological functions in vivo. DPP8 catalyzes the cleavage at the carboxyl side of the proline residue at the penultimate position. To study its structure and biochemical properties, we have overexpressed the human DPP8 protein in baculovirus infected Sf9 cells. The protein is soluble and can be purified to homogeneity. Using the chromogenic H-Gly-Pro-pNA as the substrate, a kinetic study shows that purified DPP8 is active and has a similar kcat value as that of DPP-IV, a prolyl dipeptidase that is a drug target for type II diabetes. The kinetic constants of DPP8 are also determined for other chromogenic substrates, and the results indicate that DPP8 has substrate preference at both the P1 and P2 sites. The expression system provides means of better understanding the structure, catalytic mechanism, and biological function of DPP8 protein.

  2. Prolyl Isomerase Pin1 Regulates Axon Guidance by Stabilizing CRMP2A Selectively in Distal Axons.

    PubMed

    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

    2015-10-27

    Axon guidance relies on precise translation of extracellular signal gradients into local changes in 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, a 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 levels 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

  3. Prolyl oligopeptidase binds to GAP-43 and functions without its peptidase activity.

    PubMed

    Di Daniel, Elena; Glover, Colin P; Grot, Emma; Chan, Man K; Sanderson, Thirza H; White, Julia H; Ellis, Catherine L; Gallagher, Kathleen T; Uney, James; Thomas, Julia; Maycox, Peter R; Mudge, Anne W

    2009-07-01

    Inhibitors of the enzyme prolyl oligopeptidase (PO) improve performance in rodent learning and memory tasks. PO inhibitors are also implicated in the action of drugs used to treat bipolar disorder: they reverse the effects of three mood stabilizers on the dynamic behaviour of neuronal growth cones. PO cleaves prolyl bonds in short peptides, suggesting that neuropeptides might be its brain substrates. PO is located in the cytosol, however, where it would not contact neuropeptides. Here, we show that mice with a targeted PO null-mutation have altered growth cone dynamics. The wild-type phenotype is restored by PO cDNAs encoding either native or a catalytically-dead enzyme. In addition, we show that PO binds to the growth-associated protein GAP-43, which is a key regulator of synaptic plasticity. Taken together, our results show that peptidase activity is not required for PO function in neurons and suggest that PO instead acts by binding to cytosolic proteins that control growth cone and synaptic function.

  4. Human Collagen Prolyl 4-Hydroxylase Is Activated by Ligands for Its Iron Center.

    PubMed

    Vasta, James D; Raines, Ronald T

    2016-06-14

    Collagen is the most abundant protein in animals. The posttranslational hydroxylation of proline residues in collagen contributes greatly to its conformational stability. Deficient hydroxylation is associated with a variety of disease states, including scurvy. The hydroxylation of proline residues in collagen is catalyzed by an Fe(II)- and α-ketoglutarate-dependent dioxygenase, collagen prolyl 4-hydroxylase (CP4H). CP4H has long been known to suffer oxidative inactivation during catalysis, and the cofactor ascorbate (vitamin C) is required to reactivate the enzyme by reducing its iron center from Fe(III) to Fe(II). Herein, we report on the discovery of the first synthetic activators of CP4H. Specifically, we find that 2,2'-bipyridine-4-carboxylate and 2,2'-bipyridine-5-carboxylate serve as ligands for the iron center in human CP4H that enhance the rate of ascorbate-dependent reactivation. This new mode of CP4H activation is available to other biheteroaryl compounds but does not necessarily extend to other prolyl 4-hydroxylases. As collagen is weakened in many indications, analogous activators of CP4H could have therapeutic benefits. PMID:27183028

  5. Peptide Macrocyclization Catalyzed by a Prolyl Oligopeptidase Involved in α-Amanitin Biosynthesis

    PubMed Central

    Luo, Hong; Hong, Sung-Yong; Sgambelluri, R Michael; Angelos, Evan; Li, Xuan; Walton, Jonathan D

    2014-01-01

    SUMMARY Amatoxins are ribosomally-encoded and post-translationally modified peptides (RiPPs) 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 were proposed to play a role in α- amanitin post-translational processing the exact mechanistic details are not known. Here, we show that a specific prolyl oligopeptidase (GmPOPB) is required for toxin maturation in G. marginata. Recombinant GmPOPB catalyzed two nonprocessive reactions: hydrolysis at an internal Pro to release the C-terminal 25mer from the 35mer propeptide, and transpeptidation at the second Pro to produce the cyclic octamer. On the other hand, we show that GmPOPA, the putative housekeeping POP of G. marginata, behaves like a conventional POP. PMID:25484237

  6. 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. PMID:11177261

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

    PubMed

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

    2006-03-01

    Studies in vitro have established that free tryptophan induces tna operon expression by binding to the ribosome that has just completed synthesis of TnaC-tRNA(Pro), 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-tRNA(Pro) 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-tRNA(Pro) complexes analyzed were formed in vitro; they contained either wild-type TnaC-tRNA(Pro) or its nonfunctional substitute, TnaC(W12R)-tRNA(Pro). Upon comparing these two peptidyl-tRNA-ribosome complexes, free tryptophan was found to block methylation of nucleotide A2572 of wild-type ribosome-TnaC-tRNA(Pro) complexes but not of ribosome-TnaC(W12R)-tRNA(Pro) 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-tRNA(Pro); it also inhibited binding of the antibiotic sparsomycin. These effects were not observed with TnaC(W12R)-tRNA(Pro) mutant complexes. These findings establish that Trp-12 of TnaC-tRNA(Pro) 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. A major portion of DNA gyrase inhibitor microcin B17 undergoes an N,O-peptidyl shift during synthesis.

    PubMed

    Ghilarov, Dmitry; Serebryakova, Marina; Shkundina, Irina; Severinov, Konstantin

    2011-07-29

    Microcin B17 (McB) is a 43-amino acid antibacterial peptide targeting the DNA gyrase. The McB precursor is ribosomally produced and then post-translationally modified by the McbBCD synthase. Active mature McB contains eight oxazole and thiazole heterocycles. Here, we show that a major portion of mature McB contains an additional unusual modification, a backbone ester bond connecting McB residues 51 and 52. The modification results from an N → O shift of the Ser(52) residue located immediately downstream of one of McB thiazole heterocycles. We speculate that the N,O-peptidyl shift undergone by Ser(52) is an intermediate of post-translational modification reactions catalyzed by the McbBCD synthase that normally lead to formation of McB heterocycles.

  9. TWISTED DWARF1, a Unique Plasma Membrane-anchored Immunophilin-like Protein, Interacts with Arabidopsis Multidrug Resistance-like Transporters AtPGP1 and AtPGP19

    PubMed Central

    Geisler, Markus; Kolukisaoglu, H. Üner; Bouchard, Rodolphe; Billion, Karla; Berger, Joachim; Saal, Beate; Frangne, Nathalie; Koncz-Kálmán, Zsuzsanna; Koncz, Csaba; Dudler, Robert; Blakeslee, Joshua J.; Murphy, Angus S.; Martinoia, Enrico; Schulz, Burkhard

    2003-01-01

    Null-mutations of the Arabidopsis FKBP-like immunophilin TWISTED DWARF1 (TWD1) gene cause a pleiotropic phenotype characterized by reduction of cell elongation and disorientated growth of all plant organs. Heterologously expressed TWD1 does not exhibit cis-trans-peptidylprolyl isomerase (PPIase) activity and does not complement yeast FKBP12 mutants, suggesting that TWD1 acts indirectly via protein-protein interaction. Yeast two-hybrid protein interaction screens with TWD1 identified cDNA sequences that encode the C-terminal domain of Arabidopsis multidrugresistance-like ABC transporter AtPGP1. This interaction was verified in vitro. Mapping of protein interaction domains shows that AtPGP1 surprisingly binds to the N-terminus of TWD1 harboring the cis-trans peptidyl-prolyl isomerase-like domain and not to the tetratrico-peptide repeat domain, which has been shown to mediate protein-protein interaction. Unlike all other FKBPs, TWD1 is shown to be an integral membrane protein that colocalizes with its interacting partner AtPGP1 on the plasma membrane. TWD1 also interacts with AtPGP19 (AtMDR1), the closest homologue of AtPGP1. The single gene mutation twd1-1 and double atpgp1-1/atpgp19-1 (atmdr1-1) mutants exhibit similar phenotypes including epinastic growth, reduced inflorescence size, and reduced polar auxin transport, suggesting that a functional TWD1-AtPGP1/AtPGP19 complex is required for proper plant development. PMID:14517332

  10. 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. PMID:26940742

  11. Biochemical properties and expression profile of human prolyl dipeptidase DPP9.

    PubMed

    Tang, Hung-Kuan; Tang, Hsiang-Yun; Hsu, Shu-Ching; Chu, Yue-Ru; Chien, Chia-Hui; Shu, Chin-Hang; Chen, Xin

    2009-05-15

    Dipetidyl peptidase 9 (DPP9) is a prolyl dipeptidase preferentially cleaving the peptide bond after the penultimate proline residue. The biological function of DPP9 is unknown. In this study, we have significantly improved the yield using Strep.Tactin purification system and characterized the biochemical property of DPP9. Moreover, the dimer interaction mode was investigated by introducing a mutation (F842A) at the dimer interface, which abolished the enzymatic activity without disrupting its quaternary structure. Furthermore, DPP9 was found ubiquitously expressed in fibroblasts, epithelial, and blood cells. Surprisingly, contrary to previous report, we found that the expression levels of DPP8 and DPP9 did not change upon the activation of the PBMC or Jurkat cells. These results indicate that the biochemical property of DPP9 is very similar to that of DPP8, its homologous protease. DPP9 and DPP8 are likely redundant proteins carrying out overlapping functions in vivo.

  12. 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.

  13. The prolyl hydroxylase PHD3 identifies proinflammatory macrophages and its expression is regulated by activin A.

    PubMed

    Escribese, María M; Sierra-Filardi, Elena; Nieto, Concha; Samaniego, Rafael; Sánchez-Torres, Carmen; Matsuyama, Takami; Calderon-Gómez, Elisabeth; Vega, Miguel A; Salas, Azucena; Sánchez-Mateos, Paloma; Corbí, Angel L

    2012-08-15

    Modulation of macrophage polarization underlies the onset and resolution of inflammatory processes, with polarization-specific molecules being actively sought as potential diagnostic and therapeutic tools. Based on their cytokine profile upon exposure to pathogenic stimuli, human monocyte-derived macrophages generated in the presence of GM-CSF or M-CSF are considered as proinflammatory (M1) or anti-inflammatory (M2) macrophages, respectively. We report in this study that the prolyl hydroxylase PHD3-encoding EGLN3 gene is specifically expressed by in vitro-generated proinflammatory M1(GM-CSF) human macrophages at the mRNA and protein level. Immunohistochemical analysis revealed the expression of PHD3 in CD163(+) lung macrophages under basal homeostatic conditions, whereas PHD3(+) macrophages were abundantly found in tissues undergoing inflammatory responses (e.g., Crohn's disease and ulcerative colitis) and in tumors. In the case of melanoma, PHD3 expression marked a subset of tumor-associated macrophages that exhibit a weak (e.g., CD163) or absent (e.g., FOLR2) expression of typical M2-polarization markers. EGLN3 gene expression in proinflammatory M1(GM-CSF) macrophages was found to be activin A dependent and could be prevented in the presence of an anti-activin A-blocking Ab or inhibitors of activin receptor-like kinase receptors. Moreover, EGLN3 gene expression was upregulated in response to hypoxia only in M2(M-CSF) macrophages, and the hypoxia-mediated upregulation of EGLN3 expression was significantly impaired by activin A neutralization. These results indicate that EGLN3 gene expression in macrophages is dependent on activin A both under basal and hypoxic conditions and that the expression of the EGLN3-encoded PHD3 prolyl hydroxylase identifies proinflammatory macrophages in vivo and in vitro. PMID:22778395

  14. Investigation of the dimer interface and substrate specificity of prolyl dipeptidase DPP8.

    PubMed

    Lee, Hong-Jen; Chen, Yuan-Shou; Chou, Chi-Yuan; Chien, Chia-Hui; Lin, Chun-Hung; Chang, Gu-Gang; Chen, Xin

    2006-12-15

    DPP8 belongs to the family of prolyl dipeptidases, which are capable of cleaving the peptide bond after a penultimate proline residue. Unlike DPP-IV, a drug target for type II diabetes, no information is available on the crystal structure of DPP8, the regulation of its enzymatic activity, or its substrate specificity. In this study, using analytical ultracentrifugation and native gel electrophoresis, we show that the DPP8 protein is predominantly dimeric when purified or in the cell extracts. Four conserved residues in the C-terminal loop of DPP8 (Phe(822), Val(833), Tyr(844), and His(859)), corresponding to those located at the dimer interface of DPP-IV, were individually mutated to Ala. Surprisingly, unlike DPP-IV, these single-site mutations abolished the enzymatic activity of DPP8 without disrupting its quaternary structure, indicating that dimerization itself is not sufficient for the optimal enzymatic activity of DPP8. Moreover, these mutations not only decreased k(cat), as did the corresponding DPP-IV mutations, but also dramatically increased K(m). We further show that the K(m) effect is independent of the substrate assayed. Finally, we identified the distinctive and strict substrate selectivity of DPP8 for hydrophobic or basic residues at the P2 site, which is in sharp contrast to the much less discriminative substrate specificity of DPP-IV. Our study has identified the residues absolutely required for the optimal activity of DPP8 and its unique substrate specificity. This study extends the functional importance of the C-terminal loop to the whole family of prolyl dipeptidases.

  15. Endogenous 2-oxoglutarate levels impact potencies of competitive HIF prolyl hydroxylase inhibitors.

    PubMed

    Thirstrup, Kenneth; Christensen, Søren; Møller, Henriette Aaris; Ritzén, Andreas; Bergström, Ann-Louise; Sager, Thomas Nikolaj; Jensen, Henrik Sindal

    2011-09-01

    The stability and transcriptional activity of the hypoxia-inducible factors (HIFs) are regulated by oxygen-dependent hydroxylation that is catalyzed by three HIF prolyl 4-hydroxylases (HPHs). Use of HPH inhibition as a mean for HIF-upregulation has recently gained interest as a potential treatment paradigm against neurodegenerative diseases like ischemia and Parkinson's disease. In the present investigation we report the development of a new and robust assay to measure HPH activity. The assay is based on capture of hydroxylated peptide product by the von Hippel-Lindau protein which is directly measured in a scintillation proximity assay. In addition we describe the determination of HPH subtype potencies of HPH inhibitors which either directly or indirectly inhibit the HPH enzyme. The potencies of the HPH inhibitors displayed almost identical IC(50) values toward the HPH1 and HPH2 subtype while the potency against the HPH3 subtype was increased for several of the compounds. For the most potent compound, a hydroxyl thiazole derivative, the potency against HPH2 and HPH3 was 7nM and 0.49nM, respectively corresponding to a 14-fold difference. These results suggest that HPH subtype-selective compounds may be developed. In addition we determined the 2-oxoglutarate concentration in brain tissue and neuronal cell lines as 2-oxoglutarate is an important co-factor used by the HPH enzyme during the hydroxylation reaction. The high intracellular 2-oxoglutarate concentration provides an explanation for the diminished cellular HIF activating potency of a competitive HPH inhibitor compared to its orders of magnitude higher HPH inhibiting potency. The present reported data suggest that in the development of specific Hif prolyl hydroxylase inhibitors the high 2-oxoglutarate tissue level should be taken into account as this might affect the cellular potency. Thus to specifically inhibit the intracellular HPH enzymatic reaction a competitive inhibitor with a low Ki should be developed.

  16. 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.

  17. 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

  18. Optimized expression of prolyl aminopeptidase in Pichia pastoris and its characteristics after glycosylation.

    PubMed

    Yang, Hongyu; Zhu, Qiang; Zhou, Nandi; Tian, Yaping

    2016-11-01

    Prolyl aminopeptidases are specific exopeptidases that catalyze the hydrolysis of the N-terminus proline residue of peptides and proteins. In the present study, the prolyl aminopeptidase gene (pap) from Aspergillus oryzae JN-412 was optimized through the codon usage of Pichia pastoris. Both the native and optimized pap genes were inserted into the expression vector pPIC9 K and were successfully expressed in P. pastoris. Additionally, the activity of the intracellular enzyme expressed by the recombinant optimized pap gene reached 61.26 U mL(-1), an activity that is 2.1-fold higher than that of the native gene. The recombinant enzyme was purified by one-step elution through Ni-affinity chromatography. The optimal temperature and pH of the purified PAP were 60 °C and 7.5, respectively. Additionally, the recombinant PAP was recovered at a yield greater than 65 % at an extremely broad range of pH values from 6 to 10 after treatment at 50 °C for 6 h. The molecular weight of the recombinant PAP decreased from 50 kDa to 48 kDa after treatment with a deglycosylation enzyme, indicating that the recombinant PAP was completely glycosylated. The glycosylated PAP exhibited high thermo-stability. Half of the activity remained after incubation at 50 °C for 50 h, whereas the remaining activity of PAP expressed in E. coli was only 10 % after incubation at 50 °C for 1 h. PAP could be activated by the appropriate salt concentration and exhibited salt tolerance against NaCl at a concentration up to 5 mol L(-1). PMID:27628336

  19. 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

  20. Solid-phase synthesis of peptide-4-nitroanilides.

    PubMed

    Kaspari, A; Schierhorn, A; Schutkowski, M

    1996-11-01

    A wide variety of Glu/Asp and Gln containing peptide-4-nitroanilides and other chromogenic peptidyl-arylamides could be quickly synthesized by a Fmoc-based solid-phase synthesis strategy employing the side-chain carboxyl groups for transient anchoring to the resin. Suitable synthons for this method, Fmoc-Glu-NH-Np and Fmoc-Asp-NH-Np, were prepared using a diphenylphosphinic chloride-mediated coupling reaction. Peptides of the common structure Suc-Ala-Phe-Pro-Xaa-NH-Np (Xaa = Glu/Asp, Gln) were synthesized and were shown to be substrates for the protease subtilisin Carlsberg (E.C.3.4.21.14a) and for peptidyl-prolyl cis/trans-isomerases (PPIases E.C. 5.2.1.8.). The method was extended to amino acids possessing a side chain missing an anchor for binding to the matrix. We synthesized Suc-Ala-Phe-Pro-Gln-Phe-NH-Np anchoring the dipeptide derivative Fmoc-Glu-Phe-NH-Np with the carboxyl group to Rink amide resin using standard SPPS procedures. Additionally this procedure allowed us the preparation of peptidyl-arylamides, utilizing the commercial available Fmoc-Glu-OAll as building block. A mixture of pentapeptide-4-nitroanilides with the general sequence Ala-Ala-Xaa-Pro-Gln-NH-Np was synthesized. Electrospray ionization mass spectrometry (ESI-MS) was used to evaluate the hydrolysis of the peptide mixture by the protease subtilisin Carlsberg. It could be shown that peptides with the hydrophobic amino acids Phe, Tyr, Leu and Val in the varied P3-position were most rapidly cleaved under the chosen conditions. Hydrolysis of the Gln-NH-Np bond in Ala-Ala-Pro-Pro-Gln-NH-Np has not been observed.

  1. 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

  2. Hazard identification of cis/trans-zearalenone through the looking-glass.

    PubMed

    Dellafiora, Luca; Galaverna, Gianni; Dall'Asta, Chiara; Cozzini, Pietro

    2015-12-01

    Among the food-related health issues, the presence of contaminants has a prominent role, due to the wide range of exogenous compounds that can occur in food commodities and to their large differences in structure and biological activity. A comprehensive assessment of the related risk is thus actually demanding in terms of time and facilities involved. In this context, the use of computational strategies can be an effective choice for supporting the hazard identification procedure at the early stage. In this work, we focused on the food contaminant zearalenone by comparing the trans and cis isomers, respectively the well-known mycoestrogen and its still largely understudied isomer. We estimated the possible effects exerted by human metabolism on the xenoestrogenicity of cis-ZEN by using a validated in silico strategy based on docking simulations and rescoring procedures. Similarly, the exploitation of the most promising enzymatic detoxifying routes designed for trans-ZEN - which relies on the enzyme lactono hydrolase from Clonostachys rosea - has been assessed for the cis-isomer as well. Our results showed that both isomers can act as functional analogues with respect to xenoestrogenic activity, and several cis-ZEN metabolites with high biological potential have been identified. On the contrary, in spite of the high degree of structural analogy, the cis isomer showed a pattern of interaction with the degrading enzyme in stark contrast with that observed for trans-ZEN. For these reasons, the outcomes presented herein strongly support the inclusion of cis-ZEN in further studies of occurrence, metabolism and bioactivity assessment, and suggest the need for a dedicated handling for the cis isomer in risk assessment studies. PMID:26391124

  3. 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.

  4. Extensive compensatory cis-trans regulation in the evolution of mouse gene expression

    PubMed Central

    Goncalves, Angela; Leigh-Brown, Sarah; Thybert, David; Stefflova, Klara; Turro, Ernest; Flicek, Paul; Brazma, Alvis; Odom, Duncan T.; Marioni, John C.

    2012-01-01

    Gene expression levels are thought to diverge primarily via regulatory mutations in trans within species, and in cis between species. To test this hypothesis in mammals we used RNA-sequencing to measure gene expression divergence between C57BL/6J and CAST/EiJ mouse strains and allele-specific expression in their F1 progeny. We identified 535 genes with parent-of-origin specific expression patterns, although few of these showed full allelic silencing. This suggests that the number of imprinted genes in a typical mouse somatic tissue is relatively small. In the set of nonimprinted genes, 32% showed evidence of divergent expression between the two strains. Of these, 2% could be attributed purely to variants acting in trans, while 43% were attributable only to variants acting in cis. The genes with expression divergence driven by changes in trans showed significantly higher sequence constraint than genes where the divergence was explained by variants acting in cis. The remaining genes with divergent patterns of expression (55%) were regulated by a combination of variants acting in cis and variants acting in trans. Intriguingly, the changes in expression induced by the cis and trans variants were in opposite directions more frequently than expected by chance, implying that compensatory regulation to stabilize gene expression levels is widespread. We propose that expression levels of genes regulated by this mechanism are fine-tuned by cis variants that arise following regulatory changes in trans, suggesting that many cis variants are not the primary targets of natural selection. PMID:22919075

  5. 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.

  6. Hazard identification of cis/trans-zearalenone through the looking-glass.

    PubMed

    Dellafiora, Luca; Galaverna, Gianni; Dall'Asta, Chiara; Cozzini, Pietro

    2015-12-01

    Among the food-related health issues, the presence of contaminants has a prominent role, due to the wide range of exogenous compounds that can occur in food commodities and to their large differences in structure and biological activity. A comprehensive assessment of the related risk is thus actually demanding in terms of time and facilities involved. In this context, the use of computational strategies can be an effective choice for supporting the hazard identification procedure at the early stage. In this work, we focused on the food contaminant zearalenone by comparing the trans and cis isomers, respectively the well-known mycoestrogen and its still largely understudied isomer. We estimated the possible effects exerted by human metabolism on the xenoestrogenicity of cis-ZEN by using a validated in silico strategy based on docking simulations and rescoring procedures. Similarly, the exploitation of the most promising enzymatic detoxifying routes designed for trans-ZEN - which relies on the enzyme lactono hydrolase from Clonostachys rosea - has been assessed for the cis-isomer as well. Our results showed that both isomers can act as functional analogues with respect to xenoestrogenic activity, and several cis-ZEN metabolites with high biological potential have been identified. On the contrary, in spite of the high degree of structural analogy, the cis isomer showed a pattern of interaction with the degrading enzyme in stark contrast with that observed for trans-ZEN. For these reasons, the outcomes presented herein strongly support the inclusion of cis-ZEN in further studies of occurrence, metabolism and bioactivity assessment, and suggest the need for a dedicated handling for the cis isomer in risk assessment studies.

  7. 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.

  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. Cis-trans isomerism in mononuclear nickel(II). beta. -ketoenamine complexes

    SciTech Connect

    Maverick, A.W.; Fronczek, F.R.; Martone, D.P. ); Bradbury, J.R. )

    1989-07-01

    Solution NMR and x-ray crystallographic structural studies of square-planar nickel(II) Schiff base complexes are reported. In contrast to recently prepared cofacial binuclear complexes derived from bis({beta}-ketoenamines), which have the cis arrangement of O and NH donors about each metal atom, two representative mononuclear complexes, Ni(acim){sub 2} (acimH = 4-amino-3-penten-2-one) and Ni-(bzacim){sub 2} (bzacimH = 3-amino-1-phenyl-2-buten-1-one), are produced as trans isomers. Ni(acim){sub 2} (NiC{sub 10}H{sub 16}N{sub 2}O{sub 2}) is orthorhombic, space group Ccca, a = 16.770(2), b = 15.054(2), c = 13.494(1) {angstrom}, Z = 12, R = 0.055, R{sub w} = 0.042 for 143 parameters and 1,748 reflectons with I > 1{sigma}(I). Ni(bzacim){sub 2} (NiC{sub 20}H{sub 20}n{sub 2}O{sub 2}) is monoclinic, space group P2{sub 1}/c, a = 5.9186(8), b = 13.694(2), c = 11.944(4) {angstrom}, {beta} = 112.18(2){degree}, Z = 2, R = 0.038, R{sub w} = 0.037 for 156 parameters and 1,589 reflections with I > 2{sigma}(I). Crystals of Ni(bzacim){sub 2} contain centrosymmetric, nearly planar trans molecules. The structure of Ni(acim){sub 2} contains independent molecules with 2 and 222 symmetry, but both of these are disordered, so that whether the cis or trans isomer is present cannot be determined. However, solution {sup 1}H NOE and lanthanide shift measurements indicate that the trans isomer predominates for both compounds.

  10. Improved HRGC separation of cis, trans CLA isomers as Diels-Alder adducts of alkyl esters.

    PubMed

    Blasi, F; Giua, L; Lombardi, G; Codini, M; Simonetti, M S; Damiani, P; Cossignani, L

    2011-05-01

    This paper reports the separation of four isomers of conjugated linoleic acid (CLA), c,t/t,c-8,10; c,t/t,c-9,11; c,t/t,c-10,12; c,t/t,c-11,13, after reaction of esterification with aliphatic alcohols of different chain length and adduct formation with 4-methyl-1,2,4-triazoline-3,5-dione (MTAD). The high resolution gas chromatographic analyses were carried out using a simple 50-m cyanopropyl polysiloxane capillary column both with a flame ionization detector and a mass spectrometer. The resolution between the two pair of isomers: c,t/t,c-9,11 and c,t/t,c-10,12 and between c,t/t,c-10,12 and c,t/t,c-11,13 isomers were good for all the investigated alkyl esters and increased with the chain length of alcohol esterified to carboxylic moiety of CLA isomers. The most interesting result was relative to the c,t/t,c-8,10 and c,t/t,c-9,11 isomers, critical pair of isomers also when analyzed with a 120-m cyanopropyl polysiloxane capillary column; their resolution also increased from methyl to hexyl esters of CLA isomers and reached an acceptable value (0.8) in the case of hexyl esters. The best resolutions of the four considered CLA isomers were obtained with the hexyl esters of MTAD adducts of the isomers, without excessive analysis time. This method was useful and simple to evaluate the profile of the four main c,t isomers in commercial CLA samples.

  11. Diffusion ordered spectroscopy for resolution of double bonded cis, trans-isomers

    NASA Astrophysics Data System (ADS)

    Chaudhari, Sachin Rama; Suryaprakash, N.

    2012-06-01

    NMR spectroscopic separation of double bonded cis- and trans-isomers, that have different molecular shapes but identical mass have been carried out using Diffusion Ordered Spectroscopy (DOSY). The mixtures of fumaric acid and maleic acid, that have similar hydrodynamic radii, have resolved been 'on the basis of their diffusion coefficients arising due to their different tendencies to associate with micelles or reverse micelles. Sodium dodecyl sulfate (SDS) and Dioctyl sulfosuccinate sodium salt (AOT) have been used as the media to mimic the chromatographic conditions, modify the average mobility and to achieve differential diffusion rates. The best separation of the components has been achieved by Dioctyl sulfosuccinate sodium salt (AOT) in D2O solution.

  12. A theoretical study of the stability of DNA binding with cis/trans platin.

    PubMed

    Srivastava, Seema; Khan, Irfan Ali; Srivastava, Shinoo; Gupta, Vishwambhar Dayal

    2004-12-01

    Both cis- and trans-platins are known to form intra- and interstrand cross-linking with DNA. Since the nature and strength of binding is different, it makes their efficacy as anti-tumour drug different. In the present communication, we report theoretical analysis by using an amended Zimm and Bragg theory, to explain the melting behaviour and heat capacity of DNA with and without platin binding. The sharpness of transition has been examined in terms of half width and sensitivity parameter (deltaH/sigma). The experimental measurements of Pilch et al (J Mol Biol 2000, 296, 803) and Ctirad and Brabec (J Biol Chem 2001, 276, 9655) have been used. PMID:22900359

  13. 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

  14. 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

    2016-01-01

    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

  15. 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

  16. 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

  17. 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

    2015-07-01

    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.

  18. 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

    2015-07-01

    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. PMID:26068096

  19. OsPOP5, A Prolyl Oligopeptidase Family Gene from Rice Confers Abiotic Stress Tolerance in Escherichia coli

    PubMed Central

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

    2013-01-01

    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. PMID:24152437

  1. 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.

  2. Chemical engineering of the peptidyl transferase center reveals an important role of the 2′-hydroxyl group of A2451

    PubMed Central

    Erlacher, Matthias D.; Lang, Kathrin; Shankaran, Nisha; Wotzel, Brigitte; Hüttenhofer, Alexander; Micura, Ronald; Mankin, Alexander S.; Polacek, Norbert

    2005-01-01

    The main enzymatic reaction of the large ribosomal subunit is peptide bond formation. Ribosome crystallography showed that A2451 of 23S rRNA makes the closest approach to the attacking amino group of aminoacyl-tRNA. Mutations of A2451 had relatively small effects on transpeptidation and failed to unequivocally identify the crucial functional group(s). Here, we employed an in vitro reconstitution system for chemical engineering the peptidyl transferase center by introducing non-natural nucleosides at position A2451. This allowed us to investigate the peptidyl transfer reaction performed by a ribosome that contained a modified nucleoside at the active site. The main finding is that ribosomes carrying a 2′-deoxyribose at A2451 showed a compromised peptidyl transferase activity. In variance, adenine base modifications and even the removal of the entire nucleobase at A2451 had only little impact on peptide bond formation, as long as the 2′-hydroxyl was present. This implicates a functional or structural role of the 2′-hydroxyl group at A2451 for transpeptidation. PMID:15767286

  3. Post-translationally Abnormal Collagens of Prolyl 3-Hydroxylase-2 Null Mice Offer a Pathobiological Mechanism for the High Myopia Linked to Human LEPREL1 Mutations*

    PubMed Central

    Hudson, David M.; Joeng, Kyu Sang; Werther, Rachel; Rajagopal, Abbhirami; Weis, MaryAnn; Lee, Brendan H.; Eyre, David R.

    2015-01-01

    Myopia, the leading cause of visual impairment worldwide, results from an increase in the axial length of the eyeball. Mutations in LEPREL1, the gene encoding prolyl 3-hydroxylase-2 (P3H2), have recently been identified in individuals with recessively inherited nonsyndromic severe myopia. P3H2 is a member of a family of genes that includes three isoenzymes of prolyl 3-hydroxylase (P3H), P3H1, P3H2, and P3H3. Fundamentally, it is understood that P3H1 is responsible for converting proline to 3-hydroxyproline. This limited additional knowledge also suggests that each isoenzyme has evolved different collagen sequence-preferred substrate specificities. In this study, differences in prolyl 3-hydroxylation were screened in eye tissues from P3h2-null (P3h2n/n) and wild-type mice to seek tissue-specific effects due the lack of P3H2 activity on post-translational collagen chemistry that could explain myopia. The mice were viable and had no gross musculoskeletal phenotypes. Tissues from sclera and cornea (type I collagen) and lens capsule (type IV collagen) were dissected from mouse eyes, and multiple sites of prolyl 3-hydroxylation were identified by mass spectrometry. The level of prolyl 3-hydroxylation at multiple substrate sites from type I collagen chains was high in sclera, similar to tendon. Almost every known site of prolyl 3-hydroxylation in types I and IV collagen from P3h2n/n mouse eye tissues was significantly under-hydroxylated compared with their wild-type littermates. We conclude that altered collagen prolyl 3-hydroxylation is caused by loss of P3H2. We hypothesize that this leads to structural abnormalities in multiple eye tissues, but particularly sclera, causing progressive myopia. PMID:25645914

  4. 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

  5. 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.

  6. Peptidyl-glycine alpha-amidating monooxygenase is present in islet secretory granules of the anglerfish, Lophius americanus.

    PubMed

    Mackin, R B; Flacker, J M; Mackin, J A; Noe, B D

    1987-08-01

    Anglerfish islet secretory granules have been examined for the presence of an enzyme which could perform C-terminal amidation of glucagon-like peptide II and possibly anglerfish peptide Y. Using [125I]D-Tyr-Val-Gly as substrate, a peptidyl-glycine alpha-amidating monooxygenase (PAM) was detected in islet secretory granule lysates. The enzyme is active between pH 6.0 and 8.5 and exhibits maximal activity near pH 7.0. The islet PAM requires Cu2+, ascorbate, and molecular oxygen for activity. Other divalent metal ions and redox cofactors were tested and found to be inactive in the assay. Even though added Cu2+ and ascorbate are required for detecting islet PAM activity, when these factors were incubated with substrate in the absence of secretory granule lysate, no activity was observed. It was also found that the addition of higher than optimal concentrations of either Cu2+ or ascorbate inhibited amidating activity. The results demonstrate that a PAM is present in secretory granules of anglerfish islet tissue. The characteristics of the islet PAM are similar to those of PAMs identified and characterized in other tissues which produce bioactive C-terminally amidated peptides. PMID:3305155

  7. S-peptide as a potent peptidyl linker for protein cross-linking by microbial transglutaminase from Streptomyces mobaraensis.

    PubMed

    Kamiya, Noriho; Tanaka, Tsutomu; Suzuki, Tsutomu; Takazawa, Takeshi; Takeda, Shuji; Watanabe, Kimitsuna; Nagamune, Teruyuki

    2003-01-01

    We have found that ribonuclease S-peptide can work as a novel peptidyl substrate in protein cross-linking reactions catalyzed by microbial transglutaminase (MTG) from Streptomyces mobaraensis. Enhanced green fluorescent protein tethered to S-peptide at its N-terminus (S-tag-EGFP) appeared to be efficiently cross-linked by MTG. As wild-type EGFP was not susceptible to cross-linking, the S-peptide moiety is likely to be responsible for the cross-linking. A site-directed mutation study assigned Gln15 in the S-peptide sequence as the sole acyl donor. Mass spectrometric analysis showed that two Lys residues (Lys5 and Lys11) in the S-peptide sequence functioned as acyl acceptors. We also succeeded in direct monitoring of the cross-linking process by virtue of fluorescence resonance energy transfer (FRET) between S-tag-EGFP and its blue fluorescent color variant (S-tag-EBFP). The protein cross-linking was tunable by either engineering S-peptide sequence or capping the S-peptide moiety with S-protein, the partner protein of S-peptide for the formation of ribonuclease A. The latter indicates that S-protein can be used as a specific inhibitor of S-peptide-directed protein cross-linking by MTG. The controllable protein cross-linking of S-peptide as a potent substrate of MTG will shed new light on biomolecule conjugation. PMID:12643745

  8. 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

  9. 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

  10. Initiation of Phage Infection by Partial Unfolding and Prolyl Isomerization*♦

    PubMed Central

    Hoffmann-Thoms, Stephanie; Weininger, Ulrich; Eckert, Barbara; Jakob, Roman P.; Koch, Johanna R.; Balbach, Jochen; Schmid, Franz X.

    2013-01-01

    Infection of Escherichia coli by the filamentous phage fd starts with the binding of the N2 domain of the phage gene-3-protein to an F pilus. This interaction triggers partial unfolding of the gene-3-protein, cis → trans isomerization at Pro-213, and domain disassembly, thereby exposing its binding site for the ultimate receptor TolA. The trans-proline sets a molecular timer to maintain the binding-active state long enough for the phage to interact with TolA. We elucidated the changes in structure and local stability that lead to partial unfolding and thus to the activation of the gene-3-protein for phage infection. Protein folding and TolA binding experiments were combined with real-time NMR spectroscopy, amide hydrogen exchange measurements, and phage infectivity assays. In combination, the results provide a molecular picture of how a local unfolding reaction couples with prolyl isomerization not only to generate the activated state of a protein but also to maintain it for an extended time. PMID:23486474

  11. High Myopia Caused by a Mutation in LEPREL1, Encoding Prolyl 3-Hydroxylase 2

    PubMed Central

    Mordechai, Shikma; Gradstein, Libe; Pasanen, Annika; Ofir, Rivka; El Amour, Khalil; Levy, Jaime; Belfair, Nadav; Lifshitz, Tova; Joshua, Sara; Narkis, Ginat; Elbedour, Khalil; Myllyharju, Johanna; Birk, Ohad S.

    2011-01-01

    Autosomal-recessive high-grade axial myopia was diagnosed in Bedouin Israeli consanguineous kindred. Some affected individuals also had variable expressivity of early-onset cataracts, peripheral vitreo-retinal degeneration, and secondary sight loss due to severe retinal detachments. Through genome-wide linkage analysis, the disease-associated gene was mapped to ∼1.7 Mb on chromosome 3q28 (the maximum LOD score was 11.5 at θ = 0 for marker D3S1314). Sequencing of the entire coding regions and intron-exon boundaries of the six genes within the defined locus identified a single mutation (c.1523G>T) in exon 10 of LEPREL1, encoding prolyl 3-hydroxylase 2 (P3H2), a 2-oxoglutarate-dependent dioxygenase that hydroxylates collagens. The mutation affects a glycine that is conserved within P3H isozymes. Analysis of wild-type and p.Gly508Val (c.1523G>T) mutant recombinant P3H2 polypeptides expressed in insect cells showed that the mutation led to complete inactivation of P3H2. PMID:21885030

  12. 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.

  13. HIF prolyl hydroxylase inhibitors for the treatment of renal anaemia and beyond.

    PubMed

    Maxwell, Patrick H; Eckardt, Kai-Uwe

    2016-03-01

    Small-molecule stabilizers of hypoxia inducible factor (HIF) are being developed for the treatment of renal anaemia. These molecules inhibit prolyl hydroxylase domain-containing (PHD) enzymes, resulting in HIF activation and increased production of erythropoietin. Currently, renal anaemia is treated with recombinant human erythropoietin or related analogues, referred to as conventional erythropoiesis stimulating agents (ESAs). Advantages of PHD enzyme inhibitors over conventional ESAs include their oral administration and their simpler - and potentially cheaper - production. Importantly, inhibition of PHD enzymes is likely to have a range of consequences other than increasing levels of erythropoietin, and these effects could be beneficial - for instance by reducing the need for parenteral iron - but might in some instances be harmful. Several companies are currently testing PHD enzyme inhibitors in patients with renal anaemia and have reported clear evidence of efficacy without serious safety concerns. A central question that current studies are beginning to address is whether using PHD enzyme inhibitors will influence hard end points, including mortality and the rate of cardiovascular events. In terms of approaches to therapy, the exquisite specificity of conventional ESAs is a striking contrast to the pleiotropic effects of activating HIF. Excitingly, PHD inhibitors could also be useful for conditions besides renal anaemia, such as protection from ischaemic injury. PMID:26656456

  14. 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

  15. A Prolyl-isomerase Mediates Dopamine-dependent Plasticity and Cocaine Motor Sensitization

    PubMed Central

    Park, Joo Min; Hu, Jia-Hua; Milshteyn, Aleksandr; Zhang, Ping-Wu; Moore, Chester G.; Park, Sungjin; Datko, Michael C.; Domingo, Racquel D.; Reyes, Cindy M.; Wang, Xiaodong J.; Etzkorn, Felicia A.; Xiao, Bo; Szumlinski, Karen K.; Kern, Dorothee; Linden, David J.

    2013-01-01

    Summary Synaptic plasticity induced by cocaine and other drugs underlies addiction. Here we elucidate molecular events at synapses that cause this plasticity and the resulting behavioral response to cocaine in mice. In response to D1 dopamine receptor signaling that is induced by drug administration, the glutamate receptor protein mGluR5 is phosphorylated by MAP kinase, which we show potentiates Pin1-mediated prolyl isomerization of mGluR5 in instances where the product of an activity-dependent gene, Homer1a, is present to enable Pin1-mGluR5 interaction. These biochemical events potentiate NMDA receptor-mediated currents that underlie synaptic plasticity and cocaine-evoked motor sensitization as tested in mice with relevant mutations. The findings elucidate how a coincidence of signals from the nucleus and the synapse can render mGluR5 accessible to activation with consequences for drug-induced dopamine responses, and point to depotentiation at corticostriatal synapses as a possible therapeutic target for treating addiction. PMID:23911326

  16. Novel isoindoline compounds for potent and selective inhibition of prolyl dipeptidase DPP8.

    PubMed

    Jiaang, Weir-Torn; Chen, Yuan-Shou; Hsu, Tsu; Wu, Ssu-Hui; Chien, Chia-Hui; Chang, Chung-Nien; Chang, Sheng-Ping; Lee, Shiow-Ju; Chen, Xin

    2005-02-01

    DPP8 is a prolyl dipeptidase homologous to DPP-IV, which is a drug target for Type II diabetes. The biological function of DPP8 is not known. To identify potent and selective chemical compounds against DPP8, we have synthesized a series of isoquinoline and isoindoline derivatives and have tested their inhibitory activity against DPP8, DPP-IV and DPP-II. Isoindoline derivatives were found to be more potent DPP8 inhibitors than isoquinoline derivatives. Isoindoline with a 1-(4,4'-difluor-benzhydryl)-piperazine group at the P2 site was observed to be a very potent DPP8 inhibitor, having an IC(50) value of 14nM with at least a 2500-fold selectivity over either DPP-IV or DPP-II. From SAR results, we speculate that the S1 site of DPP8 may be larger than that of DPP-IV, which would allow the accommodation of larger C-terminal residues, such as isoquinoline or isoindoline.

  17. 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

  18. 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

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

    PubMed

    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

    2014-08-12

    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 (Prep(gt/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 Prep(gt/gt) and wild-type mice. Furthermore, hyperinsulinemic euglycemic clamp studies showed no difference between Prep(gt/gt) and wild-type control mice. Central PREP regulation of insulin and glucagon secretion appears to be mediated by the autonomic nervous system because Prep(gt/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 Prep(gt/gt) mice. Taken together, our results unmask a previously unknown player in central regulation of glucose metabolism and pancreatic function.

  20. Prolyl Endopeptidase (PREP) is Associated With Male Reproductive Functions and Gamete Physiology in Mice.

    PubMed

    Dotolo, Raffaele; Kim, Jung Dae; Pariante, Paolo; Minucci, Sergio; Diano, Sabrina

    2016-03-01

    Prolyl endopeptidase (PREP) is a serine protease which has been implicated in many biological processes, such as the maturation and degradation of peptide hormones and neuropeptides, learning and memory, cell proliferation and differentiation, and glucose metabolism. A small number of reports have also suggested PREP participation in both male and female reproduction-associated processes. In the present work, we examined PREP distribution in male germ cells and studied the effects of its knockdown (Prep(gt/gt)) on testis and sperm in adult mice. The protein is expressed and localized in elongating spermatids and luminal spermatozoa of wild type (wt) mice, as well as Sertoli, Leydig, and peritubular cells. PREP is also expressed in the head and midpiece of epididymal spermatozoa, whereas the remaining tail region shows a weaker signal. Furthermore, testis weight, histology of seminiferous tubules, and epididymal sperm parameters were assessed in wt and Prep(gt/gt) mice: wild type testes have larger average tubule and lumen diameter; in addition, lumenal composition of seminiferous tubules is dissimilar between wt and Prep(gt/gt), as the percentage of spermiated tubules is much higher in wt. Finally, total sperm count, sperm motility, and normal morphology are also higher in wt than in Prep(gt/gt). These results show for the first time that the expression of PREP could be necessary for a correct reproductive function, and suggest that the enzyme may play a role in mouse spermatogenesis and sperm physiology.

  1. Identification of Selective and Potent Inhibitors of Fibroblast Activation Protein and Prolyl Oligopeptidase

    PubMed Central

    Poplawski, Sarah E.; Lai, Jack H.; Li, Youhua; Jin, Zhiping; Liu, Yuxin; Wu, Wengen; Wu, Yong; Zhou, Yuhong; Sudmeier, James L.; Sanford, David G.; Bachovchin, William W.

    2014-01-01

    Fibroblast activation protein (FAP) is a serine protease selectively expressed on reactive stromal fibroblasts of epithelial carcinomas. It is widely believed to play a role in tumor invasion and metastasis and therefore to represent a potential new drug target for cancer. Investigation into its biological function, however, has been hampered by the current unavailability of selective inhibitors. The challenge has been in identifying inhibitors that are selective for FAP over both the dipeptidyl peptidases (DPPs), with which it shares exopeptidase specificity, and prolyl oligopeptidase (PREP), with which it shares endopeptidase specificity. Here, we report the first potent FAP inhibitor with selectivity over both the DPPs and PREP, N-(pyridine-4-carbonyl)-d-Ala-boroPro (ARI-3099, 6). We also report a similarly potent and selective PREP inhibitor, N-(pyridine-3-carbonyl)-Val-boroPro (ARI-3531, 22). Both are boronic acid based inhibitors, demonstrating that high selectivity can be achieved using this electrophile. The inhibitors are stable, easy to synthesize, and should prove to be useful in helping to elucidate the biological functions of these two unique and interesting enzymes, as well as their potential as drug targets. PMID:23594271

  2. Role of prolyl hydroxylase domain proteins in the regulation of insulin secretion.

    PubMed

    Huang, Mei; Paglialunga, Sabina; Wong, Julia M-K; Hoang, Monica; Pillai, Renjitha; Joseph, Jamie W

    2016-03-01

    Type 2 diabetes is associated with impaired nutrient-regulated anaplerosis and insulin secretion in pancreatic β-cells. One key anaplerotic substrate that may be involved in regulating insulin release is α-ketoglutarate (αKG). Since prolyl hydroxylase domain proteins (PHDs) can metabolize cytosolic αKG, we sought to explore the role of this enzyme in the regulation of β-cell function. The oxygen-sensing PHDs regulate the stability of hypoxia-inducible factor 1α (HIF1α) as well as other proline-containing proteins by catalyzing the hydroxylation of proline residues. This reaction is dependent on sufficient levels of oxygen, iron, and αKG. In the present study, we utilized both pharmacological and genetic approaches to assess the impact of inhibiting PHD activity on β-cell function. We demonstrate that ethyl-3,4-dihydroxybenzoate (EDHB), a PHD inhibitor, significantly blunted glucose-stimulated insulin secretion (GSIS) from 832/13 clonal cells, rat, and human islets. EDHB reduced glucose utilization, ATP/ADP ratio, and key TCA cycle intermediates such as pyruvate, citrate, fumarate, and malate. siRNA-mediated knockdown of PHD1 and PHD3 inhibited GSIS, whereas siRNA-mediated knockdown of PHD2 had no effect on GSIS. Taken together, the current results demonstrate an important role for PHDs as mediators of islet insulin secretion. PMID:26997627

  3. A prolyl-isomerase mediates dopamine-dependent plasticity and cocaine motor sensitization.

    PubMed

    Park, Joo Min; Hu, Jia-Hua; Milshteyn, Aleksandr; Zhang, Ping-Wu; Moore, Chester G; Park, Sungjin; Datko, Michael C; Domingo, Racquel D; Reyes, Cindy M; Wang, Xiaodong J; Etzkorn, Felicia A; Xiao, Bo; Szumlinski, Karen K; Kern, Dorothee; Linden, David J; Worley, Paul F

    2013-08-01

    Synaptic plasticity induced by cocaine and other drugs underlies addiction. Here we elucidate molecular events at synapses that cause this plasticity and the resulting behavioral response to cocaine in mice. In response to D1-dopamine-receptor signaling that is induced by drug administration, the glutamate-receptor protein metabotropic glutamate receptor 5 (mGluR5) is phosphorylated by microtubule-associated protein kinase (MAPK), which we show potentiates Pin1-mediated prolyl-isomerization of mGluR5 in instances where the product of an activity-dependent gene, Homer1a, is present to enable Pin1-mGluR5 interaction. These biochemical events potentiate N-methyl-D-aspartate receptor (NMDAR)-mediated currents that underlie synaptic plasticity and cocaine-evoked motor sensitization as tested in mice with relevant mutations. The findings elucidate how a coincidence of signals from the nucleus and the synapse can render mGluR5 accessible to activation with consequences for drug-induced dopamine responses and point to depotentiation at corticostriatal synapses as a possible therapeutic target for treating addiction. PMID:23911326

  4. Prolyl 4-hydroxylase activity-responsive transcription factors: From hydroxylation to gene expression and neuroprotection

    PubMed Central

    Siddiq, Ambreena; Aminova, Leila R; Ratan, Rajiv R

    2008-01-01

    Most homeostatic processes including gene transcription occur as a result of deviations in physiological tone that threatens the survival of the organism. A prototypical homeostatic stress response includes changes in gene expression following alterations in oxygen, iron or 2-oxoglutarate levels. Each of these cofactors plays an important role in cellular metabolism. Accordingly, a family of enzymes known as the Prolyl 4-hydroxylase (PHD) enzymes are a group of dioxygenases that have evolved to sense changes in 2-oxoglutarate, oxygen and iron via changes in enzyme activity. Indeed, PHDs are a part of an established oxygen sensor system that regulates transcriptional regulation of hypoxia/stress-regulated genes and thus are an important component of events leading to cellular rescue from oxygen, iron or 2-oxoglutarate deprivations. The ability of PHD activity to regulate homeostatic responses to oxygen, iron or 2-oxoglutarate metabolism has led to the development of small molecule inhibitors of the PHDs as a strategy for activating or augmenting cellular stress responses. These small molecules are proving effective in preclinical models of stroke and Parkinson's disease. However the precise protective pathways engaged by PHD inhibition are only beginning to be defined. In the current review, we summarize the role of iron, 2-oxoglutarate and oxygen in the PHD catalyzed hydroxylation reaction and provide a brief discussion of some of the transcription factors that play an effective role in neuroprotection against oxidative stress as a result of changes in PHD activity. PMID:17981760

  5. The Ess1 prolyl isomerase is required for growth and morphogenetic switching in Candida albicans.

    PubMed Central

    Devasahayam, Gina; Chaturvedi, Vishnu; Hanes, Steven D

    2002-01-01

    Prolyl-isomerases (PPIases) are found in all organisms and are important for the folding and activity of many proteins. Of the 13 PPIases in Saccharomyces cerevisiae only Ess1, a parvulin-class PPIase, is essential for growth. Ess1 is required to complete mitosis, and Ess1 and its mammalian homolog, Pin1, interact directly with RNA polymerase II. Here, we isolate the ESS1 gene from the pathogenic fungus Candida albicans and show that it is functionally homologous to the S. cerevisiae ESS1. We generate conditional-lethal (ts) alleles of C. albicans ESS1 and use these mutations to demonstrate that ESS1 is essential for growth in C. albicans. We also show that reducing the dosage or activity of ESS1 blocks morphogenetic switching from the yeast to the hyphal and pseudohyphal forms under certain conditions. Analysis of double mutants of ESS1 and TUP1 or CPH1, two genes known to be involved in morphogenetic switching, suggests that ESS1 functions in the same pathway as CPH1 and upstream of or in parallel to TUP1. Given that switching is important for virulence of C. albicans, inhibitors of Ess1 might be useful as antifungal agents. PMID:11805043

  6. 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.

  7. 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

  8. Low molecular weight protein tyrosine phosphatase: Multifaceted functions of an evolutionarily conserved enzyme.

    PubMed

    Caselli, Anna; Paoli, Paolo; Santi, Alice; Mugnaioni, Camilla; Toti, Alessandra; Camici, Guido; Cirri, Paolo

    2016-10-01

    Originally identified as a low molecular weight acid phosphatase, LMW-PTP is actually a protein tyrosine phosphatase that acts on many phosphotyrosine-containing cellular proteins that are primarily involved in signal transduction. Differences in sequence, structure, and substrate recognition as well as in subcellular localization in different organisms enable LMW-PTP to exert many different functions. In fact, during evolution, the LMW-PTP structure adapted to perform different catalytic actions depending on the organism type. In bacteria, this enzyme is involved in the biosynthesis of group 1 and 4 capsules, but it is also a virulence factor in pathogenic strains. In yeast, LMW-PTPs dephosphorylate immunophilin Fpr3, a peptidyl-prolyl-cis-trans isomerase member of the protein chaperone family. In humans, LMW-PTP is encoded by the ACP1 gene, which is composed of three different alleles, each encoding two active enzymes produced by alternative RNA splicing. In animals, LMW-PTP dephosphorylates a number of growth factor receptors and modulates their signalling processes. The involvement of LMW-PTP in cancer progression and in insulin receptor regulation as well as its actions as a virulence factor in a number of pathogenic bacterial strains may promote the search for potent, selective and bioavailable LMW-PTP inhibitors. PMID:27421795

  9. 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.

  10. Mitochondrial Thioredoxin System as a Modulator of Cyclophilin D Redox State

    NASA Astrophysics Data System (ADS)

    Folda, Alessandra; Citta, Anna; Scalcon, Valeria; Calì, Tito; Zonta, Francesco; Scutari, Guido; Bindoli, Alberto; Rigobello, Maria Pia

    2016-03-01

    The mitochondrial thioredoxin system (NADPH, thioredoxin reductase, thioredoxin) is a major redox regulator. Here we have investigated the redox correlation between this system and the mitochondrial enzyme cyclophilin D. The peptidyl prolyl cis-trans isomerase activity of cyclophilin D was stimulated by the thioredoxin system, while it was decreased by cyclosporin A and the thioredoxin reductase inhibitor auranofin. The redox state of cyclophilin D, thioredoxin 1 and 2 and peroxiredoxin 3 was measured in isolated rat heart mitochondria and in tumor cell lines (CEM-R and HeLa) by redox Western blot analysis upon inhibition of thioredoxin reductase with auranofin, arsenic trioxide, 1-chloro-2,4-dinitrobenzene or after treatment with hydrogen peroxide. A concomitant oxidation of thioredoxin, peroxiredoxin and cyclophilin D was observed, suggesting a redox communication between the thioredoxin system and cyclophilin. This correlation was further confirmed by i) co-immunoprecipitation assay of cyclophilin D with thioredoxin 2 and peroxiredoxin 3, ii) molecular modeling and iii) depleting thioredoxin reductase by siRNA. We conclude that the mitochondrial thioredoxin system controls the redox state of cyclophilin D which, in turn, may act as a regulator of several processes including ROS production and pro-apoptotic factors release.

  11. Target Cell Cyclophilins Facilitate Human Papillomavirus Type 16 Infection

    PubMed Central

    Sapp, Martin

    2009-01-01

    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. PMID:19629175

  12. 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.

  13. Single-Domain Parvulins Constitute a Specific Marker for Recently Proposed Deep-Branching Archaeal Subgroups

    PubMed Central

    Lederer, Christoph; Heider, Dominik; van den Boom, Johannes; Hoffmann, Daniel; Mueller, Jonathan W.; Bayer, Peter

    2011-01-01

    Peptidyl-prolyl cis/trans isomerases (PPIases) are enzymes assisting protein folding and protein quality control in organisms of all kingdoms of life. In contrast to the other sub-classes of PPIases, the cyclophilins and the FK-506 binding proteins, little was formerly known about the parvulin type of PPIase in Archaea. Recently, the first solution structure of an archaeal parvulin, the PinA protein from Cenarchaeum symbiosum, was reported. Investigation of occurrence and frequency of PPIase sequences in numerous archaeal genomes now revealed a strong tendency for thermophilic microorganisms to reduce the number of PPIases. Single-domain parvulins were mostly found in the genomes of recently proposed deep-branching archaeal subgroups, the Thaumarchaeota and the ARMANs (archaeal Richmond Mine acidophilic nanoorganisms). Hence, we used the parvulin sequence to reclassify available archaeal metagenomic contigs, thereby, adding new members to these subgroups. A combination of genomic background analysis and phylogenetic approaches of parvulin sequences suggested that the assigned sequences belong to at least two distinct groups of Thaumarchaeota. Finally, machine learning approaches were applied to identify amino acid residues that separate archaeal and bacterial parvulin proteins from each other. When mapped onto the recent PinA solution structure, most of these positions form a cluster at one site of the protein possibly indicating a different functionality of the two groups of parvulin proteins. PMID:22065628

  14. Backbone and side-chain ¹H, ¹³C and ¹⁵N assignments of the PPIase domain of macrophage infectivity potentiator (Mip) protein from Coxiella burnetii.

    PubMed

    Tse, Man-Kit; Cheung, Stanley K K; Ke, Yi-hong; Lau, Candy C Y; Sze, Kong-Hung; Yuen, Kwok-Yung

    2014-04-01

    Coxiella burnetii is an obligate intracellular gram-negative bacterium uniquely evolved to thrive in the inhospitable phagolysosome of macrophage. C. burnetii causes Q fever in humans and animals, which is emerging as a global public health concern. It is highly infectious and designated as a category B biowarfare agent because of its ubiquitous nature, abundant natural reservoirs, high resistance to environmental conditions, ease of transmission and low infectious dose. The lack of knowledge and awareness of C. burnetii leads to under-reporting and under-diagnosing of Q fever cases. Therefore, further understanding of the interactions between the infected host and the bacteria is necessary. C. burnetii macrophage infectivity potentiator (cb-Mip) is a secreted protein of 230 amino acids involving in intracellular survival of the pathogen. cb-Mip belongs to the family of FK506 binding protein, which possesses peptidyl-prolyl cis/trans isomerase (PPIase) activity. Besides acting as a PPIase, Mip protein homolog has been identified as virulence factor of many intracellular pathogenic microorganisms. In the present study, we report the near complete resonance assignments of the PPIase domain-containing region of Mip protein of C. burnetii. Secondary structure prediction based on chemical shift index analysis indicates that the protein adopts a predominately beta-strand structure, which is consistent with the crystal structure of homologous Mip protein in Legionella pneumophila.

  15. Alkali- and halo-tolerant catalase from Halomonas sp. SK1: overexpression in Escherichia coli, purification, characterization, and genetic modification.

    PubMed

    Thuy, Le Huyen Ai; Phucharoen, Krisana; Ideno, Akira; Maruyama, Tadashi; Shinozawa, Takao

    2004-04-01

    A catalase gene, ohktA, from an alkali- and halo-tolerant bacterium, Halomonas sp. SK1, on the pKK223-3, was expressed in the catalase-lacking Escherichia coli strain UM2. Highly purified catalase showing a single band on SDS-PAGE was obtained by two liquid chromatography steps on DEAE-Toyopear1 and Chelating-Sepharose Fast Flow. The enzyme, oHktA, shows high catalase activity with a pH optimum at 10, and the activity was stable in 4 M KC1. This enzyme is thermo-sensitive, showing a significant loss of activity within 5 minutes at 37 degrees C. To modify the stability of the catalase, the addition of domain II of the heat stable Mn catalase from Thermus thermophilus to the C-terminus was made. When coexpressed with a chaperone (PhFKBP29) gene product, peptidyl-prolyl cis-trans isomerase, from a thermophilic bacterium, a chimeric catalase was produced in the soluble fraction. The stability of this catalase in the range of 37 degrees -45 degrees C was improved and it was stable for more than 1 h at 37 degrees C. PMID:15118308

  16. Degradation of the Tumor Suppressor PML by Pin1 Contributes to the Cancer Phenotype of Breast Cancer MDA-MB-231 Cells▿

    PubMed Central

    Reineke, Erin L.; Lam, Minh; Liu, Qing; Liu, Yu; Stanya, Kristopher J.; Chang, Kun-Sang; Means, Anthony R.; Kao, Hung-Ying

    2008-01-01

    Promyelocytic leukemia protein (PML) is an important regulator due to its role in numerous cellular processes including apoptosis, viral infection, senescence, DNA damage repair, and cell cycle regulation. Despite the role of PML in many cellular functions, little is known about the regulation of PML itself. We show that PML stability is regulated through interaction with the peptidyl-prolyl cis-trans isomerase Pin1. This interaction is mediated through four serine-proline motifs in the C terminus of PML. Binding to Pin1 results in degradation of PML in a phosphorylation-dependent manner. Furthermore, our data indicate that sumoylation of PML blocks the interaction, thus preventing degradation of PML by this pathway. Functionally, we show that in the MDA-MB-231 breast cancer cell line modulating levels of Pin1 affects steady-state levels of PML. Furthermore, degradation of PML due to Pin1 acts both to protect these cells from hydrogen peroxide-induced death and to increase the rate of proliferation. Taken together, our work defines a novel mechanism by which sumoylation of PML prevents Pin1-dependent degradation. This interaction likely occurs in numerous cell lines and may be a pathway for oncogenic transformation. PMID:18039859

  17. Proline Isomerization of the Immune Receptor-Interacting Protein RIN4 by a Cyclophilin Inhibits Effector-Triggered Immunity in Arabidopsis

    PubMed Central

    Li, Meng; Ma, Xiqing; Chiang, Yi-Hsuan; Yadeta, Koste A.; Ding, Pengfei; Dong, Liansai; Zhao, Yan; Li, Xiuming; Yu, Yufei; Zhang, Ling; Shen, Qian-Hua; Xia, Bin; Coaker, Gitta; Liu, Dong; Zhou, Jian-Min

    2016-01-01

    SUMMARY In the absence of pathogen infection, plant effector-triggered immune (ETI) receptors are maintained in a preactivation state by intermolecular interactions with other host proteins. Pathogen effector-induced alterations activate the receptor. In Arabidopsis, the ETI receptor RPM1 is activated via bacterial effector AvrB-induced phosphorylation of the RPM1-interacting protein RIN4 at Threonine 166. We find that RIN4 also interacts with the prolyl-peptidyl isomerase (PPIase) ROC1, which is reduced upon RIN4 Thr166 phosphorylation. ROC1 suppresses RPM1 immunity in a PPIase-dependent manner. Consistent with this, RIN4 Pro149 undergoes cis/trans isomerization in the presence of ROC1. While the RIN4P149V mutation abolishes RPM1 resistance, the deletion of Pro149 leads to RPM1 activation in the absence of RIN4 phosphorylation. These results support a model in which RPM1 directly senses conformational changes in RIN4 surrounding Pro149 that is controlled by ROC1. RIN4 Thr166 phosphorylation indirectly regulates RPM1 resistance by modulating the ROC1-mediated RIN4 isomerization. PMID:25299333

  18. KSHV Reactivation and Novel Implications of Protein Isomerization on Lytic Switch Control

    PubMed Central

    Guito, Jonathan; Lukac, David M.

    2015-01-01

    In Kaposi’s sarcoma-associated herpesvirus (KSHV) oncogenesis, both latency and reactivation are hypothesized to potentiate tumor growth. The KSHV Rta protein is the lytic switch for reactivation. Rta transactivates essential genes via interactions with cofactors such as the cellular RBP-Jk and Oct-1 proteins, and the viral Mta protein. Given that robust viral reactivation would facilitate antiviral responses and culminate in host cell lysis, regulation of Rta’s expression and function is a major determinant of the latent-lytic balance and the fate of infected cells. Our lab recently showed that Rta transactivation requires the cellular peptidyl-prolyl cis/trans isomerase Pin1. Our data suggest that proline‑directed phosphorylation regulates Rta by licensing binding to Pin1. Despite Pin1’s ability to stimulate Rta transactivation, unchecked Pin1 activity inhibited virus production. Dysregulation of Pin1 is implicated in human cancers, and KSHV is the latest virus known to co-opt Pin1 function. We propose that Pin1 is a molecular timer that can regulate the balance between viral lytic gene expression and host cell lysis. Intriguing scenarios for Pin1’s underlying activities, and the potential broader significance for isomerization of Rta and reactivation, are highlighted. PMID:25588053

  19. Solution structure of the Legionella pneumophila Mip-rapamycin complex

    PubMed Central

    Ceymann, Andreas; Horstmann, Martin; Ehses, Philipp; Schweimer, Kristian; Paschke, Anne-Katrin; Steinert, Michael; Faber, Cornelius

    2008-01-01

    Background Legionella pneumphila is the causative agent of Legionnaires' disease. A major virulence factor of the pathogen is the homodimeric surface protein Mip. It shows peptidyl-prolyl cis/trans isomerase activty and is a receptor of FK506 and rapamycin, which both inhibit its enzymatic function. Insight into the binding process may be used for the design of novel Mip inhibitors as potential drugs against Legionnaires' disease. Results We have solved the solution structure of free Mip77–213 and the Mip77–213-rapamycin complex by NMR spectroscopy. Mip77–213 showed the typical FKBP-fold and only minor rearrangements upon binding of rapamycin. Apart from the configuration of a flexible hairpin loop, which is partly stabilized upon binding, the solution structure confirms the crystal structure. Comparisons to the structures of free FKBP12 and the FKBP12-rapamycin complex suggested an identical binding mode for both proteins. Conclusion The structural similarity of the Mip-rapamycin and FKBP12-rapamycin complexes suggests that FKBP12 ligands may be promising starting points for the design of novel Mip inhibitors. The search for a novel drug against Legionnaires' disease may therefore benefit from the large variety of known FKBP12 inhibitors. PMID:18366641

  20. Cooperation of both, the FKBP_N-like and the DSBA-like, domains is necessary for the correct function of FTS_1067 protein involved in Francisella tularensis virulence and pathogenesis.

    PubMed

    Senitkova, Iva; Spidlova, Petra; Stulik, Jiri

    2015-08-01

    Francisella tularensis the etiological agent of tularaemia is one of the most infectious human pathogen known. Our knowledge about its key virulence factors has increased recently but it still remains a lot to explore. One of the described essential virulence factors is membrane lipoprotein FTS_1067 (nomenclature of F. tularensis subsp. holarctica strain FSC200) with homology to the protein family of disulphide oxidoreductases DsbA. Lipoprotein consists of two different domains: the C-terminal DsbA_Com1-like domain (DSBA-like) and the N-terminal FKBP-type peptidyl-prolyl cis/trans isomerases (FKBP_N-like). To uncover the biological role of these domains, we created bacterial strain with deletion of the DSBA-like domain. This defect in gene coding for lipoprotein FTS_1067 led to high in vivo attenuation associated with the ability to induce host protective immunity. Analyses performed with the truncated recombinant protein showed that the absence of DSBA-like domain revealed the loss of thiol/disulphide oxidoreductase activity and, additionally, confirmed the role of the FKBP_N-like domain in the FTS_1067 oligomerization and chaperone-like function. Finally, we verified that only full-length form of FTS_1067 recombinant protein possesses the isomerase activity. Based on our results, we proposed that for the correct FTS_1067 protein function both domains are needed.

  1. Identification of RNA targets for the nuclear multidomain cyclophilin atCyp59 and their effect on PPIase activity

    PubMed Central

    Bannikova, Olga; Zywicki, Marek; Marquez, Yamile; Skrahina, Tatsiana; Kalyna, Maria; Barta, Andrea

    2013-01-01

    AtCyp59 is a multidomain cyclophilin containing a peptidyl-prolyl cis/trans isomerase (PPIase) domain and an evolutionarily highly conserved RRM domain. Deregulation of this class of cyclophilins has been shown to affect transcription and to influence phosphorylation of the C-terminal repeat domain of the largest subunit of the RNA polymerase II. We used a genomic SELEX method for identifying RNA targets of AtCyp59. Analysis of the selected RNAs revealed an RNA-binding motif (G[U/C]N[G/A]CC[A/G]) and we show that it is evolutionarily conserved. Binding to this motif was verified by gel shift assays in vitro and by RNA immunopreciptation assays of AtCyp59 in vivo. Most importantly, we show that binding also occurs on unprocessed transcripts in vivo and that binding of specific RNAs inhibits the PPIase activity of AtCyp59 in vitro. Surprisingly, genome-wide analysis showed that the RNA motif is present in about 70% of the annotated transcripts preferentially in exons. Taken together, the available data suggest that these cyclophilins might have an important function in transcription regulation. PMID:23248006

  2. Cell cycle proteins in brain in mild cognitive impairment: insights into progression to Alzheimer disease.

    PubMed

    Keeney, Jeriel T R; Swomley, Aaron M; Harris, Jessica L; Fiorini, Ada; Mitov, Mihail I; Perluigi, Marzia; Sultana, Rukhsana; Butterfield, D Allan

    2012-10-01

    Recent studies have demonstrated the re-emergence of cell cycle proteins in brain as patients progress from the early stages of mild cognitive impairment (MCI) into Alzheimer's disease (AD). Oxidative stress markers present in AD have also been shown to be present in MCI brain suggesting that these events occur in early stages of the disease. The levels of key cell cycle proteins, such as CDK2, CDK5, cyclin G1, and BRAC1 have all been found to be elevated in MCI brain compared to age-matched control. Further, peptidyl prolyl cis-trans isomerase (Pin1), a protein that plays an important role in regulating the activity of key proteins, such as CDK5, GSK3-β, and PP2A that are involved in both the phosphorylation state of Tau and in the cell cycle, has been found to be oxidatively modified and downregulated in both AD and MCI brain. Hyperphosphorylation of Tau then results in synapse loss and the characteristic Tau aggregation as neurofibrillary tangles, an AD hallmark. In this review, we summarized the role of cell cycle dysregulation in the progression of disease from MCI to AD. Based on the current literature, it is tempting to speculate that a combination of oxidative stress and cell cycle dysfunction conceivably leads to neurodegeneration.

  3. The rotamase Pin1 is up-regulated, hypophosphorylated and required for cell cycle progression in head and neck squamous cell carcinomas.

    PubMed

    Wiegand, Susanne; Dakic, Branka; Rath, Ariane F E; Makarova, Galina; Sterz, Carolina; Meissner, Wolfgang; Bette, Michael; Adamkiewicz, Jürgen; Müller-Brüsselbach, Sabine; Müller, Rolf; Werner, Jochen A; Mandic, Robert

    2009-10-01

    The peptidyl-prolyl cis/trans isomerase Pin1 has been implicated in malignant transformation in multiple studies, however, little is known about its potential impact in head and neck cancer. This study evaluates the role of Pin1 in head and neck squamous cell carcinomas (HNSCCs). Pin1 expression and level of phosphorylation was evaluated by Western blot analysis and 2D-gel-electrophoresis. Pin1 was inhibited with juglone (5-hydroxy-1,4-naphthalenedione) or Pin1 specific siRNA and its influence on cell cycle checkpoint distribution was assessed by FACS analysis. Pin1 overexpression was found in HNSCC tissues and cell lines. 2D-gel-electrophoresis data pointed to Pin1 being hypophosphorylated in HNSCC cells which is consistent with overactivation of this rotamase. Inhibition of HNSCC cells with juglone or Pin1 siRNA induced the cell cycle inhibitor p21(WAF1/Cip1) with a concomitant reduction of cells in G2/M and an increased fraction of cells with fragmented DNA. Cell death did not correlate with significant levels of apoptosis in Pin1 depleted HNSCC cells. In summary, the data shows that Pin1 is overexpressed and hypophosphorylated in HNSCC, and that inhibition of Pin1 blocks cell cycle progression and triggers tumor cell death. Pin1 therefore could represent a new target for the development of improved HNSCC targeting drugs.

  4. 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.

  5. Assisted folding of D-glyceraldehyde-3-phosphate dehydrogenase by trigger factor.

    PubMed Central

    Huang, G. C.; Li, Z. Y.; Zhou, J. M.; Fischer, G.

    2000-01-01

    The Escherichia coli trigger factor is a peptidyl-prolyl cis-trans isomerase that catalyzes proline-limited protein folding extremely well. Here, refolding of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the presence of trigger factor was investigated. The regain of activity of GAPDH was markedly increased by trigger factor after either long- or short-term denaturation, and detectable aggregation of GAPDH intermediates was prevented. In both cases, time courses of refolding of GAPDH were decelerated by trigger factor. The reactivation yield of GAPDH showed a slow down-turn when molar ratios of trigger factor to GAPDH were above 5, due to tight binding between trigger factor and GAPDH intermediates. Such inactive bound GAPDH could be partially rescued from trigger factor by addition of reduced alphaLA as competitor, by further diluting the refolding mixture, or by disrupting hydrophobic interactions in the complexes. A model for trigger factor assisted refolding of GAPDH is proposed. We also suggest that assisted refolding of GAPDH is due mainly to the chaperone function of trigger factor. PMID:10892818

  6. A novel cyclophilin from parasitic and free-living nematodes with a unique substrate- and drug-binding domain.

    PubMed

    Ma, Dong; Nelson, Laura S; LeCoz, Krystel; Poole, Catherine; Carlow, Clotilde K S

    2002-04-26

    A highly diversified member of the cyclophilin family of peptidyl-prolyl cis-trans isomerases has been isolated from the human parasite Onchocerca volvulus (OvCYP-16). This 25-kDa cyclophilin shares 43-46% similarity to other filarial cyclophilins but does not belong to any of the groups previously defined in invertebrates or vertebrates. A homolog was also isolated from Caenorhabditis elegans (CeCYP-16). Both recombinant O. volvulus and C. elegans cyclophilins were found to possess an enzyme activity with similar substrate preference and insensitivity to cyclosporin A. They represent novel cyclophilins with important differences in the composition of the drug-binding site in particular, namely, a Glu(124) (C. elegans) or Asp(123) (O. volvulus) residue present in a critical position. Site-directed mutagenesis studies and kinetic characterization demonstrated that the single residue dictates the degree of binding to substrate and cyclosporin A. CeCYP-16::GFP-expressing lines were generated with expression in the anterior and posterior distal portions of the intestine, in all larval stages and adults. An exception was found in the dauer stage, where fluorescence was observed in both the cell bodies and processes of the ventral chord motor neurons but was absent from the intestine. These studies highlight the extensive diversification of cyclophilins in an important human parasite and a closely related model organism.

  7. Molecular chaperone activity and biological regulatory actions of the TPR-domain immunophilins FKBP51 and FKBP52.

    PubMed

    Erlejman, Alejandra G; Lagadari, Mariana; Harris, Diondra C; Cox, Marc B; Galigniana, Mario D

    2014-05-01

    Immunophilins comprise a family of intracellular proteins with peptidyl-prolyl-(cis/trans)-isomerase activity. These foldases are abundant, ubiquitous, and able to bind immunosuppressant drugs, from which the term immunophilin derives. Family members are found in abundance in virtually all organisms and subcellular compartments, and their amino acid sequences are conserved phylogenetically. Immunophilins possess the ability to function as molecular chaperones favoring the proper folding and biological regulation of their biological actions. Their ability to interact via their TPR domains with the 90-kDa heat-shock protein, and through this chaperone, with several signalling cascade factors is of particular importance. Among the family members, the highly homologous proteins FKBP51 and FKBP52 were first characterized due to their ability to interact with steroid hormone receptors. Since then, much progress has been made in understanding the mechanisms by which they regulate receptor signaling and the resulting roles they play not only in endocrine processes, but also in cell architecture, neurodifferentiation, and tumor progression. In this article we review the most relevant features of these two immunophilins and their potential as pharmacologic targets.

  8. FK506 binding protein 8 peptidylprolyl isomerase activity manages a late stage of cystic fibrosis transmembrane conductance regulator (CFTR) folding and stability.

    PubMed

    Hutt, Darren M; Roth, Daniela Martino; Chalfant, Monica A; Youker, Robert T; Matteson, Jeanne; Brodsky, Jeffrey L; Balch, William E

    2012-06-22

    Cystic fibrosis (CF) is caused by mutations in the apical chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) with 90% of patients carrying at least one deletion of the F508 (ΔF508) allele. This mutant form of CFTR is characterized by a folding and trafficking defect that prevents exit from the endoplasmic reticulum. We previously reported that ΔF508 CFTR can be recovered in a complex with Hsp90 and its co-chaperones as an on-pathway folding intermediate, suggesting that Δ508 CF disease arises due to a failure of the proteostasis network (PN), which manages protein folding and degradation in the cell. We have now examined the role of FK506-binding protein 8 (FKBP8), a component of the CFTR interactome, during the biogenesis of wild-type and ΔF508 CFTR. FKBP8 is a member of the peptidylprolyl isomerase family that mediates the cis/trans interconversion of peptidyl prolyl bonds. Our results suggest that FKBP8 is a key PN factor required at a post-Hsp90 step in CFTR biogenesis. In addition, changes in its expression level or alteration of its activity by a peptidylprolyl isomerase inhibitor alter CFTR stability and transport. We propose that CF is caused by the sequential failure of the prevailing PN pathway to stabilize ΔF508-CFTR for endoplasmic reticulum export, a pathway that can be therapeutically managed.

  9. Cyclophilin B protects SH-SY5Y human neuroblastoma cells against MPP(+)-induced neurotoxicity via JNK pathway.

    PubMed

    Oh, Yoojung; Jeong, Kwon; Kim, Kiyoon; Lee, Young-Seok; Jeong, Suyun; Kim, Sung Soo; Yoon, Kyung-Sik; Ha, Joohun; Kang, Insug; Choe, Wonchae

    2016-09-23

    Parkinson's disease (PD) is the second most common neurodegenerative disorder of aging. PD involves a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyidine (MPTP) and its toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) inhibit the complex I of the mitochondrial electron transport chain, and have been widely used to construct PD models. Cyclophilin B (CypB) is an endoplasmic reticulum protein that binds to cyclosporine A as a cyclophilin family member. CypB has peptidyl-prolyl cis-trans isomerase (PPIase) activity. We investigated the protective effects of overexpressed CypB on MPP+-induced neurocytotoxicity in SH-SY5Y human neuroblastoma cells. Overexpressed CypB decreased MPP(+)-induced oxidative stress through the modulation of antioxidant enzymes including manganese superoxide dismutase and catalase, and prevented neurocytotoxicity via mitogen-activated protein kinase, especially the c-Jun N-terminal kinase pathway. In addition, CypB inhibited the activation of MPP(+)-induced the pro-apoptotic molecules poly (ADP-ribose) polymerase, Bax, and Bcl-2, and attenuated MPP(+)-induced mitochondrial dysfunction. The data suggest that overexpressed CypB protects neuronal cells from MPP+-induced dopaminergic neuronal cell death.

  10. Pin1At regulates PIN1 polar localization and root gravitropism

    PubMed Central

    Xi, Wanyan; Gong, Ximing; Yang, Qiaoyun; Yu, Hao; Liou, Yih-Cherng

    2016-01-01

    Root gravitropism allows plants to establish root systems and its regulation depends on polar auxin transport mediated by PIN-FORMED (PIN) auxin transporters. PINOID (PID) and PROTEIN PHOSPHATASE 2A (PP2A) act antagonistically on reversible phosphorylation of PINs. This regulates polar PIN distribution and auxin transport. Here we show that a peptidyl-prolyl cis/trans isomerase Pin1At regulates root gravitropism. Downregulation of Pin1At suppresses root agravitropic phenotypes of pp2aa and 35S:PID, while overexpression of Pin1At affects root gravitropic responses and enhances the pp2aa agravitropic phenotype. Pin1At also affects auxin transport and polar localization of PIN1 in stele cells, which is mediated by PID and PP2A. Furthermore, Pin1At catalyses the conformational change of the phosphorylated Ser/Thr-Pro motifs of PIN1. Thus, Pin1At mediates the conformational dynamics of PIN1 and affects PID- and PP2A-mediated regulation of PIN1 polar localization, which correlates with the regulation of root gravitropism. PMID:26791759

  11. One novel quinoxaline derivative as a potent human cyclophilin A inhibitor shows highly inhibitory activity against mouse spleen cell proliferation.

    PubMed

    Li, Jian; Chen, Jing; Zhang, Li; Wang, Feng; Gui, Chunshan; Zhang, Li; Qin, Yu; Xu, Qiang; Liu, Hong; Nan, Fajun; Shen, Jingkang; Bai, Donglu; Chen, Kaixian; Shen, Xu; Jiang, Hualiang

    2006-08-15

    Cyclophilin A (CypA) is a ubiquitous cellular enzyme playing critical roles in many biological processes, and its inhibitor has been reported to have potential immunosuppressive activity. In this work, we reported a novel quinoxaline derivative, 2,3-di(furan-2-yl)-6-(3-N,N-diethylcarbamoyl-piperidino)carbonylamino quinoxaline (DC838, 3), which was confirmed to be a potent inhibitor against human CypA. By using the surface plasmon resonance (SPR) and fluorescence titration techniques, the kinetic analysis of CypA/DC838 interaction was quantitatively performed. CypA peptidyl prolyl cis-trans isomerase (PPIase) activity inhibition assay showed that DC838 demonstrated highly CypA PPIase inhibitory activity. In vivo assay results showed that DC838 could inhibit mouse spleen cell proliferation induced by concanavalin A (Con A). Molecular docking simulation further elucidated the specific DC838 binding to CypA at the atomic level. The current work should provide useful information in the discovery of immunosuppressor based on CypA inhibitor.

  12. Mitochondrial Thioredoxin System as a Modulator of Cyclophilin D Redox State

    PubMed Central

    Folda, Alessandra; Citta, Anna; Scalcon, Valeria; Calì, Tito; Zonta, Francesco; Scutari, Guido; Bindoli, Alberto; Rigobello, Maria Pia

    2016-01-01

    The mitochondrial thioredoxin system (NADPH, thioredoxin reductase, thioredoxin) is a major redox regulator. Here we have investigated the redox correlation between this system and the mitochondrial enzyme cyclophilin D. The peptidyl prolyl cis-trans isomerase activity of cyclophilin D was stimulated by the thioredoxin system, while it was decreased by cyclosporin A and the thioredoxin reductase inhibitor auranofin. The redox state of cyclophilin D, thioredoxin 1 and 2 and peroxiredoxin 3 was measured in isolated rat heart mitochondria and in tumor cell lines (CEM-R and HeLa) by redox Western blot analysis upon inhibition of thioredoxin reductase with auranofin, arsenic trioxide, 1-chloro-2,4-dinitrobenzene or after treatment with hydrogen peroxide. A concomitant oxidation of thioredoxin, peroxiredoxin and cyclophilin D was observed, suggesting a redox communication between the thioredoxin system and cyclophilin. This correlation was further confirmed by i) co-immunoprecipitation assay of cyclophilin D with thioredoxin 2 and peroxiredoxin 3, ii) molecular modeling and iii) depleting thioredoxin reductase by siRNA. We conclude that the mitochondrial thioredoxin system controls the redox state of cyclophilin D which, in turn, may act as a regulator of several processes including ROS production and pro-apoptotic factors release. PMID:26975474

  13. Skp1-Cullin-F-box (SCF)-type ubiquitin ligase FBXW7 negatively regulates spermatogonial stem cell self-renewal

    PubMed Central

    Kanatsu-Shinohara, Mito; Onoyama, Ichiro; Nakayama, Keiichi I.; Shinohara, Takashi

    2014-01-01

    Spermatogonial stem cells (SSCs) undergo self-renewal divisions to support spermatogenesis throughout life. Although several positive regulators of SSC self-renewal have been discovered, little is known about the negative regulators. Here, we report that F-box and WD-40 domain protein 7 (FBXW7), a component of the Skp1-Cullin-F-box–type ubiquitin ligase, is a negative regulator of SSC self-renewal. FBXW7 is expressed in undifferentiated spermatogonia in a cell cycle-dependent manner. Although peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (PIN1), essential for spermatogenesis, is thought to destroy FBXW7, Pin1 depletion decreased FBXW7 expression. Spermatogonial transplantation showed that Fbxw7 overexpression compromised SSC activity whereas Fbxw7 deficiency enhanced SSC colonization and caused accumulation of undifferentiated spermatogonia, suggesting that the level of FBXW7 is critical for self-renewal and differentiation. Screening of putative FBXW7 targets revealed that Fbxw7 deficiency up-regulated myelocytomatosis oncogene (MYC) and cyclin E1 (CCNE1). Although depletion of Myc/Mycn or Ccne1/Ccne2 compromised SSC activity, overexpression of Myc, but not Ccne1, increased colonization of SSCs. These results suggest that FBXW7 regulates SSC self-renewal in a negative manner by degradation of MYC. PMID:24879440

  14. High-resolution insights into binding of unfolded polypeptides by the PPIase chaperone SlpA.

    PubMed

    Quistgaard, Esben M; Nordlund, Pär; Löw, Christian

    2012-10-01

    SlpA is a 2-domain protein consisting of an FK506-binding protein (FKBP) domain that harbors the peptidyl-prolyl cis/trans-isomerase (PPIase) active site and a small insert-in-flap (IF) domain that endows the protein with chaperone activity. We have determined the structure of SlpA from Escherichia coli at 1.35-Å resolution. The overall structure is similar to other known structures of the FKBP-IF subfamily. However, by serendipity, the linker region of the purification tag binds in the chaperone binding groove of the IF domain, making this the first structure of an FKBP-IF protein in complex with a mimic of an unfolded chaperone substrate. The linker binds by β-sheet augmentation, thus completing the incomplete β barrel of the IF domain and shielding a considerable hydrophobic surface area from the solvent. Interestingly, a proline residue in trans configuration appears to be specifically recognized in a small pocket within the binding groove. Hence, the IF domain can preselect and prealign substrates with proline residues, which may explain how it enhances the catalytic efficiency and modulates the specificity of the FKBP domain in addition to its chaperone function. Based on pulldown results, we suggest that SlpA is likely to be involved in ribosome assembly. PMID:22735173

  15. Investigating Dynamic Interdomain Allostery in Pin1

    PubMed Central

    Peng, Jeffrey W.

    2015-01-01

    Signaling proteins often sequester complementary functional sites in separate domains. How do the different domains communicate with one another? An attractive system to address this question is the mitotic regulator, human Pin1 (Lu et al. 1996). Pin-1 consists of two tethered domains: a WW domain for substrate binding, and a catalytic domain for peptidyl-prolyl isomerase (PPIase) activity. Pin1 accelerates the cis-trans isomerization of phospho-Ser/Thr-Pro (pS/T-P) motifs within proteins regulating the cell cycle and neuronal development. The early x-ray (Ranganathan et al. 1997; Verdecia et al. 2000) and solution NMR studies (Bayer et al. 2003; Jacobs et al. 2003) of Pin1 indicated inter- and intradomain motion. We became interested in exploring how such motions might affect interdomain communication, using NMR. Our accumulated results indicate substrate binding to Pin1 WW domain changes the intra/inter domain mobility, thereby altering substrate activity in the distal PPIase domain catalytic site. Thus, Pin1 shows evidence of dynamic allostery, in the sense of Cooper and Dryden (Cooper and Dryden 1984). We highlight our results supporting this conclusion, and summarize them via a simple speculative model of conformational selection. PMID:26495045

  16. shutdown is a component of the Drosophila piRNA biogenesis machinery

    PubMed Central

    Preall, Jonathan B.; Czech, Benjamin; Guzzardo, Paloma M.; Muerdter, Felix; Hannon, Gregory J.

    2012-01-01

    In animals, the piRNA pathway preserves the integrity of gametic genomes, guarding them against the activity of mobile genetic elements. This innate immune mechanism relies on distinct genomic loci, termed piRNA clusters, to provide a molecular definition of transposons, enabling their discrimination from genes. piRNA clusters give rise to long, single-stranded precursors, which are processed into primary piRNAs through an unknown mechanism. These can engage in an adaptive amplification loop, the ping-pong cycle, to optimize the content of small RNA populations via the generation of secondary piRNAs. Many proteins have been ascribed functions in either primary biogenesis or the ping-pong cycle, though for the most part the molecular functions of proteins implicated in these pathways remain obscure. Here, we link shutdown (shu), a gene previously shown to be required for fertility in Drosophila, to the piRNA pathway. Analysis of knockdown phenotypes in both the germline and somatic compartments of the ovary demonstrate important roles for shutdown in both primary biogenesis and the ping-pong cycle. shutdown is a member of the FKBP family of immunophilins. Shu contains domains implicated in peptidyl-prolyl cis-trans isomerase activity and in the binding of HSP90-family chaperones, though the relevance of these domains to piRNA biogenesis is unknown. PMID:22753781

  17. The Hsp90 cochaperone, FKBP51, increases Tau stability and polymerizes microtubules.

    PubMed

    Jinwal, Umesh K; Koren, John; Borysov, Sergiy I; Schmid, Andreas B; Abisambra, Jose F; Blair, Laura J; Johnson, Amelia G; Jones, Jeffrey R; Shults, Cody L; O'Leary, John C; Jin, Ying; Buchner, Johannes; Cox, Marc B; Dickey, Chad A

    2010-01-13

    Imbalanced protein load within cells is a critical aspect for most diseases of aging. In particular, the accumulation of proteins into neurotoxic aggregates is a common thread for a host of neurodegenerative diseases. Our previous work demonstrated that age-related changes to the cellular chaperone repertoire contributes to abnormal buildup of the microtubule-associated protein tau that accumulates in a group of diseases termed tauopathies, the most common being Alzheimer's disease. Here, we show that the Hsp90 cochaperone, FK506-binding protein 51 (FKBP51), which possesses both an Hsp90-interacting tetratricopeptide domain and a peptidyl-prolyl cis-trans isomerase (PPIase) domain, prevents tau clearance and regulates its phosphorylation status. Regulation of the latter is dependent on the PPIase activity of FKBP51. FKB51 enhances the association of tau with Hsp90, but the FKBP51/tau interaction is not dependent on Hsp90. In vitro FKBP51 stabilizes microtubules with tau in a reaction depending on the PPIase activity of FKBP51. Based on these new findings, we propose that FKBP51 can use the Hsp90 complex to isomerize tau, altering its phosphorylation pattern and stabilizing microtubules.

  18. Involvement of lipoprotein PpiA of Streptococcus gordonii in evasion of phagocytosis by macrophages.

    PubMed

    Cho, K; Arimoto, T; Igarashi, T; Yamamoto, M

    2013-10-01

    Streptococcus gordonii is a commensal gram-positive bacterium that resides in the human oral cavity, and is one of the most common causes of infective endocarditis (IE). Bacterial surface molecules play an important role in establishing IE, and several S. gordonii proteins have been implicated in binding to host cells during the establishment of IE. In this study, we identified a putative lipoprotein, peptidyl-prolyl cis/trans isomerase (PpiA), and clarified its role in evasion of phagocytosis by macrophages. Attenuation of the gene encoding prolipoprotein diacylglyceryl transferase (Lgt) altered the localization of PpiA from the cell surface to the culture supernatant, indicating that PpiA is lipid-anchored in the cell membrane by Lgt. Both human and murine macrophages showed higher phagocytic activity towards ppiA and lgt mutants than the wild-type, indicating that the presence of PpiA suppresses phagocytosis of S. gordonii. Human macrophages treated with dextran sulfate had significantly impaired phagocytosis of S. gordonii, suggesting that class A scavenger receptors in human macrophages are involved in the phagocytosis of S. gordonii. These results provide evidence that S. gordonii lipoprotein PpiA plays an important role in inhibiting phagocytic engulfment and in evasion of the host immune response. PMID:23734737

  19. Cyclophilin D Is Involved in the Regulation of Autophagy and Affects the Lifespan of P. anserina in Response to Mitochondrial Oxidative Stress.

    PubMed

    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

  20. Identification of cyclophilin as a proinflammatory secretory product of lipopolysaccharide-activated macrophages.

    PubMed Central

    Sherry, B; Yarlett, N; Strupp, A; Cerami, A

    1992-01-01

    We have isolated an 18-kDa peptide (designated sp18, for 18-kDa secreted protein) from the conditioned medium of lipopolysaccharide-stimulated RAW 264.7 murine macrophages. Purified sp18 had in vivo inflammatory activity and in vitro chemotactic activity for human peripheral blood polymorphonuclear leukocytes and monocytes. Surprisingly, N-terminal sequencing and tryptic mapping studies revealed that sp18 and cyclophilin, an intracellular protein that binds the immunosuppressive drug cyclosporin A, are highly homologous. The in vitro chemotactic activity of sp18 on monocytes was blocked by cyclosporin A but not by cyclosporin H, a structural analog of cyclosporin A that does not bind cyclophilin. Like purified porcine cyclophilin, mouse sp18 exhibited peptidyl-prolyl cis-trans isomerase activity. Medium conditioned by lipopolysaccharide-stimulated resident peritoneal exudate macrophages isolated from C57BL/6 mice contained substantially higher levels of sp18/cyclophilin than medium conditioned by nonstimulated macrophages. The observation that sp18/cyclophilin exhibits proinflammatory activity and is secreted by macrophages in response to endotoxin suggests that this protein may function as a cytokine, and invites the hypothesis that the immunosuppressive action of cyclosporin A results in part from interaction with an extracellular form of cyclophilin released as a mediator of immune and inflammatory functions. Images PMID:1565646

  1. Cyclophilin B protects SH-SY5Y human neuroblastoma cells against MPP(+)-induced neurotoxicity via JNK pathway.

    PubMed

    Oh, Yoojung; Jeong, Kwon; Kim, Kiyoon; Lee, Young-Seok; Jeong, Suyun; Kim, Sung Soo; Yoon, Kyung-Sik; Ha, Joohun; Kang, Insug; Choe, Wonchae

    2016-09-23

    Parkinson's disease (PD) is the second most common neurodegenerative disorder of aging. PD involves a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyidine (MPTP) and its toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) inhibit the complex I of the mitochondrial electron transport chain, and have been widely used to construct PD models. Cyclophilin B (CypB) is an endoplasmic reticulum protein that binds to cyclosporine A as a cyclophilin family member. CypB has peptidyl-prolyl cis-trans isomerase (PPIase) activity. We investigated the protective effects of overexpressed CypB on MPP+-induced neurocytotoxicity in SH-SY5Y human neuroblastoma cells. Overexpressed CypB decreased MPP(+)-induced oxidative stress through the modulation of antioxidant enzymes including manganese superoxide dismutase and catalase, and prevented neurocytotoxicity via mitogen-activated protein kinase, especially the c-Jun N-terminal kinase pathway. In addition, CypB inhibited the activation of MPP(+)-induced the pro-apoptotic molecules poly (ADP-ribose) polymerase, Bax, and Bcl-2, and attenuated MPP(+)-induced mitochondrial dysfunction. The data suggest that overexpressed CypB protects neuronal cells from MPP+-induced dopaminergic neuronal cell death. PMID:27569281

  2. Elucidating role of salivary proteins in denture stomatitis using a proteomic approach.

    PubMed

    Bencharit, Sompop; Altarawneh, Sandra K; Baxter, Sarah Schwartz; Carlson, Jim; Ross, Gary F; Border, Michael B; Mack, C Russell; Byrd, Warren C; Dibble, Christopher F; Barros, Silvana; Loewy, Zvi; Offenbacher, Steven

    2012-10-30

    Denture stomatitis (DS) is the most common oral pathology among denture wearers, affecting over one-third of this group. DS is usually associated with C. albicans. However, unlike other oral candidiasis, most DS patients have intact host immunity. The presence of a denture alone is usually sufficient for DS. Saliva and its protein contents can theoretically predispose some denture wearers to DS and others resistant toward DS. Here we proposed for the first time to define salivary proteomic profiles of denture wearers with and without DS. SELDI-TOF/MS analysis suggests that there is a proteomic differentiation among control, localized and generalized DS. Based on initial SELDI-TOF/MS profiling, we further used reversed phase liquid chromatography, MALDI-TOF/MS, and LC-MS/MS to characterize the salivary proteins associated with DS. Nineteen proteins based on SELDI-TOF/MS profiling were found including cystatin-SN, statherin, kininogen-1, desmocollin-2, carbonic anhydrase-6, peptidyl-prolyl cis-trans isomerase A like peptides, cystatin C, and several immunoglobulin fragments. The proteomic content gives evidence of the interaction between host tissue, saliva, and candida. Further examination in larger populations of these proteins may help to gain a better understanding of DS pathological processes and improve DS treatments.

  3. Crystal structure of Arabidopsis cyclophilin38 reveals a previously uncharacterized immunophilin fold and a possible autoinhibitory mechanism.

    PubMed

    Vasudevan, Dileep; Fu, Aigen; Luan, Sheng; Swaminathan, Kunchithapadam

    2012-06-01

    Cyclophilin38 (CYP38) is one of the highly divergent cyclophilins from Arabidopsis thaliana. Here, we report the crystal structure of the At-CYP38 protein (residues 83 to 437 of 437 amino acids) at 2.39-Å resolution. The structure reveals two distinct domains: an N-terminal helical bundle and a C-terminal cyclophilin β-barrel, connected by an acidic loop. Two N-terminal β-strands become part of the C-terminal cyclophilin β-barrel, thereby making a previously undiscovered domain organization. This study shows that CYP38 does not possess peptidyl-prolyl cis/trans isomerase activity and identifies a possible interaction of CYP38 with the E-loop of chlorophyll protein47 (CP47), a component of photosystem II. The interaction of CYP38 with the E-loop of CP47 is mediated through its cyclophilin domain. The N-terminal helical domain is closely packed together with the putative C-terminal cyclophilin domain and establishes a strong intramolecular interaction, thereby preventing the access of the cyclophilin domain to other proteins. This was further verified by protein-protein interaction assays using the yeast two-hybrid system. Furthermore, the non-Leucine zipper N-terminal helical bundle contains several new elements for protein-protein interaction that may be of functional significance. Together, this study provides the structure of a plant cyclophilin and explains a possible mechanism for autoinhibition of its function through an intramolecular interaction.

  4. 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.

  5. 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.

  6. A proteomic approach towards understanding the cross talk between Bacteroides fragilis and Bifidobacterium longum in coculture.

    PubMed

    Rios-Covián, David; Sánchez, Borja; Martínez, Noelia; Cuesta, Isabel; Hernández-Barranco, Ana M; de Los Reyes-Gavilán, Clara G; Gueimonde, Miguel

    2016-07-01

    A better understanding of the interactions among intestinal microbes is needed to decipher the complex cross talk that takes place within the human gut. Bacteroides and Bifidobacterium genera are among the most relevant intestinal bacteria, and it has been previously reported that coculturing of these 2 microorganisms affects their survival. Therefore, coculturing of Bifidobacterium longum NB667 and Bacteroides fragilis DSMZ2151 was performed with the aim of unravelling the mechanisms involved in their interaction. To this end, we applied proteomic (2D-DIGE) analyses, and by chromatographic techniques we quantified the bacterial metabolites produced during coincubation. Coculture stimulated the growth of B. longum, retarding that of B. fragilis, with concomitant changes in the production of some proteins and metabolites of both bacteria. The combined culture promoted upregulation of the bifidobacterial pyruvate kinase and downregulation of the Bacteroides phosphoenolpyruvate carboxykinase - 2 enzymes involved in the catabolism of carbohydrates. Moreover, B. fragilis FKBP-type peptidyl-prolyl cis-trans isomerase, a protein with chaperone-like activity, was found to be overproduced in coculture, suggesting the induction of a stress response in this microorganism. This study provides mechanistic data to deepen our understanding of the interaction between Bacteroides and Bifidobacterium intestinal populations.

  7. The Rift Valley Fever virus protein NSm and putative cellular protein interactions

    PubMed Central

    2012-01-01

    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. PMID:22838834

  8. Impact of temperature dependent sampling procedures in proteomics and peptidomics--a characterization of the liver and pancreas post mortem degradome.

    PubMed

    Scholz, Birger; Sköld, Karl; Kultima, Kim; Fernandez, Celine; Waldemarson, Sofia; Savitski, Mikhail M; Söderquist, Marcus; Borén, Mats; Stella, Robert; Andrén, Per; Zubarev, Roman; James, Peter

    2011-03-01

    Little is known about the nature of post mortem degradation of proteins and peptides on a global level, the so-called degradome. This is especially true for nonneural tissues. Degradome properties in relation to sampling procedures on different tissues are of great importance for the studies of, for instance, post translational modifications and/or the establishment of clinical biobanks. Here, snap freezing of fresh (<2 min post mortem time) mouse liver and pancreas tissue is compared with rapid heat stabilization with regard to effects on the proteome (using two-dimensional differential in-gel electrophoresis) and peptidome (using label free liquid chromatography). We report several proteins and peptides that exhibit heightened degradation sensitivity, for instance superoxide dismutase in liver, and peptidyl-prolyl cis-trans isomerase and insulin C-peptides in pancreas. Tissue sampling based on snap freezing produces a greater amount of degradation products and lower levels of endogenous peptides than rapid heat stabilization. We also demonstrate that solely snap freezing related degradation can be attenuated by subsequent heat stabilization. We conclude that tissue sampling involving a rapid heat stabilization step is preferable to freezing with regard to proteomic and peptidomic sample quality.

  9. 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

  10. 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

  11. 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.

  12. Discovering conformational sub-states relevant to protein function

    SciTech Connect

    Agarwal, Pratul K; Ramanathan, Arvind

    2011-01-01

    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. 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. Overall, QAA provides a novel framework to intuitively understand the biophysical basis of conformational diversity and its relevance to protein function.

  13. 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

  14. 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.

  15. Peptidyl-CCA deacylation on the ribosome promoted by induced fit and the O3′-hydroxyl group of A76 of the unacylated A-site tRNA

    SciTech Connect

    Simonović, Miljan; Steitz, Thomas A.

    2008-11-24

    The last step in ribosome-catalyzed protein synthesis is the hydrolytic release of the newly formed polypeptide from the P-site bound tRNA. Hydrolysis of the ester link of the peptidyl-tRNA is stimulated normally by the binding of release factors (RFs). However, an unacylated tRNA or just CCA binding to the ribosomal A site can also stimulate deacylation under some nonphysiological conditions. Although the sequence of events is well described by biochemical studies, the structural basis of the mechanism underlying this process is not well understood. Two new structures of the large ribosomal subunit of Haloarcula marismortui complexed with a peptidyl-tRNA analog in the P site and two oligonucleotide mimics of unacylated tRNA, CCA and CA, in the A site show that the binding of either CA or CCA induces a very similar conformational change in the peptidyl-transferase center as induced by aminoacyl-CCA. However, only CCA positions a water molecule appropriately to attack the carbonyl carbon of the peptidyl-tRNA and stabilizes the proper orientation of the ester link for hydrolysis. We, thus, conclude that both the ability of the O3'-hydroxyl group of the A-site A76 to position the water and the A-site CCA induced conformational change of the PTC are critical for the catalysis of the deacylation of the peptidyl-tRNA by CCA, and perhaps, an analogous mechanism is used by RFs.

  16. First quantum mechanics/molecular mechanics studies of the inhibition mechanism of cruzain by peptidyl halomethyl ketones.

    PubMed

    Arafet, Kemel; Ferrer, Silvia; Moliner, Vicent

    2015-06-01

    Cruzain is a primary cysteine protease expressed by the protozoan parasite Trypanosoma cruzi during Chagas disease infection, and thus, the development of inhibitors of this protein is a promising target for designing an effective therapy against the disease. In this paper, the mechanism of inhibition of cruzain by two different irreversible peptidyl halomethyl ketones (PHK) inhibitors has been studied by means of hybrid quantum mechanics/molecular mechanics-molecular dynamics (MD) simulations to obtain a complete representation of the possible free energy reaction paths. These have been traced on free energy surfaces in terms of the potential of mean force computed at AM1d/MM and DFT/MM levels of theory. An analysis of the possible reaction mechanisms of the inhibition process has been performed showing that the nucleophilic attack of an active site cysteine, Cys25, on a carbon atom of the inhibitor and the cleavage of the halogen-carbon bond take place in a single step. PClK appears to be much more favorable than PFK from a kinetic point of view. This result would be in agreement with experimental studies in other papain-like enzymes. A deeper analysis of the results suggests that the origin of the differences between PClK and PFK can be the different stabilizing interactions established between the inhibitors and the residues of the active site of the protein. Any attempt to explore the viability of the inhibition process through a stepwise mechanism involving the formation of a thiohemiketal intermediate and a three-membered sulfonium intermediate has been unsuccessful. Nevertheless, a mechanism through a protonated thiohemiketal, with participation of His159 as a proton donor, appears to be feasible despite showing higher free energy barriers. Our results suggest that PClK can be used as a starting point to develop a proper inhibitor of cruzain.

  17. The crystal structure of BlmI as a model for nonribosomal peptide synthetase peptidyl carrier proteins

    PubMed Central

    Lohman, Jeremy R.; Ma, Ming; Cuff, Marianne E.; Bigelow, Lance; Bearden, Jessica; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.; Shen, Ben

    2014-01-01

    Carrier proteins (CPs) play a critical role in the biosynthesis of various natural products, especially in nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymology, where the CPs are referred to as peptidyl-carrier proteins (PCPs) or acyl-carrier proteins (ACPs), respectively. CPs can either be a domain in large multifunctional polypeptides or standalone proteins, termed Type I and Type II, respectively. There have been many biochemical studies of the Type I PKS and NRPS CPs, and of Type II ACPs. However, recently a number of Type II PCPs have been found and biochemically characterized. In order to understand the possible interaction surfaces for combinatorial biosynthetic efforts we crystallized the first characterized and representative Type II PCP member, BlmI, from the bleomycin biosynthetic pathway from Streptomyces verticillus ATCC 15003. The structure is similar to CPs in general but most closely resembles PCPs. Comparisons with previously determined PCP structures in complex with catalytic domains reveals a common interaction surface. This surface is highly variable in charge and shape, which likely confers specificity for interactions. Previous nuclear magnetic resonance (NMR) analysis of a prototypical Type I PCP excised from the multimodular context revealed three conformational states. Comparison of the states with the structure of BlmI and other PCPs reveals that only one of the NMR states is found in other studies, suggesting the other two states may not be relevant. The state represented by the BlmI crystal structure can therefore serve as a model for both Type I and Type II PCPs. PMID:25050442

  18. Peptidyl aldehyde NK-1.8k suppresses enterovirus 71 and enterovirus 68 infection by targeting protease 3C.

    PubMed

    Wang, Yaxin; Yang, Ben; Zhai, Yangyang; Yin, Zheng; Sun, Yuna; Rao, Zihe

    2015-05-01

    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 (3C(pro)), N69S, which conferred EV71 resistance to NK-1.8k, possibly by increasing the flexibility of the substrate binding pocket of 3C(pro). 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

  19. Time-dependent inactivation of chick-embryo prolyl 4-hydroxylase by coumalic acid. Evidence for a syncatalytic mechanism.

    PubMed Central

    Günzler, V; Hanauske-Abel, H M; Myllylä, R; Mohr, J; Kivirikko, K I

    1987-01-01

    From the structure-activity relationships of known competitive inhibitors, coumalic acid (2-oxo-1,2H-pyran-5-carboxylic acid) was deduced to be a potential syncatalytic inhibitor for chick-embryo prolyl 4-hydroxylase. The compound caused time-dependent inactivation, the reaction rate being first-order. The inactivation constant was 0.094 min-1, the Ki 17 mM and the bimolecular rate constant 0.09 M-1 X S-1. Human prolyl 4-hydroxylase and chick embryo lysyl hydroxylase were also inactivated, though to a lesser extent. Inactivation could be prevented by adding high concentrations of 2-oxoglutarate or its competitive analogues to the reaction mixture. In Lineweaver-Burk kinetics, coumalic acid displayed S-parabolic competitive inhibition with respect to 2-oxoglutarate. The inactivation reaction had cofactor requirements similar to those for the decarboxylation of 2-oxoglutarate. Enzymic activity was partially preserved in the absence of iron, but the rescue was incomplete, owing to decreased stability of the enzyme under this condition. Coumalic acid also decreased the electrophoretic mobility of the alpha-subunit, but the beta-subunit was not affected. Prolonged incubation of coumalic acid above pH 6.8 led to loss of its inactivating potency, owing to hydrolysis. It is concluded that the inactivation of prolyl 4-hydroxylase by coumalic acid is due to a syncatalytic mechanism. The data also suggest that the 2-oxoglutarate-binding site of the enzyme is located within the alpha-subunit. PMID:3036081

  20. Targeting Inhibition of Fibroblast Activation Protein-α and Prolyl Oligopeptidase Activities on Cells Common to Metastatic Tumor Microenvironments1

    PubMed Central

    Christiansen, Victoria J; Jackson, Kenneth W; Lee, Kyung N; Downs, Tamyra D; McKee, Patrick A

    2013-01-01

    Fibroblast activation protein (FAP), a membrane prolyl-specific proteinase with both dipeptidase and endopeptidase activities, is overexpressed by reactive stromal fibroblasts during epithelial-derived cancer growth. FAP digests extracellular matrix as tissue is remodeled during cancer expansion and may also promote an immunotolerant tumor microenvironment. Recent studies suggest that nonspecific FAP inhibitors suppress human cancer xenografts in mouse models. Prolyl oligopeptidase (POP), another prolyl-specific serine proteinase, is also elevated in many cancers and may have a regulatory role in angiogenesis promotion. FAP and POP cell-associated activities may be targets for diagnosis and treatment of various cancers, but their accessibilities to highly effective specific inhibitors have not been shown for cells important to cancer growth. Despite their frequent simultaneous expression in many cancers and their overlapping activities toward commonly used substrates, precise, separate measurement of FAP or POP activity has largely been ignored. To distinguish each of the two activities, we synthesized highly specific substrates and inhibitors for FAP or POP based on amino acid sequences surrounding the scissile bonds of their respective putative substrates. We found varying amounts of FAP and POP protein and activities on activated fibroblasts, mesenchymal cells, normal breast cells, and one breast cancer cell line, with some cells exhibiting more POP than FAP activity. Replicating endothelial cells (ECs) expressed POP but not FAP until tubulogenesis began. Targeting FAP-positive cells, especially mesenchymal stem cells and cancer-associated fibroblasts for inactivation or destruction, and inhibiting POP-producing EC may abrogate stromal invasion and angiogenesis simultaneously and thereby diminish cancer growth. PMID:23555181

  1. Estrogen receptor β sustains epithelial differentiation by regulating prolyl hydroxylase 2 transcription.

    PubMed

    Mak, Paul; Chang, Cheng; Pursell, Bryan; Mercurio, Arthur M

    2013-03-19

    Estrogen receptor β (ERβ) promotes the degradation of hypoxia inducible factor 1α (HIF-1α), which contributes to the ability of this hormone receptor to sustain the differentiation of epithelial and carcinoma cells. Although the loss of ERβ and consequent HIF-1 activation occur in prostate cancer with profound consequences, the mechanism by which ERβ promotes the degradation of HIF-1α is unknown. We report that ERβ regulates the ligand (3β-adiol)-dependent transcription of prolyl hydroxylase 2 (PHD2) also known as Egl nine homolog 1 (EGLN1), a 2-oxoglutarate-dependent dioxygenase that hydroxylates HIF-1α and targets it for recognition by the von Hippel-Lindau tumor suppressor and consequent degradation. ERβ promotes PHD2 transcription by interacting with a unique estrogen response element in the 5' UTR of the PHD2 gene that functions as an enhancer. PHD2 itself is critical for maintaining epithelial differentiation. Loss of PHD2 expression or inhibition of its function results in dedifferentiation with characteristics of an epithelial-mesenchymal transition, and exogenous PHD2 expression in dedifferentiated cells can restore an epithelial phenotype. Moreover, expression of HIF-1α in cells that express PHD2 does not induce dedifferentiation but expression of HIF-1α containing mutations in the proline residues that are hydroxylated by PHD2 induces dedifferentiation. These data describe a unique mechanism for the regulation of HIF-1α stability that involves ERβ-mediated transcriptional regulation of PHD2 and they highlight an unexpected role for PHD2 in maintaining epithelial differentiation.

  2. Inhibition of Human Prolyl Oligopeptidase Activity by the Cyclotide Psysol 2 Isolated from Psychotria solitudinum

    PubMed Central

    2015-01-01

    Cyclotides are head-to-tail cyclized peptides comprising a stabilizing cystine-knot motif. To date, they are well known for their diverse bioactivities such as anti-HIV and immunosuppressive properties. Yet little is known about specific molecular mechanisms, in particular the interaction of cyclotides with cellular protein targets. Native and synthetic cyclotide-like peptides from Momordica plants are potent and selective inhibitors of different serine-type proteinases such as trypsin, chymotrypsin, matriptase, and tryptase-beta. This study describes the bioactivity-guided isolation of a cyclotide from Psychotria solitudinum as an inhibitor of another serine-type protease, namely, the human prolyl oligopeptidase (POP). Analysis of the inhibitory potency of Psychotria extracts and subsequent fractionation by liquid chromatography yielded the isolated peptide psysol 2 (1), which exhibited an IC50 of 25 μM. In addition the prototypical cyclotide kalata B1 inhibited POP activity with an IC50 of 5.6 μM. The inhibitory activity appeared to be selective for POP, since neither psysol 2 nor kalata B1 were able to inhibit the proteolytic activity of trypsin or chymotrypsin. The enzyme POP is well known for its role in memory and learning processes, and it is currently being considered as a promising therapeutic target for the cognitive deficits associated with several psychiatric and neurodegenerative diseases, such as schizophrenia and Parkinson’s disease. In the context of discovery and development of POP inhibitors with beneficial ADME properties, cyclotides may be suitable starting points considering their stability in biological fluids and possible oral bioavailability. PMID:25894999

  3. In silico methods to identify meat-derived prolyl endopeptidase inhibitors.

    PubMed

    Lafarga, Tomas; O'Connor, Paula; Hayes, Maria

    2015-05-15

    According to the World Health Organization (WHO), approximately 450 million people suffer from mental or neurological disorders and five of the ten leading causes of disability and premature death worldwide are psychiatric conditions. Social, biological and neurological sciences provided extensive understanding into the role of risk and protective factors in the development of mental disorders and poor mental health. Altered activity of a number of enzymes, such as prolyl endopeptidase (PEP, EC 3.4.21.26), has been linked to the prevention and treatment of a number of mental disorders, including anxiety, depression and Alzheimer's disease. The inhibition of PEP has potential for use in the prevention and in the treatment of mental disorders. The objective of this work was to identify PEP-inhibitory peptides from meat proteins using in silico methods. In this paper, five proteins commonly found in meat by-products were evaluated as a substrate for use in the generation of PEP inhibitory peptides. These include serum albumin, collagen and myosin. These proteins were cleaved in silico using BIOPEP and ExPASy PeptideCutter and the generated peptides were compared to known PEP-inhibiting peptides in the database of BIOPEP. A number of novel PEP inhibitory peptide sequences were identified in this study, including PPL, APPH, IPP and PPG with corresponding IC50 values of 2.86, 3.95, 4.02 and 2.70 mM, respectively. This work demonstrates the usefulness of in silico analysis for predicting the release of PEP-inhibiting peptides from meat proteins.

  4. Prolyl Oligopeptidase from the Blood Fluke Schistosoma mansoni: From Functional Analysis to Anti-schistosomal Inhibitors

    PubMed Central

    Fajtová, Pavla; Štefanić, Saša; Hradilek, Martin; Dvořák, Jan; Vondrášek, Jiří; Jílková, Adéla; Ulrychová, Lenka; McKerrow, James H.; Caffrey, Conor R.; Mareš, Michael; Horn, Martin

    2015-01-01

    Background Blood flukes of the genus Schistosoma cause schistosomiasis, a parasitic disease that infects over 240 million people worldwide, and for which there is a need to identify new targets for chemotherapeutic interventions. Our research is focused on Schistosoma mansoni prolyl oligopeptidase (SmPOP) from the serine peptidase family S9, which has not been investigated in detail in trematodes. Methodology/Principal Findings We demonstrate that SmPOP is expressed in adult worms and schistosomula in an enzymatically active form. By immunofluorescence microscopy, SmPOP is localized in the tegument and parenchyma of both developmental stages. Recombinant SmPOP was produced in Escherichia coli and its active site specificity investigated using synthetic substrate and inhibitor libraries, and by homology modeling. SmPOP is a true oligopeptidase that hydrolyzes peptide (but not protein) substrates with a strict specificity for Pro at P1. The inhibition profile is analogous to those for mammalian POPs. Both the recombinant enzyme and live worms cleave host vasoregulatory, proline-containing hormones such as angiotensin I and bradykinin. Finally, we designed nanomolar inhibitors of SmPOP that induce deleterious phenotypes in cultured schistosomes. Conclusions/Significance We provide the first localization and functional analysis of SmPOP together with chemical tools for measuring its activity. We briefly discuss the notion that SmPOP, operating at the host-parasite interface to cleave host bioactive peptides, may contribute to the survival of the parasite. If substantiated, SmPOP could be a new target for the development of anti-schistosomal drugs. PMID:26039195

  5. The Pseudomonas aeruginosa PA14 ABC Transporter NppA1A2BCD Is Required for Uptake of Peptidyl Nucleoside Antibiotics

    PubMed Central

    Braun, Yvonne; Dubiley, Svetlana; Lafon, Corinne; Köhler, Thilo; Page, Malcolm G. P.; Mourez, Michael; Severinov, Konstantin

    2015-01-01

    ABSTRACT Analysis of the genome sequence of Pseudomonas aeruginosa PA14 revealed the presence of an operon encoding an ABC-type transporter (NppA1A2BCD) showing homology to the Yej transporter of Escherichia coli. The Yej transporter is involved in the uptake of the peptide-nucleotide antibiotic microcin C, a translation inhibitor that targets the enzyme aspartyl-tRNA synthetase. Furthermore, it was recently shown that the Opp transporter from P. aeruginosa PAO1, which is identical to Npp, is required for uptake of the uridyl peptide antibiotic pacidamycin, which targets the enzyme translocase I (MraY), which is involved in peptidoglycan synthesis. We used several approaches to further explore the substrate specificity of the Npp transporter. Assays of growth in defined minimal medium containing peptides of various lengths and amino acid compositions as sole nitrogen sources, as well as Biolog Phenotype MicroArrays, showed that the Npp transporter is not required for di-, tri-, and oligopeptide uptake. Overexpression of the npp operon increased susceptibility not just to pacidamycin but also to nickel chloride and the peptidyl nucleoside antibiotic blasticidin S. Furthermore, heterologous expression of the npp operon in a yej-deficient mutant of E. coli resulted in increased susceptibility to albomycin, a naturally occurring sideromycin with a peptidyl nucleoside antibiotic. Additionally, heterologous expression showed that microcin C is recognized by the P. aeruginosa Npp system. Overall, these results suggest that the NppA1A2BCD transporter is involved in the uptake of peptidyl nucleoside antibiotics by P. aeruginosa PA14. IMPORTANCE One of the world's most serious health problems is the rise of antibiotic-resistant bacteria. There is a desperate need to find novel antibiotic therapeutics that either act on new biological targets or are able to bypass known resistance mechanisms. Bacterial ABC transporters play an important role in nutrient uptake from the

  6. Cellular oxygen sensing: Importins and exportins are mediators of intracellular localisation of prolyl-4-hydroxylases PHD1 and PHD2

    SciTech Connect

    Steinhoff, Amrei; Pientka, Friederike Katharina; Moeckel, Sylvia; Kettelhake, Antje; Hartmann, Enno; Koehler, Matthias; Depping, Reinhard

    2009-10-02

    Hypoxia-inducible factors are crucial in the regulatory process of oxygen homeostasis of vertebrate cells. Inhibition of prolyl hydroxylation of HIF-{alpha} subunits by prolyl-hydroxylases (PHD1, PHD2 and PHD3) leads to transcription of a greater number of hypoxia responsive genes. We have investigated the subcellular distribution and the molecular mechanisms regulating the intracellular allocation of PHD1 and PHD2. As reported earlier we find PHD1 located exclusively in the nucleus. We demonstrate that nuclear import of PHD1 occurs importin {alpha}/{beta} dependently and relies on a nuclear localisation signal (NLS). By contrast PHD2 is cycling between nucleus and cytoplasm, and nuclear import seems to be independent of 'classical' importin {alpha}/{beta} receptors. Furthermore, we reveal that the exit of PHD2 from the nucleus requires CRM1 and the N-terminal 100 amino acids of the protein. Our findings provide new insights into the mechanisms of the regulation of the oxygen sensor cascade of PHDs in different cellular compartments.

  7. Physiological and Pathogenic Roles of Prolyl Isomerase Pin1 in Metabolic Regulations via Multiple Signal Transduction Pathway Modulations

    PubMed Central

    Nakatsu, Yusuke; Matsunaga, Yasuka; Yamamotoya, Takeshi; Ueda, Koji; Inoue, Yuki; Mori, Keiichi; Sakoda, Hideyuki; Fujishiro, Midori; Ono, Hiraku; Kushiyama, Akifumi; Asano, Tomoichiro

    2016-01-01

    Prolyl isomerases are divided into three groups, the FKBP family, Cyclophilin and the Parvulin family (Pin1 and Par14). Among these isomerases, Pin1 is a unique prolyl isomerase binding to the motif including pSer/pThr-Pro that is phosphorylated by kinases. Once bound, Pin1 modulates the enzymatic activity, protein stability or subcellular localization of target proteins by changing the cis- and trans-formations of proline. Several studies have examined the roles of Pin1 in the pathogenesis of cancers and Alzheimer’s disease. On the other hand, recent studies have newly demonstrated Pin1 to be involved in regulating glucose and lipid metabolism. Interestingly, while Pin1 expression is markedly increased by high-fat diet feeding, Pin1 KO mice are resistant to diet-induced obesity, non-alcoholic steatohepatitis and diabetic vascular dysfunction. These phenomena result from the binding of Pin1 to several key factors regulating metabolic functions, which include insulin receptor substrate-1, AMPK, Crtc2 and NF-κB p65. In this review, we focus on recent advances in elucidating the physiological roles of Pin1 as well as the pathogenesis of disorders involving this isomerase, from the viewpoint of the relationships between signal transductions and metabolic functions. PMID:27618008

  8. Cloning and expression of a novel prolyl endopeptidase from Aspergillus oryzae and its application in beer stabilization.

    PubMed

    Kang, Chao; Yu, Xiao-Wei; Xu, Yan

    2015-02-01

    A novel prolyl endopeptidase gene from Aspergillus oryzae was cloned and expressed in Pichia pastoris. Amino acid sequence analysis of the prolyl endopeptidase from Aspergillus oryzae (AO-PEP) showed that this enzyme belongs to a class serine peptide S28 family. Expression, purification and characterization of AO-PEP were analyzed. The optimum pH and temperature were pH 5.0 and 40 °C, respectively. The enzyme was activated and stabilized by metal ion Ca(2+) and inhibited by Zn(2+), Mn(2+), Al(3+), and Cu(2+). The K m and k cat values of the purified enzyme for different substrates were evaluated. The results implied that the recombinant AO-PEP possessed higher affinity for the larger substrate. A fed-batch strategy was developed for the high-cell-density fermentation and the enzyme activity reached 1,130 U/l after cultivation in 7 l fermentor. After addition of AO-PEP during the fermentation phase of beer brewing, demonstrated the potential application of AO-PEP in the non-biological stability of beer, which favor further industrial development of this new enzyme in beer stabilization, due to its reducing operational costs, as well as no beer losses unlike regeneration process and beer lost with regenerated polyvinylpolypyrrolidone system.

  9. Physiological and Pathogenic Roles of Prolyl Isomerase Pin1 in Metabolic Regulations via Multiple Signal Transduction Pathway Modulations.

    PubMed

    Nakatsu, Yusuke; Matsunaga, Yasuka; Yamamotoya, Takeshi; Ueda, Koji; Inoue, Yuki; Mori, Keiichi; Sakoda, Hideyuki; Fujishiro, Midori; Ono, Hiraku; Kushiyama, Akifumi; Asano, Tomoichiro

    2016-09-07

    Prolyl isomerases are divided into three groups, the FKBP family, Cyclophilin and the Parvulin family (Pin1 and Par14). Among these isomerases, Pin1 is a unique prolyl isomerase binding to the motif including pSer/pThr-Pro that is phosphorylated by kinases. Once bound, Pin1 modulates the enzymatic activity, protein stability or subcellular localization of target proteins by changing the cis- and trans-formations of proline. Several studies have examined the roles of Pin1 in the pathogenesis of cancers and Alzheimer's disease. On the other hand, recent studies have newly demonstrated Pin1 to be involved in regulating glucose and lipid metabolism. Interestingly, while Pin1 expression is markedly increased by high-fat diet feeding, Pin1 KO mice are resistant to diet-induced obesity, non-alcoholic steatohepatitis and diabetic vascular dysfunction. These phenomena result from the binding of Pin1 to several key factors regulating metabolic functions, which include insulin receptor substrate-1, AMPK, Crtc2 and NF-κB p65. In this review, we focus on recent advances in elucidating the physiological roles of Pin1 as well as the pathogenesis of disorders involving this isomerase, from the viewpoint of the relationships between signal transductions and metabolic functions.

  10. Suppression of Tumor Growth in Mice by Rationally Designed Pseudopeptide Inhibitors of Fibroblast Activation Protein and Prolyl Oligopeptidase1

    PubMed Central

    Jackson, Kenneth W.; Christiansen, Victoria J.; Yadav, Vivek R.; Silasi-Mansat, Robert; Lupu, Florea; Awasthi, Vibhudutta; Zhang, Roy R.; McKee, Patrick A.

    2015-01-01

    Tumor microenvironments (TMEs) are composed of cancer cells, fibroblasts, extracellular matrix, microvessels, and endothelial cells. Two prolyl endopeptidases, fibroblast activation protein (FAP) and prolyl oligopeptidase (POP), are commonly overexpressed by epithelial-derived malignancies, with the specificity of FAP expression by cancer stromal fibroblasts suggesting FAP as a possible therapeutic target. Despite overexpression in most cancers and having a role in angiogenesis, inhibition of POP activity has received little attention as an approach to quench tumor growth. We developed two specific and highly effective pseudopeptide inhibitors, M83, which inhibits FAP and POP proteinase activities, and J94, which inhibits only POP. Both suppressed human colon cancer xenograft growth > 90% in mice. By immunohistochemical stains, M83- and J94-treated tumors had fewer microvessels, and apoptotic areas were apparent in both. In response to M83, but not J94, disordered collagen accumulations were observed. Neither M83- nor J94-treated mice manifested changes in behavior, weight, or gastrointestinal function. Tumor growth suppression was more extensive than noted with recently reported efforts by others to inhibit FAP proteinase function or reduce FAP expression. Diminished angiogenesis and the accompanying profound reduction in tumor growth suggest that inhibition of either FAP or POP may offer new therapeutic approaches that directly target TMEs. PMID:25622898

  11. Physiological and Pathogenic Roles of Prolyl Isomerase Pin1 in Metabolic Regulations via Multiple Signal Transduction Pathway Modulations.

    PubMed

    Nakatsu, Yusuke; Matsunaga, Yasuka; Yamamotoya, Takeshi; Ueda, Koji; Inoue, Yuki; Mori, Keiichi; Sakoda, Hideyuki; Fujishiro, Midori; Ono, Hiraku; Kushiyama, Akifumi; Asano, Tomoichiro

    2016-01-01

    Prolyl isomerases are divided into three groups, the FKBP family, Cyclophilin and the Parvulin family (Pin1 and Par14). Among these isomerases, Pin1 is a unique prolyl isomerase binding to the motif including pSer/pThr-Pro that is phosphorylated by kinases. Once bound, Pin1 modulates the enzymatic activity, protein stability or subcellular localization of target proteins by changing the cis- and trans-formations of proline. Several studies have examined the roles of Pin1 in the pathogenesis of cancers and Alzheimer's disease. On the other hand, recent studies have newly demonstrated Pin1 to be involved in regulating glucose and lipid metabolism. Interestingly, while Pin1 expression is markedly increased by high-fat diet feeding, Pin1 KO mice are resistant to diet-induced obesity, non-alcoholic steatohepatitis and diabetic vascular dysfunction. These phenomena result from the binding of Pin1 to several key factors regulating metabolic functions, which include insulin receptor substrate-1, AMPK, Crtc2 and NF-κB p65. In this review, we focus on recent advances in elucidating the physiological roles of Pin1 as well as the pathogenesis of disorders involving this isomerase, from the viewpoint of the relationships between signal transductions and metabolic functions. PMID:27618008

  12. miR-190 Enhances HIF-Dependent Responses to Hypoxia in Drosophila by Inhibiting the Prolyl-4-hydroxylase Fatiga

    PubMed Central

    De Lella Ezcurra, Ana Laura; Bertolin, Agustina Paola; Kim, Kevin; Gándara, Lautaro; Luschnig, Stefan; Perrimon, Norbert; Melani, Mariana; Wappner, Pablo

    2016-01-01

    Cellular and systemic responses to low oxygen levels are principally mediated by Hypoxia Inducible Factors (HIFs), a family of evolutionary conserved heterodimeric transcription factors, whose alpha- and beta-subunits belong to the bHLH-PAS family. In normoxia, HIFα is hydroxylated by specific prolyl-4-hydroxylases, targeting it for proteasomal degradation, while in hypoxia the activity of these hydroxylases decreases due to low oxygen availability, leading to HIFα accumulation and expression of HIF target genes. To identify microRNAs required for maximal HIF activity, we conducted an overexpression screen in Drosophila melanogaster, evaluating the induction of a HIF transcriptional reporter. miR-190 overexpression enhanced HIF-dependent biological responses, including terminal sprouting of the tracheal system, while in miR-190 loss of function embryos the hypoxic response was impaired. In hypoxic conditions, miR-190 expression was upregulated and required for induction of HIF target genes by directly inhibiting the HIF prolyl-4-hydroxylase Fatiga. Thus, miR-190 is a novel regulator of the hypoxia response that represses the oxygen sensor Fatiga, leading to HIFα stabilization and enhancement of hypoxic responses. PMID:27223464

  13. Unusual Extra Space at the Active Site and High Activity for Acetylated Hydroxyproline of Prolyl Aminopeptidase from Serratia marcescens

    PubMed Central

    Nakajima, Yoshitaka; Ito, Kiyoshi; Sakata, Makoto; Xu, Yue; Nakashima, Kanako; Matsubara, Futoshi; Hatakeyama, Susumi; Yoshimoto, Tadashi

    2006-01-01

    The prolyl aminopeptidase complexes of Ala-TBODA [2-alanyl-5-tert-butyl-(1, 3, 4)-oxadiazole] and Sar-TBODA [2-sarcosyl-5-tert-butyl-(1, 3, 4)-oxadiazole] were analyzed by X-ray crystallography at 2.4 Å resolution. Frames of alanine and sarcosine residues were well superimposed on each other in the pyrrolidine ring of proline residue, suggesting that Ala and Sar are recognized as parts of this ring of proline residue by the presence of a hydrophobic proline pocket at the active site. Interestingly, there was an unusual extra space at the bottom of the hydrophobic pocket where proline residue is fixed in the prolyl aminopeptidase. Moreover, 4-acetyloxyproline-βNA (4-acetyloxyproline β-naphthylamide) was a better substrate than Pro-βNA. Computer docking simulation well supports the idea that the 4-acetyloxyl group of the substrate fitted into that space. Alanine scanning mutagenesis of Phe139, Tyr149, Tyr150, Phe236, and Cys271, consisting of the hydrophobic pocket, revealed that all of these five residues are involved significantly in the formation of the hydrophobic proline pocket for the substrate. Tyr149 and Cys271 may be important for the extra space and may orient the acetyl derivative of hydroxyproline to a preferable position for hydrolysis. These findings imply that the efficient degradation of collagen fragment may be achieved through an acetylation process by the bacteria. PMID:16452443

  14. Sudestada1, a Drosophila ribosomal prolyl-hydroxylase required for mRNA translation, cell homeostasis, and organ growth

    PubMed Central

    Katz, Maximiliano J.; Acevedo, Julieta M.; Loenarz, Christoph; Galagovsky, Diego; Liu-Yi, Phebee; Pérez-Pepe, Marcelo; Thalhammer, Armin; Sekirnik, Rok; Ge, Wei; Melani, Mariana; Thomas, María G.; Simonetta, Sergio; Boccaccio, Graciela L.; Schofield, Christopher J.; Cockman, Matthew E.; Ratcliffe, Peter J.; Wappner, Pablo

    2014-01-01

    Genome sequences predict the presence of many 2-oxoglutarate (2OG)-dependent oxygenases of unknown biochemical and biological functions in Drosophila. Ribosomal protein hydroxylation is emerging as an important 2OG oxygenase catalyzed pathway, but its biological functions are unclear. We report investigations on the function of Sudestada1 (Sud1), a Drosophila ribosomal oxygenase. As with its human and yeast homologs, OGFOD1 and Tpa1p, respectively, we identified Sud1 to catalyze prolyl-hydroxylation of the small ribosomal subunit protein RPS23. Like OGFOD1, Sud1 catalyzes a single prolyl-hydroxylation of RPS23 in contrast to yeast Tpa1p, where Pro-64 dihydroxylation is observed. RNAi-mediated Sud1 knockdown hinders normal growth in different Drosophila tissues. Growth impairment originates from both reduction of cell size and diminution of the number of cells and correlates with impaired translation efficiency and activation of the unfolded protein response in the endoplasmic reticulum. This is accompanied by phosphorylation of eIF2α and concomitant formation of stress granules, as well as promotion of autophagy and apoptosis. These observations, together with those on enzyme homologs described in the companion articles, reveal conserved biochemical and biological roles for a widely distributed ribosomal oxygenase. PMID:24550463

  15. Modifcation of the Prolyl Ring of Val-Pro and the Impact of this Modification on b2 Ion Structure

    NASA Astrophysics Data System (ADS)

    Bernier, Matthew C.; Wysocki, Vicki H.; Gucinski, Ashley; Chamot-Rooke, Julia

    2013-06-01

    Here we present b2 ion studies on one tripeptide (VPA) and show how the addition of a fluorine or a hydroxyl group on the 3rd position of the proline ring can affect the b2 ion formation. Action IRMPD results of ValHyp (Hyp=hydroxyproline), ValFlp (Flp=trans-fluoroproline), and Valflp (flp=cis-fluoroproline) all show the presence of a strong oxazolone band in the CO region at 1900 cm-1. The presence of peaks in the diketopiperazine region between 1700 and 1800 cm-1 varies depending on which substituent is placed on the prolyl ring. Recently published data from our group showed a pair of medium sized diketopiperazine bands at 1760 and 1701 cm-1 for ValPro and we observed similarly intense bands for Valflp at 1752 and 1689 cm-1. ValHyp and ValFlp fail to show any significant diketopiperazine bands, but if zoomed in x10 a small band can be observed at 1756 cm-1 for ValHyp. From this data it is apparent that substitution of the second position prolyl ring can alter the formation of the b2 diketopiperazine ion.

  16. PIN1 in hepatocellular carcinoma is associated with TP53 gene status.

    PubMed

    Bae, Jun Sang; Noh, Sang Jae; Kim, Kyoung Min; Jang, Kyu Yun; Park, Ho Sung; Chung, Myoung Ja; Park, Byung-Hyun; Moon, Woo Sung

    2016-10-01

    Phosphorylation of proteins on serine/threonine residues that precede proline (pSer/Thr-Pro) is specifically catalyzed by the peptidyl-prolyl cis-trans isomerase PIN1. PIN1-mediated prolyl-isomerization induces cell cycle arrest and growth inhibition through the regulation of target proteins, including TP53. We examined whether PIN1 acts in a different manner according to TP53 gene status in hepatocellular carcinoma (HCC). We investigated the expression of PIN1 and TP53 proteins in 119 HCC tissue samples. We also analyzed PIN1 expression in combination with TP53 gene mutation and its correlation with the clinical outcome. In addition, we used synthetic small interfering RNA to silence PIN1 gene expression in TP53 wild-type and TP53 mutant HCC cell lines, and then evaluated cell proliferation, migration and invasion. Expression of PIN1 was strongly associated with expression of TP53 protein or TP53 mutation of HCC samples. PIN1 and TP53 expression in TP53 mutant HCC cell lines was higher than that in TP53 wild-type HCC cell lines. Silencing of PIN1 in HLE cells containing mutant TP53 significantly decreased cell proliferation, migration and invasion. In contrast to PIN1 silencing in HLE cells, PIN1 silencing in HepG2 cells containing functional wild-type TP53 resulted in enhanced tumor cell proliferation. HCC patients bearing PIN1 expression with wild-type TP53 were predicted to demonstrate favorable relapse-free survival. Our results suggest that PIN1 plays a role in cancer cell proliferation, migration and invasion in a different manner according to the TP53 gene mutation status in HCC. In particular, interaction of PIN1 with mutant TP53 can act as a tumor promoter and increase its oncogenic activities in HCC. PMID:27499097

  17. Structure-based design, synthesis, and SAR evaluation of a new series of 8-hydroxyquinolines as HIF-1alpha prolyl hydroxylase inhibitors.

    PubMed

    Warshakoon, Namal C; Wu, Shengde; Boyer, Angelique; Kawamoto, Richard; Sheville, Justin; Renock, Sean; Xu, Kevin; Pokross, Matthew; Zhou, Songtao; Winter, Carol; Walter, Richard; Mekel, Marlene; Evdokimov, Artem G

    2006-11-01

    A new series of potent 8-hydroxyquinolines was designed based on the newly resolved X-ray crystal structure of EGLN-1. Both alkyl and aryl 8-hydroxyquinoline-7-carboxyamides were good HIF-1alpha prolyl hydroxylase (EGLN) inhibitors. In subsequent VEGF induction assays, these exhibited potent VEGF activity. In addition, this class of compounds did show the ability to stabilize HIF-1alpha.

  18. Hypoxia inducible factor prolyl hydroxylases as targets for neuroprotection by “antioxidant” metal chelators: from ferroptosis to stroke

    PubMed Central

    Speer, Rachel E.; Karuppagounder, Saravanan S.; Basso, Manuela; Sleiman, Sama; Kumar, Amit; Brand, David; Smirnova, Natalya; Gazaryan, Irina; Khim, Soah J.; Ratan, Rajiv R.

    2015-01-01

    Neurologic conditions including stroke, Alzheimer’s disease, Parkinson’s disease and Huntington’s disease are leading causes of death and long-term disability in the United States, and efforts to develop novel therapeutics for these conditions have historically had poor success in translating from bench to bedside. Hypoxia Inducible Factor-1alpha (HIF-1α) mediates a broad, evolutionarily conserved, endogenous adaptive program to hypoxia, and manipulation of components of the HIF pathway are neuroprotective in a number of human neurological diseases and experimental models. In this review, we discuss molecular components of one aspect of hypoxic adpatation in detail, and provide perspective on which targets within this pathway appear to be ripest for preventing and repairing neurodegeneration. Further, we highlight the role of HIF prolyl hydroxylases as emerging targets for the salutary effects of metal chelators on ferroptosis in vitro as well in animal models of neurological diseases. PMID:23376032

  19. The prolyl 4-hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate generates effective iron deficiency in cultured cells.

    PubMed

    Wang, Jian; Buss, Joan L; Chen, Guohua; Ponka, Prem; Pantopoulos, Kostas

    2002-10-01

    Ethyl-3,4-dihydroxybenzoate (EDHB) is commonly utilized as a substrate analog and competitive inhibitor of prolyl 4-hydroxylases. These iron-dependent enzymes have received a lot of attention for their involvement in crucial biochemical pathways such as collagen maturation and oxygen sensing. Since EDHB is also capable of chelating the enzyme-bound iron, we study here its function as a chelator. We show that the affinity of EDHB for ferric iron is significantly lower than that of desferrioxamine. Nevertheless, EDHB is sufficient to promote effective iron deficiency in cells, reflected in the activation of the iron-responsive element/iron regulatory protein regulatory network. Thus, treatment of B6 fibroblasts with EDHB results in slow activation of iron regulatory protein 1 accompanied by an increase in transferrin receptor levels and reduction of the ferritin pool.

  20. [Multicomponent antithrombotic effect of the neuroprotective prolyl dipeptide GVS-111 and its major metabolite cyclo-L-prolylglycine].

    PubMed

    Ostrovskaia, R U; Liapina, L A; Pastorova, V E; Mirzoev, T Kh; Gudasheva, T A; Seredenin, S B; Ashmarin, I P

    2002-01-01

    The experiments in vivo showed that the new nootropic prolyl-containing GVS-111 produces an antithrombotic effect, influencing various stages of the blood coagulation process. GVS-111 exhibits anticoagulant and fibrinolytic properties and enhances fibrin destabilization by reducing the XIIIa factor activity. These effects are manifested upon both intraperitoneal (1 mg/kg) and peroral (10 mg/kg) administration of GVS-111 (in both cases, a single daily treatment over a period of 10 days). The same effects (anticoagulant, fibrinolytic, antifibrin-stabilizing) were observed in in vitro experiments with both GVS-111 (10(-3)-10(-6) M) and its main metabolite cyclo-L-prolylglycine (up to 10(-10) M). In addition, the latter metabolite exhibited an antiaggregant effect. The antithrombotic activity of GVS-111, together with previously established neuroprotector properties, low toxicity, and the absence of complications, makes this compound a promising antistroke drug. PMID:12109290

  1. Expression, Purification, Crystallization And Preliminary X-Ray Studies of a Prolyl-4-Hydroxylase Protein From Bacillus Anthracis

    SciTech Connect

    Miller, M.A.; Scott, E.E.; Limburg, J.

    2009-05-26

    Collagen prolyl-4-hydroxylase (C-P4H) catalyzes the hydroxylation of specific proline residues in procollagen, which is an essential step in collagen biosynthesis. A new form of P4H from Bacillus anthracis (anthrax-P4H) that shares many characteristics with the type I C-P4H from human has recently been characterized. The structure of anthrax-P4H could provide important insight into the chemistry of C-P4Hs and into the function of this unique homodimeric P4H. X-ray diffraction data of selenomethionine-labeled anthrax-P4H recombinantly expressed in Escherichia coli have been collected to 1.4 {angstrom} resolution.

  2. Prolyl Oligopeptidase Inhibition Attenuates Steatosis in the L02 Human Liver Cell Line

    PubMed Central

    Zhou, Da; Li, Bing-Hang; Wang, Jing; Ding, Yong-Nian; Dong, Yan; Chen, Yuan-Wen; Fan, Jian-Gao

    2016-01-01

    Background Prolyl oligopeptidase (POP) is a serine endopeptidase that is widely distributed in vivo, particularly in the liver. Significant changes in functional mitochondrial proteins involved with mitochondrial oxidoreductases/transporters and nucleic acid binding proteins were observed after POP inhibition in the liver, which suggested a role of POP in regulating liver energy metabolism. Steatosis in nonalcoholic fatty liver disease (NAFLD) is associated with disturbances in lipid and energy metabolism in hepatocytes. Here, we aimed to study the effect of POP on hepatocyte steatosis. Methods The human liver cell line L02 was used to investigate the biological effects of POP. An in vitro cell model of steatosis was successfully induced with oleic acid and palmitic acid. L02 cells were also subjected to S17092 (a POP inhibitor) at different concentrations for 24 or 48 h. Ac-SDKP levels and POP activity were measured to assess the rate of inhibition of POP by S17092. The POP gene and protein expression levels were detected using real-time PCR and Western blots, respectively. Oil red O staining was performed and the triglyceride levels in the L02 cells were also measured. Cell proliferation and apoptosis were detected using CCK-8 and flow cytometry, respectively. The expression of genes involved in lipid metabolism was detected using real-time PCR. The effects of POP inhibition on LC3B II were detected by Western blot. Results Compared with the control, the POP mRNA levels increased by approximately 30%, and the POP protein levels increased by almost 60% in the steatotic L02 cells. After S17092 (0.026~130 μM) incubation for 24 or 48 h, cell proliferation was significantly decreased in the free fatty acid (FFA)-treated cells at 26–130 μM; however, S17092 did not affect the proliferation of L02 cells after 24 h of incubation with S17092 at 0.026–65 μM without FFA treatment. S17092 treatment (13 and 26 μM) also elicited no significant effect on apoptosis in

  3. Tuning the Transcriptional Response to Hypoxia by Inhibiting Hypoxia-inducible Factor (HIF) Prolyl and Asparaginyl Hydroxylases*

    PubMed Central

    Chan, Mun Chiang; Ilott, Nicholas E.; Schödel, Johannes; Sims, David; Tumber, Anthony; Lippl, Kerstin; Mole, David R.; Pugh, Christopher W.; Ratcliffe, Peter J.; Ponting, Chris P.; Schofield, Christopher J.

    2016-01-01

    The hypoxia-inducible factor (HIF) system orchestrates cellular responses to hypoxia in animals. HIF is an α/β-heterodimeric transcription factor that regulates the expression of hundreds of genes in a tissue context-dependent manner. The major hypoxia-sensing component of the HIF system involves oxygen-dependent catalysis by the HIF hydroxylases; in humans there are three HIF prolyl hydroxylases (PHD1–3) and an asparaginyl hydroxylase (factor-inhibiting HIF (FIH)). PHD catalysis regulates HIFα levels, and FIH catalysis regulates HIF activity. How differences in HIFα hydroxylation status relate to variations in the induction of specific HIF target gene transcription is unknown. We report studies using small molecule HIF hydroxylase inhibitors that investigate the extent to which HIF target gene expression is induced by PHD or FIH inhibition. The results reveal substantial differences in the role of prolyl and asparaginyl hydroxylation in regulating hypoxia-responsive genes in cells. PHD inhibitors with different structural scaffolds behave similarly. Under the tested conditions, a broad-spectrum 2-oxoglutarate dioxygenase inhibitor is a better mimic of the overall transcriptional response to hypoxia than the selective PHD inhibitors, consistent with an important role for FIH in the hypoxic transcriptional response. Indeed, combined application of selective PHD and FIH inhibitors resulted in the transcriptional induction of a subset of genes not fully responsive to PHD inhibition alone. Thus, for the therapeutic regulation of HIF target genes, it is important to consider both PHD and FIH activity, and in the case of some sets of target genes, simultaneous inhibition of the PHDs and FIH catalysis may be preferable. PMID:27502280

  4. TsPAP1 encodes a novel plant prolyl aminopeptidase whose expression is induced in response to suboptimal growth conditions

    SciTech Connect

    Szawlowska, Urszula; Grabowska, Agnieszka; Zdunek-Zastocka, Edyta; Bielawski, Wieslaw

    2012-03-02

    Highlights: Black-Right-Pointing-Pointer A cDNA encoding a novel plant prolyl aminopeptidase, TsPAP1, was obtained from triticale. Black-Right-Pointing-Pointer The cloned TsPAP1 cDNA is 1387 bp long and encodes a protein of 390 amino acids. Black-Right-Pointing-Pointer The deduced TsPAP1 protein revealed characteristics of the monomeric bacterial PAPs. Black-Right-Pointing-Pointer The TsPAP1 mRNA level increased under drought, salinity and in the presence of metal ions. -- Abstract: A triticale cDNA encoding a prolyl aminopeptidase (PAP) was obtained by RT-PCR and has been designated as TsPAP1. The cloned cDNA is 1387 bp long and encodes a protein of 390 amino acids with a calculated molecular mass of 43.9 kDa. The deduced TsPAP1 protein exhibits a considerable sequence identity with the biochemically characterized bacterial and fungal PAP proteins of small molecular masses ({approx}35 kDa). Moreover, the presence of conserved regions that are characteristic for bacterial monomeric PAP enzymes (the GGSWG motif, the localization of the catalytic triad residues and the segment involved in substrate binding) has also been noted. Primary structure analysis and phylogenetic analysis revealed that TsPAP1 encodes a novel plant PAP protein that is distinct from the multimeric proteins that have thus far been characterized in plants and whose counterparts have been recognized only in bacteria and fungi. A significant increase in the TsPAP1 transcript level in the shoots of triticale plants was observed under drought and saline conditions as well as in the presence of cadmium and aluminium ions in the nutrient medium. This paper is the first report describing changes in the transcript levels of any plant PAP in response to suboptimal growth conditions.

  5. A cytoplasmic prolyl hydroxylation and glycosylation pathway modifies Skp1 and regulates O2-dependent development in Dictyostelium.

    PubMed

    West, Christopher M; Wang, Zhuo A; van der Wel, Hanke

    2010-02-01

    The soil amoeba Dictyostelium is an obligate aerobe that monitors O(2) for informational purposes in addition to utilizing it for oxidative metabolism. Whereas low O(2) suffices for proliferation, a higher level is required for slugs to culminate into fruiting bodies, and O(2) influences slug polarity, slug migration, and cell-type proportioning. Dictyostelium expresses a cytoplasmic prolyl 4-hydroxylase (P4H1) known to mediate O(2)-sensing in animals, but lacks HIFalpha, a major hydroxylation target whose accumulation directly induces animal hypoxia-dependent transcriptional changes. The O(2)-requirement for culmination is increased by P4H1-gene disruption and reduced by P4H1 overexpression. A target of Dictyostelium P4H1 is Skp1, a subunit of the SCF-class of E3-ubiquitin ligases related to the VBC-class that mediates hydroxylation-dependent degradation of animal HIFalpha. Skp1 is a target of a novel cytoplasmic O-glycosylation pathway that modifies HyPro143 with a pentasaccharide, and glycosyltransferase mutants reveal that glycosylation intermediates have antagonistic effects toward P4H1 in O(2)-signaling. Current evidence indicates that Skp1 is the only glycosylation target in cells, based on metabolic labeling, biochemical complementation, and enzyme specificity studies. Bioinformatics studies suggest that the HyPro-modification pathway existed in the ancestral eukaryotic lineage and was retained in selected modern day unicellular organisms whose life cycles experience varying degrees of hypoxia. It is proposed that, in Dictyostelium and other protists including the agent for human toxoplasmosis Toxoplasma gondii, prolyl hydroxylation and glycosylation mediate O(2)-signaling in hierarchical fashion via Skp1 to control the proteome, directly via degradation rather than indirectly via transcription as found in animals.

  6. The effect of aminoacyl- or peptidyl-tRNA at the A-site on the arrangement of deacylated tRNA at the ribosomal P-site.

    PubMed

    Babkina, G T; Bausk, E V; Graifer, D M; Karpova, G G; Matasova, N B

    1984-05-21

    Photoreactive derivatives of E. coli tRNAPhe bearing arylazido groups on guanine residues (azido-tRNA) were used for affinity labelling of E. coli ribosomes in the region of the P-site when the A-site was either free or occupied by aminoacyl- or peptidyl-tRNA. Corresponding complexes of azido-tRNA with ribosomes and poly(U) were obtained both nonenzymatically and with the use of elongation factors. UV-irradiation of the complexes resulted in labelling of ribosomal proteins (preferentially of 30 S subunit). Proteins S9 and S21 were labelled only when the A-site was free; S14 - only when it was occupied; S11, S13, S19 - in both cases; S5, S7, S12, S20 - in some states.

  7. Mutation of active site serine residue with cysteine displays change in acyl-acceptor preference of β-peptidyl aminopeptidase from Pseudomonas aeruginosa PAO1.

    PubMed

    Arima, Jiro; Tanaka, Ayumi; Morimoto, Masazumi; Mori, Nobuhiro

    2014-02-01

    A β-peptidyl aminopeptidase, a peptidase belonging to the P1 family, catalyzes aminolysis in accordance with its hydrolytic activity. We specifically examined β-peptidyl aminopeptidase of Pseudomonas aeruginosa PAO1 (BapF) to assess the effects of mutation of catalytic Ser with Cys or Thr on its catalytic ability. Recombinant BapF and its S237C mutant exhibited p-nitroaniline release activity toward β-homo-Gly-p-nitroanilide (βhGly-pNA), but the products of the enzyme reaction differed completely from one another. Wild-type BapF showed βhGly-βhGly-pNA synthetic activity, but the product vanished in a few minutes and converted to free βhGly. In contrast, the product βhGly-βhGly-pNA was synthesized by S237C BapF efficiently without degradation, indicating that because of the mutation, the enzyme came to recognize only the amine group as an acyl acceptor instead of water. Furthermore, a difference in acyl acceptor preference between that of wild type and S237C BapF was observed. When using cysteamine as an acyl acceptor, βhGly-cysteamine was synthesized only in the reaction using S237C BapF. In contrast, S237C BapF was unable to synthesize βhGly-cystamine when using cystamine as an acyl acceptor, although it was synthesized by wild-type BapF. Such a dynamic change in the acyl acceptor by the mutation of catalytic Ser with Cys is regarded as a unique feature of family P1 peptidases.

  8. Site-selective chemical modification of chymotrypsin using peptidyl derivatives bearing optically active diphenyl 1-amino-2-phenylethylphosphonate: Stereochemical effect of the diphenyl phosphonate moiety.

    PubMed

    Ono, Shin; Nakai, Takahiko; Kuroda, Hirofumi; Miyatake, Ryuta; Horino, Yoshikazu; Abe, Hitoshi; Umezaki, Masahito; Oyama, Hiroshi

    2016-11-01

    Diphenyl (α-aminoalkyl)phosphonates act as mechanism-based inhibitors against serine proteases by forming a covalent bond with the hydroxy group of the active center Ser residue. Because the covalent bond was found to be broken and replaced by 2-pyridinaldoxime methiodide (2PAM), we employed a peptidyl derivative bearing diphenyl 1-amino-2-phenylethylphosphonate moiety (Phe(p) (OPh)2 ) to target the active site of chymotrypsin and to selectively anchor to Lys175 in the vicinity of the active site. Previously, it was reported that the configuration of the α-carbon of phosphorus in diphenyl (α-aminoalkyl)phosphonates affects the inactivation reaction of serine proteases, i.e., the (R)-enantiomeric diphenyl phosphonate is comparable to l-amino acids and it effectively reacts with serine proteases, whereas the (S)-enantiomeric form does not. In this study, we evaluated the stereochemical effect of the phosphonate moiety on the selective chemical modification. Epimeric dipeptidyl derivatives, Ala-(R or S)-Phe(p) (OPh)2 , were prepared by separation with RP-HPLC. A tripeptidyl (R)-epimer (Ala-Ala-(R)-Phe(p) (OPh)2 ) exhibited a more potent inactivation ability against chymotrypsin than the (S)-epimer. The enzyme inactivated by the (R)-epimer was more effectively reactivated with 2PAM than the enzyme inactivated by the (S)-epimer. Finally, N-succinimidyl (NHS) active ester derivatives, NHS-Suc-Ala-Ala- (R or S)-Phe(p) (OPh)2 , were prepared, and we evaluated their action when modifying Lys175 in chymotrypsin. We demonstrated that the epimeric NHS derivative that possessed the diphenyl phosphonate moiety with the (R)-configuration effectively modified Lys175 in chymotrypsin, whereas that with the (S)-configuration did not. These results demonstrate the utility of peptidyl derivatives that bear an optically active diphenyl phosphonate moiety as affinity labeling probes in protein bioconjugation. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 521-530, 2016.

  9. Prolyl hydroxylation by EglN2 destabilizes FOXO3a by blocking its interaction with the USP9x deubiquitinase.

    PubMed

    Zheng, Xingnan; Zhai, Bo; Koivunen, Peppi; Shin, Sandra J; Lu, Gang; Liu, Jiayun; Geisen, Christoph; Chakraborty, Abhishek A; Moslehi, Javid J; Smalley, David M; Wei, Xin; Chen, Xian; Chen, Zhengming; Beres, Justine M; Zhang, Jing; Tsao, Jen Lan; Brenner, Mitchell C; Zhang, Yuqing; Fan, Cheng; DePinho, Ronald A; Paik, Jihye; Gygi, Steven P; Kaelin, William G; Zhang, Qing

    2014-07-01

    The three EglN prolyl hydroxylases (EglN1, EglN2, and EglN3) regulate the stability of the HIF transcription factor. We recently showed that loss of EglN2, however, also leads to down-regulation of Cyclin D1 and decreased cell proliferation in a HIF-independent manner. Here we report that EglN2 can hydroxylate FOXO3a on two specific prolyl residues in vitro and in vivo. Hydroxylation of these sites prevents the binding of USP9x deubiquitinase, thereby promoting the proteasomal degradation of FOXO3a. FOXO transcription factors can repress Cyclin D1 transcription. Failure to hydroxylate FOXO3a promotes its accumulation in cells, which in turn suppresses Cyclin D1 expression. These findings provide new insights into post-transcriptional control of FOXO3a and provide a new avenue for pharmacologically altering Cyclin D1 activity.

  10. Prolyl hydroxylation by EglN2 destabilizes FOXO3a by blocking its interaction with the USP9x deubiquitinase

    PubMed Central

    Zheng, Xingnan; Zhai, Bo; Koivunen, Peppi; Shin, Sandra J.; Lu, Gang; Liu, Jiayun; Geisen, Christoph; Chakraborty, Abhishek A.; Moslehi, Javid J.; Smalley, David M.; Wei, Xin; Chen, Xian; Chen, Zhengming; Beres, Justine M.; Tsao, Jen Lan; Brenner, Mitchell C.; Fan, Cheng; DePinho, Ronald A.; Paik, Jihye; Gygi, Steven P.; Kaelin, William G.; Zhang, Qing

    2014-01-01

    The three EglN prolyl hydroxylases (EglN1, EglN2, and EglN3) regulate the stability of the HIF transcription factor. We recently showed that loss of EglN2, however, also leads to down-regulation of Cyclin D1 and decreased cell proliferation in a HIF-independent manner. Here we report that EglN2 can hydroxylate FOXO3a on two specific prolyl residues in vitro and in vivo. Hydroxylation of these sites prevents the binding of USP9x deubiquitinase, thereby promoting the proteasomal degradation of FOXO3a. FOXO transcription factors can repress Cyclin D1 transcription. Failure to hydroxylate FOXO3a promotes its accumulation in cells, which in turn suppresses Cyclin D1 expression. These findings provide new insights into post-transcriptional control of FOXO3a and provide a new avenue for pharmacologically altering Cyclin D1 activity. PMID:24990963

  11. Lipohexin, a new inhibitor of prolyl endopeptidase from Moeszia lindtneri (HKI-0054) and Paecilomyces sp. (HKI-0055; HKI-0096). I. Screening, isolation and structure elucidation.

    PubMed

    Heinze, S; Ritzau, M; Ihn, W; Hülsmann, H; Schlegel, B; Dornberger, K; Fleck, W F; Zerlin, M; Christner, C; Gräfe, U; Küllertz, G; Fischer, G

    1997-05-01

    Lipohexin was isolated as a novel lipohexapeptide (I) (C39H68N6O9) from three fungal strains, Moeszia lindtneri HKI-0054, Paecilomyces sp. HKI-0055 and Paecilomyces sp. HKI-0096. The structure was elucidated by detailed mass spectrometric and NMR experiments. The proline-containing peptide displays moderate antibacterial activity against Bacillus subtilis ATCC 6633 and inhibits competitively the prolyl endopeptidase from human placenta.

  12. Molecular cloning of the. alpha. -subunit of human prolyl 4-hydroxylase: The complete cDNA-derived amino acid sequence and evidence for alternative splicing of RNA transcripts

    SciTech Connect

    Helaakoski, T.; Vuori, K.; Myllylae, R.; Kivirikko, K.I.; Pihlajaniemi, T. )

    1989-06-01

    Prolyl 4-hydroxylase an {alpha}{sub 2}{beta}{sub 2} tetramer, catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. The authors report here on the isolation of cDNA clones encoding the {alpha}-subunit of the enzyme from human tumor HT-1080, placenta, and fibroblast cDNA libraries. Eight overlapping clones covering almost all of the corresponding 3,000-nucleotide mRNA, including all the coding sequences, were characterized. These clones encode a polypeptide of 517 amino acid residues and a signal peptide of 17 amino acids. Previous characterization of cDNA clones for the {beta}-subunit of prolyl 4-hydroxylase has indicated that its C terminus has the amino acid sequence Lys-Asp-Gly-Leu, which, it has been suggested, is necessary for the retention of a polypeptide within the lumen of the endoplasmic reticulum. The {alpha}-subunit does not have this C-terminal sequence, and thus one function of the {beta}-subunit in the prolyl 4-hydroxylase tetramer appears to be to retain the enzyme within this cell organelle. Southern blot analyses of human genomic DNA with a cDNA probe for the {alpha}-subunit suggested the presence of only one gene encoding the two types of mRNA, which appear to result from mutually exclusive alternative splicing of primary transcripts of one gene.

  13. Photophysics of the Red Chromophore of HcRed: Evidence for Cis-Trans Isomerization and Protonation-State Changes

    SciTech Connect

    Cotlet, M.; Mudalige, K.; Habuchi, S.; Goodwin, P.M.; Pai, R.K.; De Schryver, F.

    2010-03-15

    HcRed is a dimeric intrinsically fluorescent protein with origins in the sea anemone Heteractis crispa. This protein exhibits deep red absorption and emission properties. Using a combination of ensemble and single molecule methods and by varying environmental parameters such as temperature and pH, we found spectroscopic evidence for the presence of two ground state conformers, trans and cis chromophores that are in thermal equilibrium and that follow different excited-state pathways upon exposure to light. The photocycle of HcRed appears to be a combination of both kindling proteins and bright emitting GFP/GFP-like proteins: the trans chromophore undergoes light driven isomerization followed by radiative relaxation with a fluorescence lifetime of 0.5 ns. The cis chromophore exhibits a photocycle similar to bright GFPs and GFP-like proteins such as enhanced GFP, enhanced YFP or DsRed, with radiative relaxation with a fluorescence lifetime of 1.5 ns, singlet-triplet deactivation on a microsecond time scale and solvent controlled protonation/deprotonation in tens of microseconds. Using single molecule spectroscopy, we identify trans and cis conformers at the level of individual moieties and show that it is possible that the two conformers can coexist in a single protein due to the dimeric nature of HcRed.

  14. Cis-trans isomerism in a square-planar platinum(II) complex bearing bulky fluorinated phosphane ligands.

    PubMed

    Bernès, Sylvain; Meléndez, Francisco J; Torrens, Hugo

    2016-04-01

    Transition-metal complexes bearing fluorinated phosphane and thiolate ligands has been an area of study in recent years and the chemical context of the current work is related to the metal-assisted functionalization of fluorinated derivatives. The cis and trans isomers of the square-planar complex bis[(pentafluorophenyl)diphenylphosphane-κP]bis(2,3,5,6-tetrafluorobenzenethiolato-κS)platinum(II), [Pt(C6HF4S)2{P(C6H5)2(C6F5)}2], have been crystallized from a single chromatographic fraction and characterized by X-ray diffraction analysis. The stabilization of the cis isomer results from weak intramolecular π-stacking interactions and possibly from the formation of a C-F...Pt contact, characterized by an F...Pt separation of 2.957 (6) Å. The natural bond orbital analysis (NBO) for this isomer confirms that the corresponding F → Pt charge transfer accounts for 6.92 kcal mol(-1) in the isomer stabilization. Such interactions are not present in the centrosymmetric trans isomer.

  15. Cis-trans isomerism in a square-planar platinum(II) complex bearing bulky fluorinated phosphane ligands.

    PubMed

    Bernès, Sylvain; Meléndez, Francisco J; Torrens, Hugo

    2016-04-01

    Transition-metal complexes bearing fluorinated phosphane and thiolate ligands has been an area of study in recent years and the chemical context of the current work is related to the metal-assisted functionalization of fluorinated derivatives. The cis and trans isomers of the square-planar complex bis[(pentafluorophenyl)diphenylphosphane-κP]bis(2,3,5,6-tetrafluorobenzenethiolato-κS)platinum(II), [Pt(C6HF4S)2{P(C6H5)2(C6F5)}2], have been crystallized from a single chromatographic fraction and characterized by X-ray diffraction analysis. The stabilization of the cis isomer results from weak intramolecular π-stacking interactions and possibly from the formation of a C-F...Pt contact, characterized by an F...Pt separation of 2.957 (6) Å. The natural bond orbital analysis (NBO) for this isomer confirms that the corresponding F → Pt charge transfer accounts for 6.92 kcal mol(-1) in the isomer stabilization. Such interactions are not present in the centrosymmetric trans isomer. PMID:27045175

  16. Investigation of cis/trans proline isomerism in a multiply occurring peptide fragment from human salivary proline-rich glycoprotein.

    PubMed

    Loomis, R E; Gonzalez, M; Loomis, P M

    1991-11-01

    The solution-state conformations of eight proline-containing peptide fragments found in human salivary proline-rich glycoprotein (PRG) were investigated in 2 x distilled water (treated with metal ion chelating resin) using 13C-nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. The peptide sequences and acronyms were as follows: PRG9-2 = NH2-G(1)-P(2)-CONH2, PRG9-3 = NH2-G(1)P(2)-P(3)-CONH2, PRG9-4 = NH2-G(1)-P(2)-P(3)-P(4)-CONH2, PRG9-5 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-CONH2, PRG9-6 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-CONH2, PRG9-7 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-CONH2, PRG9-8 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-CONH2 and PRG9-9 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-P(9)-CONH2. Sequence-specific resonance assignments from the 13C-NMR spectra indicated that the trans proline isomer dominated the conformations of the peptides. CD results clearly showed the presence of the poly-L-proline II helix as the major conformation in PRG9-3----PRG9-5, supplemented by beta- and/or gamma-turns in PRG9-6----PRG9-9. These data suggest that in "metal free" water, native PRG could contain several small poly-L-proline II helices along with beta- and/or gamma-turns. Since proline is the major amino acid present in native PRG, these localized conformations may contribute to PRG's global conformation and act as a primary force in determining its biological activities.

  17. "There's no chasing involved": cis/trans relationships, "tranny chasers," and the future of a sex-positive trans politics.

    PubMed

    Tompkins, Avery Brooks

    2014-01-01

    This article adds to a small, but growing, body of work on trans sexualities and partnerships, and provides a much-needed inquiry into the complex and contested politics of desire when we take trans identities, bodies, and sexualities into account. Using digital ethnographic data from YouTube videos along with in-person observational data from LGBTQ and trans conferences in the U.S., Tompkins argues that a sex-positive trans politics cannot emerge in trans and trans-allied communities if the rhetoric of the "tranny chaser" continues to inform discourses of desire and attraction to trans people.

  18. The Prolyl Isomerase Pin1 Promotes the Herpesvirus-Induced Phosphorylation-Dependent Disassembly of the Nuclear Lamina Required for Nucleocytoplasmic Egress

    PubMed Central

    Milbradt, Jens; Hutterer, Corina; Bahsi, Hanife; Wagner, Sabrina; Sonntag, Eric; Kaufer, Benedikt B.; Mori, Yasuko; Sticht, Heinrich; Fossen, Torgils; Marschall, Manfred

    2016-01-01

    The nuclear lamina lines the inner nuclear membrane providing a structural framework for the nucleus. Cellular processes, such as nuclear envelope breakdown during mitosis or nuclear export of large ribonucleoprotein complexes, are functionally linked to the disassembly of the nuclear lamina. In general, lamina disassembly is mediated by phosphorylation, but the precise molecular mechanism is still not completely understood. Recently, we suggested a novel mechanism for lamina disassembly during the nuclear egress of herpesviral capsids which involves the cellular isomerase Pin1. In this study, we focused on mechanistic details of herpesviral nuclear replication to demonstrate the general importance of Pin1 for lamina disassembly. In particular, Ser22-specific lamin phosphorylation consistently generates a Pin1-binding motif in cells infected with human and animal alpha-, beta-, and gammaherpesviruses. Using nuclear magnetic resonance spectroscopy, we showed that binding of Pin1 to a synthetic lamin peptide induces its cis/trans isomerization in vitro. A detailed bioinformatic evaluation strongly suggests that this structural conversion induces large-scale secondary structural changes in the lamin N-terminus. Thus, we concluded that a Pin1-induced conformational change of lamins may represent the molecular trigger responsible for lamina disassembly. Consistent with this concept, pharmacological inhibition of Pin1 activity blocked lamina disassembly in herpesvirus-infected fibroblasts and consequently impaired virus replication. In addition, a phospho-mimetic Ser22Glu lamin mutant was still able to form a regular lamina structure and overexpression of a Ser22-phosphorylating kinase did not induce lamina disassembly in Pin1 knockout cells. Intriguingly, this was observed in absence of herpesvirus infection proposing a broader importance of Pin1 for lamina constitution. Thus, our results suggest a functional model of similar events leading to disassembly of the nuclear

  19. The Prolyl Isomerase Pin1 Promotes the Herpesvirus-Induced Phosphorylation-Dependent Disassembly of the Nuclear Lamina Required for Nucleocytoplasmic Egress.

    PubMed

    Milbradt, Jens; Hutterer, Corina; Bahsi, Hanife; Wagner, Sabrina; Sonntag, Eric; Horn, Anselm H C; Kaufer, Benedikt B; Mori, Yasuko; Sticht, Heinrich; Fossen, Torgils; Marschall, Manfred

    2016-08-01

    The nuclear lamina lines the inner nuclear membrane providing a structural framework for the nucleus. Cellular processes, such as nuclear envelope breakdown during mitosis or nuclear export of large ribonucleoprotein complexes, are functionally linked to the disassembly of the nuclear lamina. In general, lamina disassembly is mediated by phosphorylation, but the precise molecular mechanism is still not completely understood. Recently, we suggested a novel mechanism for lamina disassembly during the nuclear egress of herpesviral capsids which involves the cellular isomerase Pin1. In this study, we focused on mechanistic details of herpesviral nuclear replication to demonstrate the general importance of Pin1 for lamina disassembly. In particular, Ser22-specific lamin phosphorylation consistently generates a Pin1-binding motif in cells infected with human and animal alpha-, beta-, and gammaherpesviruses. Using nuclear magnetic resonance spectroscopy, we showed that binding of Pin1 to a synthetic lamin peptide induces its cis/trans isomerization in vitro. A detailed bioinformatic evaluation strongly suggests that this structural conversion induces large-scale secondary structural changes in the lamin N-terminus. Thus, we concluded that a Pin1-induced conformational change of lamins may represent the molecular trigger responsible for lamina disassembly. Consistent with this concept, pharmacological inhibition of Pin1 activity blocked lamina disassembly in herpesvirus-infected fibroblasts and consequently impaired virus replication. In addition, a phospho-mimetic Ser22Glu lamin mutant was still able to form a regular lamina structure and overexpression of a Ser22-phosphorylating kinase did not induce lamina disassembly in Pin1 knockout cells. Intriguingly, this was observed in absence of herpesvirus infection proposing a broader importance of Pin1 for lamina constitution. Thus, our results suggest a functional model of similar events leading to disassembly of the nuclear

  20. The Prolyl Isomerase Pin1 Promotes the Herpesvirus-Induced Phosphorylation-Dependent Disassembly of the Nuclear Lamina Required for Nucleocytoplasmic Egress.

    PubMed

    Milbradt, Jens; Hutterer, Corina; Bahsi, Hanife; Wagner, Sabrina; Sonntag, Eric; Horn, Anselm H C; Kaufer, Benedikt B; Mori, Yasuko; Sticht, Heinrich; Fossen, Torgils; Marschall, Manfred

    2016-08-01

    The nuclear lamina lines the inner nuclear membrane providing a structural framework for the nucleus. Cellular processes, such as nuclear envelope breakdown during mitosis or nuclear export of large ribonucleoprotein complexes, are functionally linked to the disassembly of the nuclear lamina. In general, lamina disassembly is mediated by phosphorylation, but the precise molecular mechanism is still not completely understood. Recently, we suggested a novel mechanism for lamina disassembly during the nuclear egress of herpesviral capsids which involves the cellular isomerase Pin1. In this study, we focused on mechanistic details of herpesviral nuclear replication to demonstrate the general importance of Pin1 for lamina disassembly. In particular, Ser22-specific lamin phosphorylation consistently generates a Pin1-binding motif in cells infected with human and animal alpha-, beta-, and gammaherpesviruses. Using nuclear magnetic resonance spectroscopy, we showed that binding of Pin1 to a synthetic lamin peptide induces its cis/trans isomerization in vitro. A detailed bioinformatic evaluation strongly suggests that this structural conversion induces large-scale secondary structural changes in the lamin N-terminus. Thus, we concluded that a Pin1-induced conformational change of lamins may represent the molecular trigger responsible for lamina disassembly. Consistent with this concept, pharmacological inhibition of Pin1 activity blocked lamina disassembly in herpesvirus-infected fibroblasts and consequently impaired virus replication. In addition, a phospho-mimetic Ser22Glu lamin mutant was still able to form a regular lamina structure and overexpression of a Ser22-phosphorylating kinase did not induce lamina disassembly in Pin1 knockout cells. Intriguingly, this was observed in absence of herpesvirus infection proposing a broader importance of Pin1 for lamina constitution. Thus, our results suggest a functional model of similar events leading to disassembly of the nuclear

  1. Membrane-Active Sequences within gp41 Membrane Proximal External Region (MPER) Modulate MPER-Containing Peptidyl Fusion Inhibitor Activity and the Biosynthesis of HIV-1 Structural Proteins

    PubMed Central

    Zhang, Si Min; Jejcic, Alenka; Tam, James P.; Vahlne, Anders

    2015-01-01

    The membrane proximal external region (MPER) is a highly conserved membrane-active region located at the juxtamembrane positions within class I viral fusion glycoproteins and essential for membrane fusion events during viral entry. The MPER in the human immunodeficiency virus type I (HIV-1) envelope protein (Env) interacts with the lipid bilayers through a cluster of tryptophan (Trp) residues and a C-terminal cholesterol-interacting motif. The inclusion of the MPER N-terminal sequence contributes to the membrane reactivity and anti-viral efficacy of the first two anti-HIV peptidyl fusion inhibitors T20 and T1249. As a type I transmembrane protein, Env also interacts with the cellular membranes during its biosynthesis and trafficking. Here we investigated the roles of MPER membrane-active sequences during both viral entry and assembly, specifically, their roles in the design of peptidyl fusion inhibitors and the biosynthesis of viral structural proteins. We found that elimination of the membrane-active elements in MPER peptides, namely, penta Trp→alanine (Ala) substitutions and the disruption of the C-terminal cholesterol-interacting motif through deletion inhibited the anti-viral effect against the pseudotyped HIV-1. Furthermore, as compared to C-terminal dimerization, N-terminal dimerization of MPER peptides and N-terminal extension with five helix-forming residues enhanced their anti-viral efficacy substantially. The secondary structure study revealed that the penta-Trp→Ala substitutions also increased the helical content in the MPER sequence, which prompted us to study the biological relevance of such mutations in pre-fusion Env. We observed that Ala mutations of Trp664, Trp668 and Trp670 in MPER moderately lowered the intracellular and intraviral contents of Env while significantly elevating the content of another viral structural protein, p55/Gag and its derivative p24/capsid. The data suggest a role of the gp41 MPER in the membrane-reactive events during

  2. HIF prolyl 4-hydroxylase-2 inhibition improves glucose and lipid metabolism and protects against obesity and metabolic dysfunction.

    PubMed

    Rahtu-Korpela, Lea; Karsikas, Sara; Hörkkö, Sohvi; Blanco Sequeiros, Roberto; Lammentausta, Eveliina; Mäkelä, Kari A; Herzig, Karl-Heinz; Walkinshaw, Gail; Kivirikko, Kari I; Myllyharju, Johanna; Serpi, Raisa; Koivunen, Peppi

    2014-10-01

    Obesity is a major public health problem, predisposing subjects to metabolic syndrome, type 2 diabetes, and cardiovascular diseases. Specific prolyl 4-hydroxylases (P4Hs) regulate the stability of the hypoxia-inducible factor (HIF), a potent governor of metabolism, with isoenzyme 2 being the main regulator. We investigated whether HIF-P4H-2 inhibition could be used to treat obesity and its consequences. Hif-p4h-2-deficient mice, whether fed normal chow or a high-fat diet, had less adipose tissue, smaller adipocytes, and less adipose tissue inflammation than their littermates. They also had improved glucose tolerance and insulin sensitivity. Furthermore, the mRNA levels of the HIF-1 targets glucose transporters, glycolytic enzymes, and pyruvate dehydrogenase kinase-1 were increased in their tissues, whereas acetyl-CoA concentration was decreased. The hepatic mRNA level of the HIF-2 target insulin receptor substrate-2 was higher, whereas that of two key enzymes of fatty acid synthesis was lower. Serum cholesterol levels and de novo lipid synthesis were decreased, and the mice were protected against hepatic steatosis. Oral administration of an HIF-P4H inhibitor, FG-4497, to wild-type mice with metabolic dysfunction phenocopied these beneficial effects. HIF-P4H-2 inhibition may be a novel therapy that not only protects against the development of obesity and its consequences but also reverses these conditions.

  3. Inhibition of HIF prolyl-4-hydroxylases by FG-4497 reduces brain tissue injury and edema formation during ischemic stroke.

    PubMed

    Reischl, Stefan; Li, Lexiao; Walkinshaw, Gail; Flippin, Lee A; Marti, Hugo H; Kunze, Reiner

    2014-01-01

    Ischemic stroke results in disruption of the blood-brain barrier (BBB), edema formation and neuronal cell loss. Some neuroprotective factors such as vascular endothelial growth factor (VEGF) favor edema formation, while others such as erythropoietin (Epo) can mitigate it. Both factors are controlled by hypoxia inducible transcription factors (HIF) and the activity of prolyl hydroxylase domain proteins (PHD). We hypothesize that activation of the adaptive hypoxic response by inhibition of PHD results in neuroprotection and prevention of vascular leakage. Mice, subjected to cerebral ischemia, were pre- or post-treated with the novel PHD inhibitor FG-4497. Inhibition of PHD activity resulted in HIF-1α stabilization, increased expression of VEGF and Epo, improved outcome from ischemic stroke and reduced edema formation by maintaining BBB integrity. Additional in vitro studies using brain endothelial cells and primary astrocytes confirmed that FG-4497 induces the HIF signaling pathway, leading to increased VEGF and Epo expression. In an in vitro ischemia model, using combined oxygen and glucose deprivation, FG-4497 promoted the survival of neurons. Furthermore, FG-4497 prevented the ischemia-induced rearrangement and gap formation of the tight junction proteins zonula occludens 1 and occludin, both in cultured endothelial cells and in infarcted brain tissue in vivo. These results indicate that FG-4497 has the potential to prevent cerebral ischemic damage by neuroprotection and prevention of vascular leakage.

  4. Analyzing slowly exchanging protein conformations by ion mobility mass spectrometry: study of the dynamic equilibrium of prolyl oligopeptidase.

    PubMed

    López, Abraham; Vilaseca, Marta; Madurga, Sergio; Varese, Monica; Tarragó, Teresa; Giralt, Ernest

    2016-07-01

    Ion mobility mass spectrometry (IMMS) is a biophysical technique that allows the separation of isobaric species on the basis of their size and shape. The high separation capacity, sensitivity and relatively fast time scale measurements confer IMMS great potential for the study of proteins in slow (µs-ms) conformational equilibrium in solution. However, the use of this technique for examining dynamic proteins is still not generalized. One of the major limitations is the instability of protein ions in the gas phase, which raises the question as to what extent the structures detected reflect those in solution. Here, we addressed this issue by analyzing the conformational landscape of prolyl oligopeptidase (POP) - a model of a large dynamic enzyme in the µs-ms range - by native IMMS and compared the results obtained in the gas phase with those obtained in solution. In order to interpret the experimental results, we used theoretical simulations. In addition, the stability of POP gaseous ions was explored by charge reduction and collision-induced unfolding experiments. Our experiments disclosed two species of POP in the gas phase, which correlated well with the open and closed conformations in equilibrium in solution; moreover, a gas-phase collapsed form of POP was also detected. Therefore, our findings not only support the potential of IMMS for the study of multiple co-existing conformations of large proteins in slow dynamic equilibrium in solution but also stress the need for careful data analysis to avoid artifacts. Copyright © 2016 John Wiley & Sons, Ltd.

  5. Structure of Prolyl-tRNA Synthetase-Halofuginone Complex Provides Basis for Development of Drugs against Malaria and Toxoplasmosis.

    PubMed

    Jain, Vitul; Yogavel, Manickam; Oshima, Yoshiteru; Kikuchi, Haruhisa; Touquet, Bastien; Hakimi, Mohamed-Ali; Sharma, Amit

    2015-05-01

    The Chinese herb Dichroa febrifuga has traditionally treated malaria-associated fever. Its active component febrifugine (FF) and derivatives such as halofuginone (HF) are potent anti-malarials. Here, we show that FF-based derivatives arrest parasite growth by direct interaction with and inhibition of the protein translation enzyme prolyl-tRNA synthetase (PRS). Dual administration of inhibitors that target different tRNA synthetases suggests high utility of these drug targets. We reveal the ternary complex structure of PRS-HF and adenosine 5'-(β,γ-imido)triphosphate where the latter facilitates HF integration into the PRS active site. Structural analyses also highlight spaces within the PRS architecture for HF derivatization of its quinazolinone, but not piperidine, moiety. We also show a remarkable ability of HF to kill the related human parasite Toxoplasma gondii, suggesting wider HF efficacy against parasitic PRSs. Hence, our cell-, enzyme-, and structure-based data on FF-based inhibitors strengthen the case for their inclusion in anti-malarial and anti-toxoplasmosis drug development efforts.

  6. Origin and Evolution of Glutamyl-prolyl tRNA Synthetase WHEP Domains Reveal Evolutionary Relationships within Holozoa

    PubMed Central

    Ray, Partho Sarothi; Fox, Paul L.

    2014-01-01

    Repeated domains in proteins that have undergone duplication or loss, and sequence divergence, are especially informative about phylogenetic relationships. We have exploited divergent repeats of the highly structured, 50-amino acid WHEP domains that join the catalytic subunits of bifunctional glutamyl-prolyl tRNA synthetase (EPRS) as a sequence-informed repeat (SIR) to trace the origin and evolution of EPRS in holozoa. EPRS is the only fused tRNA synthetase, with two distinct aminoacylation activities, and a non-canonical translation regulatory function mediated by the WHEP domains in the linker. Investigating the duplications, deletions and divergence of WHEP domains, we traced the bifunctional EPRS to choanozoans and identified the fusion event leading to its origin at the divergence of ichthyosporea and emergence of filozoa nearly a billion years ago. Distribution of WHEP domains from a single species in two or more distinct clades suggested common descent, allowing the identification of linking organisms. The discrete assortment of choanoflagellate WHEP domains with choanozoan domains as well as with those in metazoans supported the phylogenetic position of choanoflagellates as the closest sister group to metazoans. Analysis of clustering and assortment of WHEP domains provided unexpected insights into phylogenetic relationships amongst holozoan taxa. Furthermore, observed gaps in the transition between WHEP domain groupings in distant taxa allowed the prediction of undiscovered or extinct evolutionary intermediates. Analysis based on SIR domains can provide a phylogenetic counterpart to palaentological approaches of discovering “missing links” in the tree of life. PMID:24968216

  7. Inhibition of HIF prolyl-4-hydroxylases by FG-4497 reduces brain tissue injury and edema formation during ischemic stroke.

    PubMed

    Reischl, Stefan; Li, Lexiao; Walkinshaw, Gail; Flippin, Lee A; Marti, Hugo H; Kunze, Reiner

    2014-01-01

    Ischemic stroke results in disruption of the blood-brain barrier (BBB), edema formation and neuronal cell loss. Some neuroprotective factors such as vascular endothelial growth factor (VEGF) favor edema formation, while others such as erythropoietin (Epo) can mitigate it. Both factors are controlled by hypoxia inducible transcription factors (HIF) and the activity of prolyl hydroxylase domain proteins (PHD). We hypothesize that activation of the adaptive hypoxic response by inhibition of PHD results in neuroprotection and prevention of vascular leakage. Mice, subjected to cerebral ischemia, were pre- or post-treated with the novel PHD inhibitor FG-4497. Inhibition of PHD activity resulted in HIF-1α stabilization, increased expression of VEGF and Epo, improved outcome from ischemic stroke and reduced edema formation by maintaining BBB integrity. Additional in vitro studies using brain endothelial cells and primary astrocytes confirmed that FG-4497 induces the HIF signaling pathway, leading to increased VEGF and Epo expression. In an in vitro ischemia model, using combined oxygen and glucose deprivation, FG-4497 promoted the survival of neurons. Furthermore, FG-4497 prevented the ischemia-induced rearrangement and gap formation of the tight junction proteins zonula occludens 1 and occludin, both in cultured endothelial cells and in infarcted brain tissue in vivo. These results indicate that FG-4497 has the potential to prevent cerebral ischemic damage by neuroprotection and prevention of vascular leakage. PMID:24409307

  8. Retention of prolyl hydroxylase PHD2 in the cytoplasm prevents PHD2-induced anchorage-independent carcinoma cell growth

    SciTech Connect

    Jokilehto, Terhi; Hoegel, Heidi; Heikkinen, Pekka; Rantanen, Krista; Elenius, Klaus; Sundstroem, Jari; Jaakkola, Panu M.

    2010-04-15

    Cellular oxygen tension is sensed by a family of prolyl hydroxylases (PHD1-3) that regulate the degradation of hypoxia-inducible factors (HIF-1{alpha} and -2{alpha}). The PHD2 isoform is considered as the main downregulator of HIF in normoxia. Our previous results have shown that nuclear translocation of PHD2 associates with poorly differentiated tumor phenotype implying that nuclear PHD2 expression is advantageous for tumor growth. Here we show that a pool of PHD2 is shuttled between the nucleus and the cytoplasm. In line with this, accumulation of wild type PHD2 in the nucleus was detected in human colon adenocarcinomas and in cultured carcinoma cells. The PHD2 isoforms showing high nuclear expression increased anchorage-independent carcinoma cell growth. However, retention of PHD2 in the cytoplasm inhibited the anchorage-independent cell growth. A region that inhibits the nuclear localization of PHD2 was identified and the deletion of the region promoted anchorage-independent growth of carcinoma cells. Finally, the cytoplasmic PHD2, as compared with the nuclear PHD2, less efficiently downregulated HIF expression. Forced HIF-1{alpha} or -2{alpha} expression decreased and attenuation of HIF expression increased the anchorage-independent cell growth. However, hydroxylase-inactivating mutations in PHD2 had no effect on cell growth. The data imply that nuclear PHD2 localization promotes malignant cancer phenotype.

  9. The Cytoplasmic Prolyl-tRNA Synthetase of the Malaria Parasite is a Dual-Stage Target for Drug Development

    PubMed Central

    Herman, Jonathan D.; Pepper, Lauren R.; Cortese, Joseph F.; Estiu, Guillermina; Galinsky, Kevin; Zuzarte-Luis, Vanessa; Derbyshire, Emily R.; Ribacke, Ulf; Lukens, Amanda K.; Santos, Sofia A.; Patel, Vishal; Clish, Clary B.; Sullivan, William J.; Zhou, Huihao; Bopp, Selina E.; Schimmel, Paul; Lindquist, Susan; Clardy, Jon; Mota, Maria M.; Keller, Tracy L.; Whitman, Malcolm; Wiest, Olaf; Wirth, Dyann F.; Mazitschek, Ralph

    2015-01-01

    The emergence of drug resistance is a major limitation of current antimalarials. The discovery of new druggable targets and pathways including those that are critical for multiple life cycle stages of the malaria parasite is a major goal for the development of the next-generation of antimalarial drugs. Using an integrated chemogenomics approach that combined drug-resistance selection, whole genome sequencing and an orthogonal yeast model, we demonstrate that the cytoplasmic prolyl-tRNA synthetase (PfcPRS) of the malaria parasite Plasmodium falciparum is a biochemical and functional target of febrifugine and its synthetic derivatives such as halofuginone. Febrifugine is the active principle of a traditional Chinese herbal remedy for malaria. We show that treatment with febrifugine derivatives activated the amino acid starvation response in both P. falciparum and a transgenic yeast strain expressing PfcPRS. We further demonstrate in the P. berghei mouse model of malaria that halofuginol, a new halofuginone analog that we developed, is highly active against both liver and asexual blood stages of the malaria parasite. Halofuginol, unlike halofuginone and febrifugine, is well tolerated at efficacious doses, and represents a promising lead for the development of dual-stage next generation antimalarials. PMID:25995223

  10. Inhibition of HIF-prolyl-4-hydroxylases prevents mitochondrial impairment and cell death in a model of neuronal oxytosis

    PubMed Central

    Neitemeier, S; Dolga, A M; Honrath, B; Karuppagounder, S S; Alim, I; Ratan, R R; Culmsee, C

    2016-01-01

    Mitochondrial impairment induced by oxidative stress is a main characteristic of intrinsic cell death pathways in neurons underlying the pathology of neurodegenerative diseases. Therefore, protection of mitochondrial integrity and function is emerging as a promising strategy to prevent neuronal damage. Here, we show that pharmacological inhibition of hypoxia-inducible factor prolyl-4-hydroxylases (HIF-PHDs) by adaptaquin inhibits lipid peroxidation and fully maintains mitochondrial function as indicated by restored mitochondrial membrane potential and ATP production, reduced formation of mitochondrial reactive oxygen species (ROS) and preserved mitochondrial respiration, thereby protecting neuronal HT-22 cells in a model of glutamate-induced oxytosis. Selective reduction of PHD1 protein using CRISPR/Cas9 technology also reduced both lipid peroxidation and mitochondrial impairment, and attenuated glutamate toxicity in the HT-22 cells. Regulation of activating transcription factor 4 (ATF4) expression levels and related target genes may mediate these beneficial effects. Overall, these results expose HIF-PHDs as promising targets to protect mitochondria and, thereby, neurons from oxidative cell death. PMID:27148687

  11. Lipophilic modification enhances anti-colitic properties of rosmarinic acid by potentiating its HIF-prolyl hydroxylases inhibitory activity.

    PubMed

    Jeong, Seongkeun; Park, Huijeong; Hong, Sungchae; Yum, Soohwan; Kim, Wooseong; Jung, Yunjin

    2015-01-15

    Inhibition of hypoxia inducible factor-prolyl hydroxylase-2 (HPH), leading to activation of hypoxia inducible factor (HIF)-1 is a potential therapeutic strategy for the treatment of colitis. Rosmarinic acid (RA), an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid is a naturally occurring polyphenolic compound with two catechols, a or inhibition of HPH. To improve accessibility of highly hydrophilic RA to HPH, an intracellular target, RA was chemically modified to decrease hydrophilicity. Of the less-hydrophilic derivatives, rosmarinic acid methyl ester (RAME) most potently inhibited HPH. Accordingly, RAME prevented hydroxylation of HIF-1α and consequently stabilized HIF-1α protein in cells. RAME inhibition of HPH and induction of HIF-1α were diminished by elevated doses of the required factors of HPH, 2-ketoglutarate and ascorbate. RAME induction of HIF-1α led to activation of an ulcer healing pathway, HIF-1-vascular endothelial growth factor (VEGF), in human colon carcinoma cells. RAME administered rectally ameliorated TNBS-induced rat colitis and substantially decreased the levels of pro-inflammatory mediators in the inflamed colonic tissue. In parallel with the cellular effects of RAME, RAME up-regulated HIF-1α and VEGF in the inflamed colonic tissue. Thus, lipophilic modification of RA improves its ability to inhibit HPH, leading to activation of the HIF-1-VEGF pathway. RAME, a lipophilic RA derivative, may exert anti-colitic effects via activation of the ulcer healing pathway. PMID:25483211

  12. Collagen-derived dipeptide prolyl-hydroxyproline promotes differentiation of MC3T3-E1 osteoblastic cells

    SciTech Connect

    Kimira, Yoshifumi; Ogura, Kana; Taniuchi, Yuri; Kataoka, Aya; Inoue, Naoki; Sugihara, Fumihito; Nakatani, Sachie; Shimizu, Jun; Wada, Masahiro; Mano, Hiroshi

    2014-10-24

    Highlights: • Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization. • Pro-Hyp significantly increased alkaline phosphatase activity. • Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. - Abstract: Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. The objective of this study was to investigate the effects of Pro-Hyp on the proliferation and differentiation of MC3T3-E1 osteoblastic cells. Addition of Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization but alkaline phosphatase activity was significantly increased. Furthermore, cells treated with Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. These results indicate that Pro-Hyp promotes osteoblast differentiation. This study demonstrates for the first time that Pro-Hyp has a positive effect on osteoblast differentiation with upregulation of Runx2, Osterix, and Collα1 gene expression.

  13. Essential roles of insulin, AMPK signaling and lysyl and prolyl hydroxylases in the biosynthesis and multimerization of adiponectin.

    PubMed

    Zhang, Lin; Li, Ming-Ming; Corcoran, Marie; Zhang, Shaoping; Cooper, Garth J S

    2015-01-01

    Post-translational modifications (PTMs) of the adiponectin molecule are essential for its full bioactivity, and defects in PTMs leading to its defective production and multimerization have been linked to the mechanisms of insulin resistance, obesity, and type-2 diabetes. Here we observed that, in differentiated 3T3-L1 adipocytes, decreased insulin signaling caused by blocking of insulin receptors (InsR) with an anti-InsR blocking antibody, increased rates of adiponectin secretion, whereas concomitant elevations in insulin levels counteracted this effect. Adenosine monophosphate-activated protein kinase (AMPK) signaling regulated adiponectin production by modulating the expression of adiponectin receptors, the secretion of adiponectin, and eventually the expression of adiponectin itself. We found that lysyl hydroxylases (LHs) and prolyl hydroxylases (PHs) were expressed in white-adipose tissue of ob/ob mice, wherein LH3 levels were increased compared with controls. In differentiated 3T3-L1 adipocytes, both non-specific inhibition of LHs and PHs by dipyridyl, and specific inhibition of LHs by minoxidil and of P4H with ethyl-3,4-dihydroxybenzoate, caused significant suppression of adiponectin production, more particularly of the higher-order isoforms. Transient gene knock-down of LH3 (Plod3) caused a suppressive effect, especially on the high molecular-weight (HMW) isoforms. These data indicate that PHs and LHs are both required for physiological adiponectin production and in particular are essential for the formation/secretion of the HMW isoforms. PMID:25240468

  14. Prolyl Hydroxylase Domain-Containing Protein 2 (Phd2) Regulates Chondrocyte Differentiation and Secondary Ossification in Mice

    PubMed Central

    Cheng, Shaohong; Aghajanian, Patrick; Pourteymoor, Sheila; Alarcon, Catrina; Mohan, Subburaman

    2016-01-01

    Endochondral ossification plays an important role in the formation of the primary ossification centers (POCs) and secondary ossification centers (SOCs) of mammalian long bones. However, the molecular mechanisms that regulate POC and SOC formation are different. We recently demonstrated that Prolyl Hydroxylase Domain-containing Protein 2 (Phd2) is a key mediator of vitamin C effects on bone. We investigated the role of Phd2 on endochondral ossification of the epiphyses by conditionally deleting the Phd2 gene in osteoblasts and chondrocytes. We found that the deletion of Phd2 in osteoblasts did not cause changes in bone parameters in the proximal tibial epiphyses in 5 week old mice. In contrast, deletion of Phd2 in chondrocytes resulted in increased bone mass and bone formation rate (normalized to tissue volume) in long bone epiphyses, indicating that Phd2 expressed in chondrocytes, but not osteoblasts, negatively regulates secondary ossification of epiphyses. Phd2 deletion in chondrocytes elevated mRNA expression of hypoxia-inducible factor (HIF) signaling molecules including Hif-1α, Hif-2α, Vegfa, Vegfb, and Epo, as well as markers for chondrocyte hypertrophy and mineralization such as Col10, osterix, alkaline phosphatase, and bone sialoprotein. These data suggest that Phd2 expressed in chondrocytes inhibits endochondral ossification at the epiphysis by suppressing HIF signaling pathways. PMID:27775044

  15. Cyclic dipeptide cyclo(l-leucyl-l-prolyl) from marine Bacillus amyloliquefaciens mitigates biofilm formation and virulence in Listeria monocytogenes.

    PubMed

    Gowrishankar, Shanmugaraj; Sivaranjani, Murugesan; Kamaladevi, Arumugam; Ravi, Arumugam Veera; Balamurugan, Krishnaswamy; Karutha Pandian, Shunmugiah

    2016-06-01

    This study was intentionally focused on cyclo(l-leucyl-l-prolyl) (CLP), a cyclic dipeptide with myriad pharmaceutical significance, to explore its antivirulence efficacy against the predominant foodborne pathogen,Listeria monocytogenes(LM). Minimum inhibitory concentration (MIC) of CLP against LM ATCC 19111 was found to be 512 μg mL(-1) CLP at sub-MICs (64 128, 256 μg mL(-1)) demonstrated a profound non-bactericidal dose-dependent antibiofilm efficacy (on polystyrene and glass) against LM, which was further confirmed through confocal and scanning electron microscopic analysis (on stainless steel surface).In vitrobioassays divulged the phenomenal inhibitory efficacy of CLP towards various virulence traits of LM, specifically its overwhelming suppression of swimming and swarming motility. Data ofin vivoassay usingCaenorhabditis eleganssignified that the plausible mechanism of CLP could be by impeding the pathogen's initial adhesion and thereby attenuating the biofilm assemblage and its associated virulence. This was further confirmed by significant decrease in extracellular polymeric substance, autoaggregation, hydrophobicity index and extracellular DNA of the CLP-treated LM cells. Collectively, this study unveils the antivirulence efficacy of CLP against the Gram-positive foodborne pathogen and the strainBacillus amyloliquefaciensaugurs well to be a promising probiotic in controlling infections associated with LM. PMID:26945590

  16. Molecular cloning and heterologous expression in Pichia pastoris of X-prolyl-dipeptidyl aminopeptidase from basidiomycete Ustilago maydis.

    PubMed

    Juárez-Montiel, Margarita; Ibarra, J Antonio; Chávez-Camarillo, Griselda; Hernández-Rodríguez, César; Villa-Tanaca, Lourdes

    2014-03-01

    Dipeptidyl aminopeptidases are enzymes involved in the posttranslational control of bioactive peptides. Here we identified the gene dapUm in Ustilago maydis by homology with other fungal dipeptidyl aminopeptidases. Analysis of the dapUm-deduced amino acid sequence indicated that it encodes for membrane-type serine protease with a characteristic prolyl oligopeptidase catalytic motif triad: Ser, Asp, His. In order to overexpress the DapUm, the gene encoding for it was cloned and transformed into Pichia. Using this system, we observed a ∼ 125-kDa recombinant protein with an optimal enzymatic activity at pH 6.0 and at 40 °C for the Ala-Pro-p-nitroanilide substrate and an experimental pH of 6.9. U. maydis DapUm was specifically inhibited by phenylmethylsulfonyl fluoride and Pefabloc, confirming the presence of a serine residue in the active site. To our knowledge, this study is the first report on the cloning and expression of a DPP IV dipeptidyl aminopeptidase from a basidiomycete organism. Moreover, the use of recombinant DapUm will allow us to further study and characterize this enzyme, in addition to testing chemical compounds for pharmaceutical purposes.

  17. Deficiency of prolyl oligopeptidase in mice disturbs synaptic plasticity and reduces anxiety-like behaviour, body weight, and brain volume.

    PubMed

    Höfling, Corinna; Kulesskaya, Natalia; Jaako, Külli; Peltonen, Iida; Männistö, Pekka T; Nurmi, Antti; Vartiainen, Nina; Morawski, Markus; Zharkovsky, Alexander; Võikar, Vootele; Roßner, Steffen; García-Horsman, J Arturo

    2016-06-01

    Prolyl oligopeptidase (PREP) has been implicated in neurodegeneration and neuroinflammation and has been considered a drug target to enhance memory in dementia. However, the true physiological role of PREP is not yet understood. In this paper, we report the phenotyping of a mouse line where the PREP gene has been knocked out. This work indicates that the lack of PREP in mice causes reduced anxiety but also hyperactivity. The cortical volumes of PREP knockout mice were smaller than those of wild type littermates. Additionally, we found increased expression of diazepam binding inhibitor protein in the cortex and of the somatostatin receptor-2 in the hippocampus of PREP knockout mice. Furthermore, immunohistochemistry and tail suspension test revealed lack of response of PREP knockout mice to lipopolysaccharide insult. Further analysis revealed significantly increased levels of polysialylated-neural cell adhesion molecule in PREP deficient mice. These findings might be explained as possible alteration in brain plasticity caused by PREP deficiency, which in turn affect behaviour and brain development.

  18. Statistical optimization of cell disruption techniques for releasing intracellular X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis spp. lactis.

    PubMed

    Üstün-Aytekin, Özlem; Arısoy, Sevda; Aytekin, Ali Özhan; Yıldız, Ece

    2016-03-01

    X-prolyl dipeptidyl aminopeptidase (PepX) is an intracellular enzyme from the Gram-positive bacterium Lactococcus lactis spp. lactis NRRL B-1821, and it has commercial importance. The objective of this study was to compare the effects of several cell disruption methods on the activity of PepX. Statistical optimization methods were performed for two cavitation methods, hydrodynamic (high-pressure homogenization) and acoustic (sonication), to determine the more appropriate disruption method. Two level factorial design (2FI), with the parameters of number of cycles and pressure, and Box-Behnken design (BBD), with the parameters of cycle, sonication time, and power, were used for the optimization of the high-pressure homogenization and sonication methods, respectively. In addition, disruption methods, consisting of lysozyme, bead milling, heat treatment, freeze-thawing, liquid nitrogen, ethylenediaminetetraacetic acid (EDTA), Triton-X, sodium dodecyl sulfate (SDS), chloroform, and antibiotics, were performed and compared with the high-pressure homogenization and sonication methods. The optimized values of high-pressure homogenization were one cycle at 130 MPa providing activity of 114.47 mU ml(-1), while sonication afforded an activity of 145.09 mU ml(-1) at 28 min with 91% power and three cycles. In conclusion, sonication was the more effective disruption method, and its optimal operation parameters were manifested for the release of intracellular enzyme from a L. lactis spp. lactis strain, which is a Gram-positive bacterium. PMID:26584994

  19. Statistical optimization of cell disruption techniques for releasing intracellular X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis spp. lactis.

    PubMed

    Üstün-Aytekin, Özlem; Arısoy, Sevda; Aytekin, Ali Özhan; Yıldız, Ece

    2016-03-01

    X-prolyl dipeptidyl aminopeptidase (PepX) is an intracellular enzyme from the Gram-positive bacterium Lactococcus lactis spp. lactis NRRL B-1821, and it has commercial importance. The objective of this study was to compare the effects of several cell disruption methods on the activity of PepX. Statistical optimization methods were performed for two cavitation methods, hydrodynamic (high-pressure homogenization) and acoustic (sonication), to determine the more appropriate disruption method. Two level factorial design (2FI), with the parameters of number of cycles and pressure, and Box-Behnken design (BBD), with the parameters of cycle, sonication time, and power, were used for the optimization of the high-pressure homogenization and sonication methods, respectively. In addition, disruption methods, consisting of lysozyme, bead milling, heat treatment, freeze-thawing, liquid nitrogen, ethylenediaminetetraacetic acid (EDTA), Triton-X, sodium dodecyl sulfate (SDS), chloroform, and antibiotics, were performed and compared with the high-pressure homogenization and sonication methods. The optimized values of high-pressure homogenization were one cycle at 130 MPa providing activity of 114.47 mU ml(-1), while sonication afforded an activity of 145.09 mU ml(-1) at 28 min with 91% power and three cycles. In conclusion, sonication was the more effective disruption method, and its optimal operation parameters were manifested for the release of intracellular enzyme from a L. lactis spp. lactis strain, which is a Gram-positive bacterium.

  20. The K+-dependent GTPase Nug1 is implicated in the association of the helicase Dbp10 to the immature peptidyl transferase centre during ribosome maturation

    PubMed Central

    Manikas, Rizos-Georgios; Thomson, Emma; Thoms, Matthias; Hurt, Ed

    2016-01-01

    Ribosome synthesis employs a number of energy-consuming enzymes in both eukaryotes and prokaryotes. One such enzyme is the conserved circularly permuted GTPase Nug1 (nucleostemin in human). Nug1 is essential for 60S subunit assembly and nuclear export, but its role and time of action during maturation remained unclear. Based on in vitro enzymatic assays using the Chaetomium thermophilum (Ct) orthologue, we show that Nug1 exhibits a low intrinsic GTPase activity that is stimulated by potassium ions, rendering Nug1 a cation-dependent GTPase. In vivo we observe 60S biogenesis defects upon depletion of yeast Nug1 or expression of a Nug1 nucleotide-binding mutant. Most prominently, the RNA helicase Dbp10 was lost from early pre-60S particles, which suggested a physical interaction that could be reconstituted in vitro using CtNug1 and CtDbp10. In vivo rRNA–protein crosslinking revealed that Nug1 and Dbp10 bind at proximal and partially overlapping sites on the 60S pre-ribosome, most prominently to H89 that will constitute part of the peptidyl transferase center (PTC). The binding sites of Dbp10 are the same as those identified for the prokaryotic helicase DbpA bound to the 50S subunit. We suggest that Dbp10 and DbpA are performing a conserved role during PTC formation in all organisms. PMID:26823502

  1. The K⁺-dependent GTPase Nug1 is implicated in the association of the helicase Dbp10 to the immature peptidyl transferase centre during ribosome maturation.

    PubMed

    Manikas, Rizos-Georgios; Thomson, Emma; Thoms, Matthias; Hurt, Ed

    2016-02-29

    Ribosome synthesis employs a number of energy-consuming enzymes in both eukaryotes and prokaryotes. One such enzyme is the conserved circularly permuted GTPase Nug1 (nucleostemin in human). Nug1 is essential for 60S subunit assembly and nuclear export, but its role and time of action during maturation remained unclear. Based on in vitro enzymatic assays using the Chaetomium thermophilum (Ct) orthologue, we show that Nug1 exhibits a low intrinsic GTPase activity that is stimulated by potassium ions, rendering Nug1 a cation-dependent GTPase. In vivo we observe 60S biogenesis defects upon depletion of yeast Nug1 or expression of a Nug1 nucleotide-binding mutant. Most prominently, the RNA helicase Dbp10 was lost from early pre-60S particles, which suggested a physical interaction that could be reconstituted in vitro using CtNug1 and CtDbp10. In vivo rRNA-protein crosslinking revealed that Nug1 and Dbp10 bind at proximal and partially overlapping sites on the 60S pre-ribosome, most prominently to H89 that will constitute part of the peptidyl transferase center (PTC). The binding sites of Dbp10 are the same as those identified for the prokaryotic helicase DbpA bound to the 50S subunit. We suggest that Dbp10 and DbpA are performing a conserved role during PTC formation in all organisms. PMID:26823502

  2. Abrogation of collagen-induced arthritis by a peptidyl arginine deiminase inhibitor is associated with modulation of T cell-mediated immune responses

    PubMed Central

    Kawalkowska, Joanna; Quirke, Anne-Marie; Ghari, Fatemeh; Davis, Simon; Subramanian, Venkataraman; Thompson, Paul R.; Williams, Richard O.; Fischer, Roman; La Thangue, Nicholas B.; Venables, Patrick J.

    2016-01-01

    Proteins containing citrulline, a post-translational modification of arginine, are generated by peptidyl arginine deiminases (PAD). Citrullinated proteins have pro-inflammatory effects in both innate and adaptive immune responses. Here, we examine the therapeutic effects in collagen-induced arthritis of the second generation PAD inhibitor, BB-Cl-amidine. Treatment after disease onset resulted in the reversal of clinical and histological changes of arthritis, associated with a marked reduction in citrullinated proteins in lymph nodes. There was little overall change in antibodies to collagen or antibodies to citrullinated peptides, but a shift from pro-inflammatory Th1 and Th17-type responses to pro-resolution Th2-type responses was demonstrated by serum cytokines and antibody subtypes. In lymph node cells from the arthritic mice treated with BB-Cl-amidine, there was a decrease in total cell numbers but an increase in the proportion of Th2 cells. BB-Cl-amidine had a pro-apoptotic effect on all Th subsets in vitro with Th17 cells appearing to be the most sensitive. We suggest that these immunoregulatory effects of PAD inhibition in CIA are complex, but primarily mediated by transcriptional regulation. We suggest that targeting PADs is a promising strategy for the treatment of chronic inflammatory disease. PMID:27210478

  3. A SAM-dependent methyltransferase cotranscribed with arsenate reductase alters resistance to peptidyl transferase center-binding antibiotics in Azospirillum brasilense Sp7.

    PubMed

    Singh, Sudhir; Singh, Chhaya; Tripathi, Anil Kumar

    2014-05-01

    The genome of Azospirillum brasilense harbors a gene encoding S-adenosylmethionine-dependent methyltransferase, which is located downstream of an arsenate reductase gene. Both genes are cotranscribed and translationally coupled. When they were cloned and expressed individually in an arsenate-sensitive strain of Escherichia coli, arsenate reductase conferred tolerance to arsenate; however, methyltransferase failed to do so. Sequence analysis revealed that methyltransferase was more closely related to a PrmB-type N5-glutamine methyltransferase than to the arsenate detoxifying methyltransferase ArsM. Insertional inactivation of prmB gene in A. brasilense resulted in an increased sensitivity to chloramphenicol and resistance to tiamulin and clindamycin, which are known to bind at the peptidyl transferase center (PTC) in the ribosome. These observations suggested that the inability of prmB:km mutant to methylate L3 protein might alter hydrophobicity in the antibiotic-binding pocket of the PTC, which might affect the binding of chloramphenicol, clindamycin, and tiamulin differentially. This is the first report showing the role of PrmB-type N5-glutamine methyltransferases in conferring resistance to tiamulin and clindamycin in any bacterium.

  4. Abnormal type I collagen post-translational modification and crosslinking in a cyclophilin B KO mouse model of recessive osteogenesis imperfecta.

    PubMed

    Cabral, Wayne A; Perdivara, Irina; Weis, MaryAnn; Terajima, Masahiko; Blissett, Angela R; Chang, Weizhong; Perosky, Joseph E; Makareeva, Elena N; Mertz, Edward L; Leikin, Sergey; Tomer, Kenneth B; Kozloff, Kenneth M; Eyre, David R; Yamauchi, Mitsuo; Marini, Joan C

    2014-06-01

    Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib-/- mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2-11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib-/- fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered crosslink

  5. Abnormal Type I Collagen Post-translational Modification and Crosslinking in a Cyclophilin B KO Mouse Model of Recessive Osteogenesis Imperfecta

    PubMed Central

    Cabral, Wayne A.; Perdivara, Irina; Weis, MaryAnn; Terajima, Masahiko; Blissett, Angela R.; Chang, Weizhong; Perosky, Joseph E.; Makareeva, Elena N.; Mertz, Edward L.; Leikin, Sergey; Tomer, Kenneth B.; Kozloff, Kenneth M.; Eyre, David R.; Yamauchi, Mitsuo; Marini, Joan C.

    2014-01-01

    Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib−/− mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2–11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib−/− fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered

  6. Prolyl 4-hydroxylase from Volvox carteri. A low-Mr enzyme antigenically related to the alpha subunit of the vertebrate enzyme.

    PubMed Central

    Kaska, D D; Myllylä, R; Günzler, V; Gibor, A; Kivirikko, K I

    1988-01-01

    Prolyl 4-hydroxylase was isolated in a highly purified form from a multi-cellular green alga, Volvox carteri, by a procedure consisting of ion-exchange chromatography and affinity chromatography on poly(L-hydroxyproline) coupled to Sepharose. Two other affinity-column procedures were also developed, one involving 3,4-dihydroxyphenylacetate and the other 3,4-dihydroxyphenylpropionate linked to Sepharose. The Km values of the Volvox enzyme for the co-substrates and the peptide substrate, as well as the inhibition constants for selected 2-oxoglutarate analogues, were similar to those of the enzyme from Chlamydomonas reinhardii, except that the Km for 2-oxoglutarate with the Volvox enzyme was 6-fold greater. The temperature optimum of the Volvox enzyme was also 10 degrees C higher. The apparent Mr of the Volvox enzyme by gel filtration was about 40,000, being similar to that reported for the Chlamydomonas enzyme but markedly lower than that of the vertebrate enzymes. A similar apparent Mr of about 40,000 was also found for prolyl 4-hydroxylase from the green alga Enteromorpha intestinalis, whereas the enzyme from various vascular plants gave an apparent Mr greater than 300,000. SDS/polyacrylamide-gel electrophoresis demonstrated in the highly purified Volvox enzyme the presence of a major protein band doublet with a Mr of about 65,000 and a minor doublet of Mr about 55,000-57,000. A polyclonal antiserum, prepared against the Mr-65,000 doublet, stained in immunoblotting the Mr-65,000 doublet as well as the alpha subunit, but not the beta subunit, of the vertebrate prolyl 4-hydroxylase. An antiserum against the beta subunit of the vertebrate enzyme stained in immunoblotting a Mr-50,000 polypeptide in a partially purified Volvox enzyme preparation, but did not stain either the Mr-65,000 or the Mr-55,000-57,000 doublet of the highly purified enzyme. The data thus suggest that the active Volvox carteri prolyl 4-hydroxylase is an enzyme monomer antigenically related to the

  7. Poststatin, a new inhibitor of prolyl endopeptidase, produced by Streptomyces viridochromogenes MH534-30F3. I. Taxonomy, production, isolation, physico-chemical properties and biological activities.

    PubMed

    Aoyagi, T; Nagai, M; Ogawa, K; Kojima, F; Okada, M; Ikeda, T; Hamada, M; Takeuchi, T

    1991-09-01

    Poststatin, a new inhibitor of prolyl endopeptidase (PEP) was discovered in the fermentation broth of Streptomyces viridochromogenes MH534-30F3. It was purified by Diaion HP-20, Sephadex LH-20 and YMC-gel (ODS-A) column chromatography and then isolated as a colorless powder. Poststatin has the molecular formula C26H47N5O7. The IC50 value of poststatin against the PEP of partially purified porcine kidney was 0.03 microgram/ml. It has low acute toxicity. No deaths occured after iv injection of 250 mg/kg of this agent to mice. PMID:1938617

  8. Anaerobic respiration sustains mitochondrial membrane potential in a prolyl hydroxylase pathway-activated cancer cell line in a hypoxic microenvironment.

    PubMed

    Takahashi, Eiji; Sato, Michihiko

    2014-02-15

    To elucidate how tumor cells produce energy in oxygen-depleted microenvironments, we studied the possibility of mitochondrial electron transport without oxygen. We produced well-controlled oxygen gradients (ΔO2) in monolayer-cultured cells. We then visualized oxygen levels and mitochondrial membrane potential (ΔΦm) in individual cells by using the red shift of green fluorescent protein (GFP) fluorescence and a cationic fluorescent dye, respectively. In this two-dimensional tissue model, ΔΦm was abolished in cells >500 μm from the oxygen source [the anoxic front (AF)], indicating limitations in diffusional oxygen delivery. This result perfectly matched GFP-determined ΔO2. In cells pretreated with dimethyloxaloylglycine (DMOG), a prolyl hydroxylase domain-containing protein (PHD) inhibitor, the AF was expanded to 1,500-2,000 μm from the source. In these cells, tissue ΔO2 was substantially decreased, indicating that PHD pathway activation suppressed mitochondrial respiration. The expansion of the AF and the reduction of ΔO2 were much more prominent in a cancer cell line (Hep3B) than in the equivalent fibroblast-like cell line (COS-7). Hence, the results indicate that PHD pathway-activated cells can sustain ΔΦm, despite significantly decreased electron flux to complex IV. Complex II inhibition abolished the effect of DMOG in expanding the AF, although tissue ΔO2 remained shallow. Separate experiments demonstrated that complex II plays a substantial role in sustaining ΔΦm in DMOG-pretreated Hep3B cells with complex III inhibition. From these results, we conclude that PHD pathway activation can sustain ΔΦm in an otherwise anoxic microenvironment by decreasing tissue ΔO2 while activating oxygen-independent electron transport in mitochondria.

  9. Molecular Cloning, Characterization, and Expression Analysis of a Prolyl 4-Hydroxylase from the Marine Sponge Chondrosia reniformis.

    PubMed

    Pozzolini, Marina; Scarfì, Sonia; Mussino, Francesca; Ferrando, Sara; Gallus, Lorenzo; Giovine, Marco

    2015-08-01

    Prolyl 4-hydroxylase (P4H) catalyzes the hydroxylation of proline residues in collagen. P4H has two functional subunits, α and β. Here, we report the cDNA cloning, characterization, and expression analysis of the α and β subunits of the P4H derived from the marine sponge Chondrosia reniformis. The amino acid sequence of the α subunit is 533 residues long with an M r of 59.14 kDa, while the β subunit counts 526 residues with an M r of 58.75 kDa. Phylogenetic analyses showed that αP4H and βP4H are more related to the mammalian sequences than to known invertebrate P4Hs. Western blot analysis of sponge lysate protein cross-linking revealed a band of 240 kDa corresponding to an α2β2 tetramer structure. This result suggests that P4H from marine sponges shares the same quaternary structure with vertebrate homologous enzymes. Gene expression analyses showed that αP4H transcript is higher in the choanosome than in the ectosome, while the study of factors affecting its expression in sponge fragmorphs revealed that soluble silicates had no effect on the αP4H levels, whereas ascorbic acid strongly upregulated the αP4H mRNA. Finally, treatment with two different tumor necrosis factor (TNF)-alpha inhibitors determined a significant downregulation of αP4H gene expression in fragmorphs demonstrating, for the first time in Porifera, a positive involvement of TNF in sponge matrix biosynthesis. The molecular characterization of P4H genes involved in collagen hydroxylation, including the mechanisms that regulate their expression, is a key step for future recombinant sponge collagen production and may be pivotal to understand pathological mechanisms related to extracellular matrix deposition in higher organisms.

  10. Suppression of vascular network formation by chronic hypoxia and prolyl-hydroxylase 2 (phd2) deficiency during vertebrate development.

    PubMed

    Metikala, Sanjeeva; Neuhaus, Herbert; Hollemann, Thomas

    2016-04-01

    In the adult, new vessels and red blood cells form in response to hypoxia. Here, the oxygen-sensing system (PHD-HIF) has recently been put into focus, since the prolyl-hydroxylase domain proteins (PHD) and hypoxia-inducible factors (HIF) are considered as potential therapeutic targets to treat ischemia, cancers or age-related macula degeneration. While the oxygen-sensing system (PHD-HIF) has been studied intensively in this respect, only little is known from developing vertebrate embryos since mutations within this pathway led to an early decease of embryos due to placental defects. During vertebrate embryogenesis, a progenitor cell called hemangioblast is assumed to give rise to blood cells and blood vessels in a process called hematopoiesis and vasculogenesis, respectively. Xenopus provides an ideal experimental system to address these processes in vivo, as its development does not depend on a functional placenta and thus allows analyzing the role of oxygen directly. To this end, we adopted a computer-controlled four-channel system, which allowed us to culture Xenopus embryos under defined oxygen concentrations. Our data show that the development of vascular structures and blood cells is strongly impaired under hypoxia, while general development is less compromised. Interestingly, suppression of Phd2 function using specific antisense morpholinos or a chemical inhibitor resulted in mostly overlapping vascular defects; nevertheless, blood cell was formed almost normally. Our results provide the first evidence that oxygen via Phd2 has a decisive influence on the formation of the vascular network during vertebrate embryogenesis. These findings may be considered in certain potential treatment concepts. PMID:26678600

  11. Serum activities of adenosine deaminase, dipeptidyl peptidase IV and prolyl endopeptidase in patients with fibromyalgia: diagnostic implications.

    PubMed

    Čulić, Ognjen; Cordero, Mario D; Žanić-Grubišić, Tihana; Somborac-Bačura, Anita; Pučar, Lara Batičić; Detel, Dijana; Varljen, Jadranka; Barišić, Karmela

    2016-10-01

    Fibromyalgia (FM) is a chronic pain syndrome with number of symptoms that present challenge in terms of diagnosis and treatment. Patients with FM show abnormal profile of purines in plasma. In this work, we measured serum activities of enzymes involved in purine metabolism, namely total adenosine deaminase (ADE) and its isoforms (ADE1 and ADE2), ecto-ATPase, and 5'-nucleotidase (5'-NT). We also measured activity of dipeptidyl peptidase IV (DPPIV) and prolyl endopeptidase (PEP). Spectrophotometric and fluorometric methods were used for enzyme activity determinations. Enzyme activities were measured in sera of 24 patients with FM that were not undergoing pharmacological treatment during the study. Control group comprised 32 healthy control subjects. Significantly higher activities of total ADE (P = 0.025) and ADE2 (P = 0.011) were observed in FM patients, while no significant differences in ADE1, ecto-ATPase, and 5'-NT activities (P > 0.05) were found when compared to healthy controls. Moreover, increase in the activity of DPPIV (P = 0.015) and lower activity of PEP (P = 0.011) were also found in the FM group. ROC analysis pointed to different diagnostic sensitivities/specificities for individual enzyme activities measured as follows: ADE (50.0/87.5), ADE2 (41.7/90.6), DPPIV (62.5/71.9), and PEP (83.3/62.5). ADE2 and PEP were shown to be independent predictors of FM, while combination of the two gives AUC of 0.786 (95 % confidence interval of 0.656-0.885, P < 0.05). Our results are showing that serum activities of ADE2 and PEP could be useful as biomarkers for FM diagnosis. However, relatively low diagnostic sensitivity of ADE2 and specificity of PEP must be taken into account.

  12. Extended ischemia prevents HIF1alpha degradation at reoxygenation by impairing prolyl-hydroxylation: role of Krebs cycle metabolites.

    PubMed

    Serra-Pérez, Anna; Planas, Anna M; Núñez-O'Mara, Analía; Berra, Edurne; García-Villoria, Judit; Ribes, Antònia; Santalucía, Tomàs

    2010-06-11

    Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor that activates the cellular response to hypoxia. The HIF1alpha subunit is constantly synthesized and degraded under normoxia, but degradation is rapidly inhibited when oxygen levels drop. Oxygen-dependent hydroxylation by prolyl-4-hydroxylases (PHD) mediates HIF1alpha proteasome degradation. Brain ischemia limits the availability not only of oxygen but also of glucose. We hypothesized that this circumstance could have a modulating effect on HIF. We assessed the separate involvement of oxygen and glucose in HIF1alpha regulation in differentiated neuroblastoma cells subjected to ischemia. We report higher transcriptional activity and HIF1alpha expression under oxygen deprivation in the presence of glucose (OD), than in its absence (oxygen and glucose deprivation, OGD). Unexpectedly, HIF1alpha was not degraded at reoxygenation after an episode of OGD. This was not due to impairment of proteasome function, but was associated with lower HIF1alpha hydroxylation. Krebs cycle metabolites fumarate and succinate are known inhibitors of PHD, while alpha-ketoglutarate is a co-substrate of the reaction. Lack of HIF1alpha degradation in the presence of oxygen was accompanied by a very low alpha-ketoglutarate/fumarate ratio. Furthermore, treatment with a fumarate analogue prevented HIF1alpha degradation under normoxia. In all, our data suggest that postischemic metabolic alterations in Krebs cycle metabolites impair HIF1alpha degradation in the presence of oxygen by decreasing its hydroxylation, and highlight the involvement of metabolic pathways in HIF1alpha regulation besides the well known effects of oxygen.

  13. Modulation of agonist binding to human dopamine receptor subtypes by L-prolyl-L-leucyl-glycinamide and a peptidomimetic analog.

    PubMed

    Verma, Vaneeta; Mann, Amandeep; Costain, Willard; Pontoriero, Giuseppe; Castellano, Jessica M; Skoblenick, Kevin; Gupta, Suresh K; Pristupa, Zdenek; Niznik, Hyman B; Johnson, Rodney L; Nair, Venugopalan D; Mishra, Ram K

    2005-12-01

    The present study was undertaken to investigate the role of the hypothalamic tripeptide L-prolyl-L-leucyl-glycinamide (PLG) and its conformationally constrained analog 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA) in modulating agonist binding to human dopamine (DA) receptor subtypes using human neuroblastoma SH-SY5Y cells stably transfected with respective cDNAs. Both PLG and PAOPA enhanced agonist [3H]N-propylnorapomorphine (NPA) and [3H]quinpirole binding in a dose-dependent manner to the DA D2L,D2S, and D4 receptors. However, agonist binding to the D1 and D3 receptors and antagonist binding to the D2L receptors by PLG were not significantly affected. Scatchard analysis of [3H]NPA binding to membranes in the presence of PLG revealed a significant increase in affinity of the agonist binding sites for the D2L, D2S, and D4 receptors. Analysis of agonist/antagonist competition curves revealed that PLG and PAOPA increased the population and affinity of the high-affinity form of the D2L receptor and attenuated guanosine 5'-(beta,gamma-imido)-triphosphate-induced inhibition of high-affinity agonist binding sites for the DA D2L receptor. Furthermore, direct NPA binding with D2L cell membranes pretreated with suramin, a compound that can uncouple receptor/G protein complexes, and incubated with and without DA showed that both PLG and PAOPA had only increased agonist binding in membranes pretreated with both suramin and DA, suggesting that PLG requires the D2L receptor/G protein complex to increase agonist binding. These results suggest that PLG possibly modulates DA D2S, D2L, and D4 receptors in an allosteric manner and that the coupling of D2 receptors to the G protein is essential for this modulation to occur. PMID:16126839

  14. Virus induced gene silencing of three putative prolyl 4-hydroxylases enhances plant growth in tomato (Solanum lycopersicum).

    PubMed

    Fragkostefanakis, Sotirios; Sedeek, Khalid E M; Raad, Maya; Zaki, Marwa Samir; Kalaitzis, Panagiotis

    2014-07-01

    Proline hydroxylation is a major posttranslational modification of hydroxyproline-rich glycoproteins (HRGPs) that is catalyzed by prolyl 4-hydroxylases (P4Hs). HRGPs such as arabinogalactan proteins (AGPs) and extensios play significant roles on cell wall structure and function and their implication in cell division and expansion has been reported. We used tobacco rattle virus (TRV)-based virus induced gene silencing to investigate the role of three tomato P4Hs, out of ten present in the tomato genome, in growth and development. Eight-days old tomato seedlings were infected with the appropriate TRV vectors and plants were allowed to grow under standard conditions for 6 weeks. Lower P4H mRNA levels were associated with lower hydroxyproline content in root and shoot tissues indicating successful gene silencing. P4H-silenced plants had longer roots and shoots and larger leaves. The increased leaf area can be attributed to increased cell division as indicated by the higher leaf epidermal cell number in SlP4H1- and SlP4H9-silenced plants. In contrast, SlP4H7-silenced plants had larger leaves due to enhanced cell expansion. Western blot analysis revealed that silencing of SlP4H7 and SlP4H9 was associated with reduced levels of JIM8-bound AGP and JIM11-bound extensin epitopes, while silencing of SlP4H1 reduced only the levels of AGP proteins. Collectively these results show that P4Hs have significant and distinct roles in cell division and expansion of tomato leaves.

  15. The Proteome Response to Amyloid Protein Expression In Vivo

    PubMed Central

    Gomes, Ricardo A.; Franco, Catarina; Da Costa, Gonçalo; Planchon, Sébastien; Renaut, Jenny; Ribeiro, Raquel M.; Pinto, Francisco; Silva, Marta Sousa; Coelho, Ana Varela; Freire, Ana Ponces; Cordeiro, Carlos

    2012-01-01

    Protein misfolding disorders such as Alzheimer, Parkinson and transthyretin amyloidosis are characterized by the formation of protein amyloid deposits. Although the nature and location of the aggregated proteins varies between different diseases, they all share similar molecular pathways of protein unfolding, aggregation and amyloid deposition. Most effects of these proteins are likely to occur at the proteome level, a virtually unexplored reality. To investigate the effects of an amyloid protein expression on the cellular proteome, we created a yeast expression system using human transthyretin (TTR) as a model amyloidogenic protein. We used Saccharomyces cerevisiae, a living test tube, to express native TTR (non-amyloidogenic) and the amyloidogenic TTR variant L55P, the later forming aggregates when expressed in yeast. Differential proteome changes were quantitatively analyzed by 2D-differential in gel electrophoresis (2D-DIGE). We show that the expression of the amyloidogenic TTR-L55P causes a metabolic shift towards energy production, increased superoxide dismutase expression as well as of several molecular chaperones involved in protein refolding. Among these chaperones, members of the HSP70 family and the peptidyl-prolyl-cis-trans isomerase (PPIase) were identified. The latter is highly relevant considering that it was previously found to be a TTR interacting partner in the plasma of ATTR patients but not in healthy or asymptomatic subjects. The small ubiquitin-like modifier (SUMO) expression is also increased. Our findings suggest that refolding and degradation pathways are activated, causing an increased demand of energetic resources, thus the metabolic shift. Additionally, oxidative stress appears to be a consequence of the amyloidogenic process, posing an enhanced threat to cell survival. PMID:23185553

  16. Overexpression of OsCYP19-4 increases tolerance to cold stress and enhances grain yield in rice (Oryza sativa).

    PubMed

    Yoon, Dae Hwa; Lee, Sang Sook; Park, Hyun Ji; Lyu, Jae Il; Chong, Won Seog; Liu, Jang Ryol; Kim, Beom-Gi; Ahn, Jun Cheul; Cho, Hye Sun

    2016-01-01

    AtCYP19-4 (also known as CYP5) was previously identified as interacting in vitro with GNOM, a member of a large family of ARF guanine nucleotide exchange factors that is required for proper polar localization of the auxin efflux carrier PIN1. The present study demonstrated that OsCYP19-4, a gene encoding a putative homologue of AtCYP19-4, was up-regulated by several stresses and showed over 10-fold up-regulation in response to cold. The study further demonstrated that the promoter of OsCYP19-4 was activated in response to cold stress. An OsCYP19-4-GFP fusion protein was targeted to the outside of the plasma membrane via the endoplasmic reticulum as determined using brefeldin A, a vesicle trafficking inhibitor. An in vitro assay with a synthetic substrate oligomer confirmed that OsCYP19-4 had peptidyl-prolyl cis-trans isomerase activity, as was previously reported for AtCYP19-4. Rice plants overexpressing OsCYP19-4 showed cold-resistance phenotypes with significantly increased tiller and spike numbers, and consequently enhanced grain weight, compared with wild-type plants. Based on these results, the authors suggest that OsCYP19-4 is required for developmental acclimation to environmental stresses, especially cold. Furthermore, the results point to the potential of manipulating OsCYP19-4 expression to enhance cold tolerance or to increase biomass. PMID:26453745

  17. The Crystal Structure of PPIL1 Bound to Cyclosporine A Suggests a Binding Mode for a Linear Epitope of the SKIP Protein

    PubMed Central

    Stegmann, Christian M.; Lührmann, Reinhard; Wahl, Markus C.

    2010-01-01

    Background The removal of introns from pre-mRNA is carried out by a large macromolecular machine called the spliceosome. The peptidyl-prolyl cis/trans isomerase PPIL1 is a component of the human spliceosome and binds to the spliceosomal SKIP protein via a binding site distinct from its active site. Principal Findings Here, we have studied the PPIL1 protein and its interaction with SKIP biochemically and by X-ray crystallography. A minimal linear binding epitope derived from the SKIP protein could be determined using a peptide array. A 36-residue region of SKIP centred on an eight-residue epitope suffices to bind PPIL1 in pull-down experiments. The crystal structure of PPIL1 in complex with the inhibitor cyclosporine A (CsA) was obtained at a resolution of 1.15 Å and exhibited two bound Cd2+ ions that enabled SAD phasing. PPIL1 residues that have previously been implicated in binding of SKIP are involved in the coordination of Cd2+ ions in the present crystal structure. Employing the present crystal structure, the determined minimal binding epitope and previously published NMR data [1], a molecular docking study was performed. In the docked model of the PPIL1·SKIP interaction, a proline residue of SKIP is buried in a hydrophobic pocket of PPIL1. This hydrophobic contact is encircled by several hydrogen bonds between the SKIP peptide and PPIL1. Conclusion We characterized a short, linear epitope of SKIP that is sufficient to bind the PPIL1 protein. Our data indicate that this SKIP peptide could function in recruiting PPIL1 into the core of the spliceosome. We present a molecular model for the binding mode of SKIP to PPIL1 which emphasizes the versatility of cyclophilin-type PPIases to engage in additional interactions with other proteins apart from active site contacts despite their limited surface area. PMID:20368803

  18. Multiple redox and non-redox interactions define 2-Cys peroxiredoxin as a regulatory hub in the chloroplast.

    PubMed

    Muthuramalingam, Meenakumari; Seidel, Thorsten; Laxa, Miriam; Nunes de Miranda, Susana M; Gärtner, Florian; Ströher, Elke; Kandlbinder, Andrea; Dietz, Karl-Josef

    2009-11-01

    In plants, the highly abundant 2-cysteine peroxiredoxin (2-CysPrx) is associated with the chloroplast and involved in protecting photosynthesis. This work addresses the multiple interactions of the 2-CysPrx in the chloroplast, which depend on its redox state. Transcript co-regulation analysis showed a strong linkage to the peptidyl-prolyl-cis/trans isomerase Cyclophilin 20-3 (Cyp20-3) and other components of the photosynthetic apparatus. Co-expression in protoplasts and quantification of fluorescence resonance energy transfer (FRET) efficiency in vivo confirmed protein interactions of 2-CysPrx with Cyp20-3 as well as NADPH-dependent thioredoxin reductase C (NTRC), while thioredoxin x (Trx-x) did not form complexes that could enable FRET. Likewise, changes in FRET of fluorescently labeled 2-CysPrx in vitro and in vivo proved redox dependent dynamics of 2-CysPrx. Addition of Cyp20-3 to an in vitro peroxidase assay with 2-CysPrx had no significant effect on peroxide reduction. Also, in the presence of NTRC, addition of Cyp20-3 did not further enhance peroxide reduction. In addition, 2-CysPrx functioned as chaperone and inhibited aggregation of citrate synthase during heat treatment. This activity was partly inhibited by Cyp20-3. As a new interaction partner of decameric 2-CysPrx, photosystem II could be identified after chloroplast fractionation and in pull-down assays after reconstitution. In summary, the data indicate a dynamic function of plant 2-CysPrx as redox sensor, chaperone, and regulator in the chloroplast with diverse functions beyond its role as thiol peroxidase.

  19. Parvulin 17-catalyzed Tubulin Polymerization Is Regulated by Calmodulin in a Calcium-dependent Manner.

    PubMed

    Burgardt, Noelia Inés; Schmidt, Andreas; Manns, Annika; Schutkowski, Alexandra; Jahreis, Günther; Lin, Yi-Jan; Schulze, Bianca; Masch, Antonia; Lücke, Christian; Weiwad, Matthias

    2015-07-01

    Recently we have shown that the peptidyl-prolyl cis/trans isomerase parvulin 17 (Par17) interacts with tubulin in a GTP-dependent manner, thereby promoting the formation of microtubules. Microtubule assembly is regulated by Ca(2+)-loaded calmodulin (Ca(2+)/CaM) both in the intact cell and under in vitro conditions via direct interaction with microtubule-associated proteins. Here we provide the first evidence that Ca(2+)/CaM interacts also with Par17 in a physiologically relevant way, thus preventing Par17-promoted microtubule assembly. In contrast, parvulin 14 (Par14), which lacks only the first 25 N-terminal residues of the Par17 sequence, does not interact with Ca(2+)/CaM, indicating that this interaction is exclusive for Par17. Pulldown experiments and chemical shift perturbation analysis with (15)N-labeled Par17 furthermore confirmed that calmodulin (CaM) interacts in a Ca(2+)-dependent manner with the Par17 N terminus. The reverse experiment with (15)N-labeled Ca(2+)/CaM demonstrated that the N-terminal Par17 segment binds to both CaM lobes simultaneously, indicating that Ca(2+)/CaM undergoes a conformational change to form a binding channel between its two lobes, apparently similar to the structure of the CaM-smMLCK(796-815) complex. In vitro tubulin polymerization assays furthermore showed that Ca(2+)/CaM completely suppresses Par17-promoted microtubule assembly. The results imply that Ca(2+)/CaM binding to the N-terminal segment of Par17 causes steric hindrance of the Par17 active site, thus interfering with the Par17/tubulin interaction. This Ca(2+)/CaM-mediated control of Par17-assisted microtubule assembly may provide a mechanism that couples Ca(2+) signaling with microtubule function.

  20. Novel Cycloheximide Derivatives Targeting the Moonlighting Protein Mip Exhibit Specific Antimicrobial Activity Against Legionella pneumophila.

    PubMed

    Rasch, Janine; Theuerkorn, Martin; Ünal, Can; Heinsohn, Natascha; Tran, Stefan; Fischer, Gunter; Weiwad, Matthias; Steinert, Michael

    2015-01-01

    Macrophage infectivity potentiator (Mip) and Mip-like proteins are virulence factors in a wide range of pathogens including Legionella pneumophila. These proteins belong to the FK506 binding protein (FKBP) family of peptidyl-prolyl-cis/trans-isomerases (PPIases). In L. pneumophila, the PPIase activity of Mip is required for invasion of macrophages, transmigration through an in vitro lung-epithelial barrier, and full virulence in the guinea pig infection model. Additionally, Mip is a moonlighting protein that binds to collagen IV in the extracellular matrix. Here, we describe the development and synthesis of cycloheximide derivatives with adamantyl moieties as novel FKBP ligands, and analyze their effect on the viability of L. pneumophila and other bacteria. All compounds efficiently inhibited PPIase activity of the prototypic human FKBP12 as well as Mip with IC50-values as low as 180 nM and 1.7 μM, respectively. Five of these derivatives inhibited the growth of L. pneumophila at concentrations of 30-40 μM, but exhibited no effect on other tested bacterial species indicating a specific spectrum of antibacterial activity. The derivatives carrying a 3,5-dimethyladamantan-1-[yl]acetamide substitution (MT_30.32), and a 3-ethyladamantan-1-[yl]acetamide substitution (MT_30.51) had the strongest effects in PPIase- and liquid growth assays. MT_30.32 and MT_30.51 were also inhibitory in macrophage infection studies without being cytotoxic. Accordingly, by applying a combinatorial approach, we were able to generate novel, hybrid inhibitors consisting of cycloheximide and adamantane, two known FKBP inhibitors that interact with different parts of the PPIase domain, respectively. Interestingly, despite the proven Mip-inhibitory activity, the viability of a Mip-deficient strain was affected to the same degree as its wild type. Hence, we also propose that cycloheximide derivatives with adamantyl moieties are potent PPIase inhibitors with multiple targets in L. pneumophila.

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

    PubMed

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

    2011-11-01

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

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

    PubMed Central

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

    2011-01-01

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

  3. Overexpression of OsCYP19-4 increases tolerance to cold stress and enhances grain yield in rice (Oryza sativa).

    PubMed

    Yoon, Dae Hwa; Lee, Sang Sook; Park, Hyun Ji; Lyu, Jae Il; Chong, Won Seog; Liu, Jang Ryol; Kim, Beom-Gi; Ahn, Jun Cheul; Cho, Hye Sun

    2016-01-01

    AtCYP19-4 (also known as CYP5) was previously identified as interacting in vitro with GNOM, a member of a large family of ARF guanine nucleotide exchange factors that is required for proper polar localization of the auxin efflux carrier PIN1. The present study demonstrated that OsCYP19-4, a gene encoding a putative homologue of AtCYP19-4, was up-regulated by several stresses and showed over 10-fold up-regulation in response to cold. The study further demonstrated that the promoter of OsCYP19-4 was activated in response to cold stress. An OsCYP19-4-GFP fusion protein was targeted to the outside of the plasma membrane via the endoplasmic reticulum as determined using brefeldin A, a vesicle trafficking inhibitor. An in vitro assay with a synthetic substrate oligomer confirmed that OsCYP19-4 had peptidyl-prolyl cis-trans isomerase activity, as was previously reported for AtCYP19-4. Rice plants overexpressing OsCYP19-4 showed cold-resistance phenotypes with significantly increased tiller and spike numbers, and consequently enhanced grain weight, compared with wild-type plants. Based on these results, the authors suggest that OsCYP19-4 is required for developmental acclimation to environmental stresses, especially cold. Furthermore, the results point to the potential of manipulating OsCYP19-4 expression to enhance cold tolerance or to increase biomass.

  4. Overexpression of PIN1 Enhances Cancer Growth and Aggressiveness with Cyclin D1 Induction in EBV-Associated Nasopharyngeal Carcinoma

    PubMed Central

    Xu, Meng; Cheung, Chartia Ching-Mei; Chow, Chit; Lun, Samantha Wei-Man; Cheung, Siu-Tim; Lo, Kwok-Wai

    2016-01-01

    Background Nasopharyngeal carcinoma (NPC) is a peculiar Epstein Barr virus (EBV)-associated malignancy that is prevalent in South-East Asia. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) isomerizes specific phosphorylated amino acid residues, which makes it an important regulator in cell survival and apoptosis. In this study, we investigated the contribution made by PIN1 in NPC tumorigenesis and PIN1’s potential role as a therapeutic target. Methods The expression of PIN1 was examined in a panel of NPC cell lines, xenografts and primary tumors. The functional roles of PIN1 in NPC cells were elucidated by the knockdown and overexpression of PIN1 in in vitro and in vivo nude mice models by siRNA and lenti-viral transfection, respectively. The antitumor effects of the PIN1 inhibitor Juglone in NPC cells were also evaluated. Results We revealed the consistent overexpression of PIN1 in almost all EBV-associated NPC cell lines, xenografts and primary tumors. PIN1 suppression was capable of inhibiting cyclin D1 expression and activating caspase-3 in NPC cells. It positively regulated NPC cell proliferation, colony formation and anchorage-independent growth. The inhibition of PIN1 suppressed tumor growth in vitro and in vivo. Conclusions This study demonstrates the oncogenic role of PIN1 in NPC tumorigenesis, and shows that its overexpression can enhance tumor cell growth via the upregulation of cyclinD1. Our findings inform the development of novel treatments targeting PIN1 for NPC patients. PMID:27258148

  5. The oncometabolite R-2-hydroxyglutarate activates NF-κB-dependent tumor-promoting stromal niche for acute myeloid leukemia cells.

    PubMed

    Chen, Jing-Yi; Lai, You-Syuan; Tsai, Hui-Jen; Kuo, Cheng-Chin; Yen, B Linju; Yeh, Su-Peng; Sun, H Sunny; Hung, Wen-Chun

    2016-01-01

    Mutations of isocitrate dehydrogenase 1 (IDH1) and IDH2 in acute myeloid leukemia (AML) cells produce the oncometabolite R-2-hydroxyglutarate (R-2HG) to induce epigenetic alteration and block hematopoietic differentiation. However, the effect of R-2HG released by IDH-mutated AML cells on the bone marrow microenvironment is unclear. Here, we report that R-2HG induces IκB kinase-independent activation of NF-κB in bone marrow stromal cells. R-2HG acts via a reactive oxygen species/extracellular signal-regulated kinase (ERK)-dependent pathway to phosphorylate NF-κB on the Thr254 residue. This phosphorylation enhances the interaction of NF-κB and the peptidyl-prolyl cis-trans isomerase PIN1 and increases the protein stability and transcriptional activity of NF-κB. As a consequence, R-2HG enhances NF-κB-dependent expression of cytokines including IL-6, IL-8 and complement 5a to stimulate proliferation of AML cells. In addition, R-2HG also upregulates vascular endothelial adhesion molecule 1 and CXCR4 in stromal cells to enhance the contact between AML and stromal cells and attenuates chemotherapy-induced apoptosis. More importantly, we validated the R-2HG-activated gene signature in the primary bone marrow stromal cells isolated from IDH-mutated AML patients. Collectively, our results suggest that AML cell-derived R-2HG may be helpful for the establishment of a supportive bone marrow stromal niche to promote AML progression via paracrine stimulation. PMID:27577048

  6. Disentangling mechanisms involved in collagen pyridinoline cross-linking: The immunophilin FKBP65 is critical for dimerization of lysyl hydroxylase 2.

    PubMed

    Gjaltema, Rutger A F; van der Stoel, Miesje M; Boersema, Miriam; Bank, Ruud A

    2016-06-28

    Collagens are subjected to extensive posttranslational modifications, such as lysine hydroxylation. Bruck syndrome (BS) is a connective tissue disorder characterized at the molecular level by a loss of telopeptide lysine hydroxylation, resulting in reduced collagen pyridinoline cross-linking. BS results from mutations in the genes coding for lysyl hydroxylase (LH) 2 or peptidyl-prolyl cis-trans isomerase (PPIase) FKBP65. Given that the immunophilin FKBP65 does not exhibit LH activity, it is likely that LH2 activity is somehow dependent on FKPB65. In this report, we provide insights regarding the interplay between LH2 and FKBP65. We found that FKBP65 forms complexes with LH2 splice variants LH2A and LH2B but not with LH1 and LH3. Ablating the catalytic activity of FKBP65 or LH2 did not affect complex formation. Both depletion of FKBP65 and inhibition of FKBP65 PPIase activity reduced the dimeric (active) form of LH2 but did not affect the binding of monomeric (inactive) LH2 to procollagen Iα1. Furthermore, we show that LH2A and LH2B cannot form heterodimers with each other but are able to form heterodimers with LH1 and LH3. Collectively, our results indicate that FKBP65 is linked to pyridinoline cross-linking by specifically mediating the dimerization of LH2. Moreover, FKBP65 does not interact with LH1 and LH3, explaining why in BS triple-helical hydroxylysines are not affected. Our results provide a mechanistic link between FKBP65 and the loss of pyridinolines and may hold the key to future treatments for diseases related to collagen cross-linking anomalies, such as fibrosis and cancer. PMID:27298363

  7. Parvulin 17-catalyzed Tubulin Polymerization Is Regulated by Calmodulin in a Calcium-dependent Manner*

    PubMed Central

    Burgardt, Noelia Inés; Schmidt, Andreas; Manns, Annika; Schutkowski, Alexandra; Jahreis, Günther; Lin, Yi-Jan; Schulze, Bianca; Masch, Antonia; Lücke, Christian; Weiwad, Matthias

    2015-01-01

    Recently we have shown that the peptidyl-prolyl cis/trans isomerase parvulin 17 (Par17) interacts with tubulin in a GTP-dependent manner, thereby promoting the formation of microtubules. Microtubule assembly is regulated by Ca2+-loaded calmodulin (Ca2+/CaM) both in the intact cell and under in vitro conditions via direct interaction with microtubule-associated proteins. Here we provide the first evidence that Ca2+/CaM interacts also with Par17 in a physiologically relevant way, thus preventing Par17-promoted microtubule assembly. In contrast, parvulin 14 (Par14), which lacks only the first 25 N-terminal residues of the Par17 sequence, does not interact with Ca2+/CaM, indicating that this interaction is exclusive for Par17. Pulldown experiments and chemical shift perturbation analysis with 15N-labeled Par17 furthermore confirmed that calmodulin (CaM) interacts in a Ca2+-dependent manner with the Par17 N terminus. The reverse experiment with 15N-labeled Ca2+/CaM demonstrated that the N-terminal Par17 segment binds to both CaM lobes simultaneously, indicating that Ca2+/CaM undergoes a conformational change to form a binding channel between its two lobes, apparently similar to the structure of the CaM-smMLCK796–815 complex. In vitro tubulin polymerization assays furthermore showed that Ca2+/CaM completely suppresses Par17-promoted microtubule assembly. The results imply that Ca2+/CaM binding to the N-terminal segment of Par17 causes steric hindrance of the Par17 active site, thus interfering with the Par17/tubulin interaction. This Ca2+/CaM-mediated control of Par17-assisted microtubule assembly may provide a mechanism that couples Ca2+ signaling with microtubule function. PMID:25940090

  8. Nuclear Translocation of β-Catenin during Mesenchymal Stem Cells Differentiation into Hepatocytes Is Associated with a Tumoral Phenotype

    PubMed Central

    Herencia, Carmen; Martínez-Moreno, Julio M.; Herrera, Concepción; Corrales, Fernando; Santiago-Mora, Raquel; Espejo, Isabel; Barco, Monserrat; Almadén, Yolanda; de la Mata, Manuel; Rodríguez-Ariza, Antonio; Muñoz-Castañeda, Juan R.

    2012-01-01

    Wnt/β-catenin pathway controls biochemical processes related to cell differentiation. In committed cells the alteration of this pathway has been associated with tumors as hepatocellular carcinoma or hepatoblastoma. The present study evaluated the role of Wnt/β-catenin activation during human mesenchymal stem cells differentiation into hepatocytes. The differentiation to hepatocytes was achieved by the addition of two different conditioned media. In one of them, β-catenin nuclear translocation, up-regulation of genes related to the Wnt/β-catenin pathway, such as Lrp5 and Fzd3, as well as the oncogenes c-myc and p53 were observed. While in the other protocol there was a Wnt/β-catenin inactivation. Hepatocytes with nuclear translocation of β-catenin also had abnormal cellular proliferation, and expressed membrane proteins involved in hepatocellular carcinoma, metastatic behavior and cancer stem cells. Further, these cells had also increased auto-renewal capability as shown in spheroids formation assay. Comparison of both differentiation protocols by 2D-DIGE proteomic analysis revealed differential expression of 11 proteins with altered expression in hepatocellular carcinoma. Cathepsin B and D, adenine phosphoribosyltransferase, triosephosphate isomerase, inorganic pyrophosphatase, peptidyl-prolyl cis-trans isomerase A or lactate dehydrogenase β-chain were up-regulated only with the protocol associated with Wnt signaling activation while other proteins involved in tumor suppression, such as transgelin or tropomyosin β-chain were down-regulated in this protocol. In conclusion, our results suggest that activation of the Wnt/β-catenin pathway during human mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotype. PMID:22506042

  9. Nuclear translocation of β-catenin during mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotype.

    PubMed

    Herencia, Carmen; Martínez-Moreno, Julio M; Herrera, Concepción; Corrales, Fernando; Santiago-Mora, Raquel; Espejo, Isabel; Barco, Monserrat; Almadén, Yolanda; de la Mata, Manuel; Rodríguez-Ariza, Antonio; Muñoz-Castañeda, Juan R

    2012-01-01

    Wnt/β-catenin pathway controls biochemical processes related to cell differentiation. In committed cells the alteration of this pathway has been associated with tumors as hepatocellular carcinoma or hepatoblastoma. The present study evaluated the role of Wnt/β-catenin activation during human mesenchymal stem cells differentiation into hepatocytes. The differentiation to hepatocytes was achieved by the addition of two different conditioned media. In one of them, β-catenin nuclear translocation, up-regulation of genes related to the Wnt/β-catenin pathway, such as Lrp5 and Fzd3, as well as the oncogenes c-myc and p53 were observed. While in the other protocol there was a Wnt/β-catenin inactivation. Hepatocytes with nuclear translocation of β-catenin also had abnormal cellular proliferation, and expressed membrane proteins involved in hepatocellular carcinoma, metastatic behavior and cancer stem cells. Further, these cells had also increased auto-renewal capability as shown in spheroids formation assay. Comparison of both differentiation protocols by 2D-DIGE proteomic analysis revealed differential expression of 11 proteins with altered expression in hepatocellular carcinoma. Cathepsin B and D, adenine phosphoribosyltransferase, triosephosphate isomerase, inorganic pyrophosphatase, peptidyl-prolyl cis-trans isomerase A or lactate dehydrogenase β-chain were up-regulated only with the protocol associated with Wnt signaling activation while other proteins involved in tumor suppression, such as transgelin or tropomyosin β-chain were down-regulated in this protocol. In conclusion, our results suggest that activation of the Wnt/β-catenin pathway during human mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotype.

  10. The cyclophilin A DIAGEOTROPICA gene affects auxin transport in both root and shoot to control lateral root formation.

    PubMed

    Ivanchenko, Maria G; Zhu, Jinsheng; Wang, Bangjun; Medvecká, Eva; Du, Yunlong; Azzarello, Elisa; Mancuso, Stefano; Megraw, Molly; Filichkin, Sergei; Dubrovsky, Joseph G; Friml, Jiří; Geisler, Markus

    2015-02-15

    Cyclophilin A is a conserved peptidyl-prolyl cis-trans isomerase (PPIase) best known as the cellular receptor of the immunosuppressant cyclosporine A. Despite significant effort, evidence of developmental functions of cyclophilin A in non-plant systems has remained obscure. Mutations in a tomato (Solanum lycopersicum) cyclophilin A ortholog, DIAGEOTROPICA (DGT), have been shown to abolish the organogenesis of lateral roots; however, a mechanistic explanation of the phenotype is lacking. Here, we show that the dgt mutant lacks auxin maxima relevant to priming and specification of lateral root founder cells. DGT is expressed in shoot and root, and localizes to both the nucleus and cytoplasm during lateral root organogenesis. Mutation of ENTIRE/IAA9, a member of the auxin-responsive Aux/IAA protein family of transcriptional repressors, partially restores the inability of dgt to initiate lateral root primordia but not the primordia outgrowth. By comparison, grafting of a wild-type scion restores the process of lateral root formation, consistent with participation of a mobile signal. Antibodies do not detect movement of the DGT protein into the dgt rootstock; however, experiments with radiolabeled auxin and an auxin-specific microelectrode demonstrate abnormal auxin fluxes. Functional studies of DGT in heterologous yeast and tobacco-leaf auxin-transport systems demonstrate that DGT negatively regulates PIN-FORMED (PIN) auxin efflux transporters by affecting their plasma membrane localization. Studies in tomato support complex effects of the dgt mutation on PIN expression level, expression domain and plasma membrane localization. Our data demonstrate that DGT regulates auxin transport in lateral root formation.

  11. Computational insight into small molecule inhibition of cyclophilins.

    PubMed

    Sambasivarao, Somisetti V; Acevedo, Orlando

    2011-02-28

    Cyclophilins (Cyp) are a family of cellular enzymes possessing peptidyl-prolyl isomerase activity, which catalyze the cis-trans interconversion of proline-containing peptide bonds. The two most abundant family members, CypA and CypB, have been identified as valid drug targets for a wide range of diseases, including HCV, HIV, and multiple cancers. However, the development of small molecule inhibitors that possess nM potency and high specificity for a particular Cyp is difficult given the complete conservation of all active site residues between the enzymes. Monte Carlo statistical sampling coupled to free energy perturbation theory (MC/FEP) calculations have been carried out to elucidate the origin of the experimentally observed nM inhibition of CypA by acylurea-based derivatives and the >200-fold in vitro selectivity between CypA and CypB from aryl 1-indanylketone-based μM inhibitors. The computed free-energies of binding were in close accord with those derived from experiments. Binding affinity values for the inhibitors were determined to be dependent upon the stabilization strength of the nonbonded interactions provided toward two catalytic residues: Arg55 and Asn102 in CypA and the analogous Arg63 and Asn110 residues in CypB. Fine-tuning of the hydrophobic interactions allowed for enhanced potency among derivatives. The aryl 1-indanylketones are predicted to differentiate between the cyclophilins by using distinct binding motifs that exploit subtle differences in the active site arrangements. Ideas for the development of new selective compounds with the potential for advancement to low-nanomolar inhibition are presented. PMID:21194235

  12. Classification of rice (oryza sativa l. japonica nipponbare) immunophilins (fkbps, cyps) and expression patterns under water stress

    PubMed Central

    2010-01-01

    Background FK506 binding proteins (FKBPs) and cyclophilins (CYPs) are abundant and ubiquitous proteins belonging to the peptidyl-prolyl cis/trans isomerase (PPIase) superfamily, which regulate much of metabolism through a chaperone or an isomerization of proline residues during protein folding. They are collectively referred to as immunophilin (IMM), being present in almost all cellular organs. In particular, a number of IMMs relate to environmental stresses. Results FKBP and CYP proteins in rice (Oryza sativa cv. Japonica) were identified and classified, and given the appropriate name for each IMM, considering the ortholog-relation with Arabidopsis and Chlamydomonas or molecular weight of the proteins. 29 FKBP and 27 CYP genes can putatively be identified in rice; among them, a number of genes can be putatively classified as orthologs of Arabidopsis IMMs. However, some genes were novel, did not match with those of Arabidopsis and Chlamydomonas, and several genes were paralogs by genetic duplication. Among 56 IMMs in rice, a significant number are regulated by salt and/or desiccation stress. In addition, their expression levels responding to the water-stress have been analyzed in different tissues, and some subcellular IMMs located by means of tagging with GFP protein. Conclusion Like other green photosynthetic organisms such as Arabidopsis (23 FKBPs and 29 CYPs) and Chlamydomonas (23 FKBs and 26 CYNs), rice has the highest number of IMM genes among organisms reported so far, suggesting that the numbers relate closely to photosynthesis. Classification of the putative FKBPs and CYPs in rice provides the information about their evolutional/functional significance when comparisons are drawn with the relatively well studied genera, Arabidopsis and Chlamydomonas. In addition, many of the genes upregulated by water stress offer the possibility of manipulating the stress responses in rice. PMID:21087465

  13. Cyclophilin A is a new M cell marker of bovine intestinal epithelium.

    PubMed

    Hondo, Tetsuya; Someya, Shunsuke; Nagasawa, Yuya; Terada, Shunsuke; Watanabe, Hitoshi; Chen, Xiangning; Watanabe, Kouichi; Ohwada, Shyuichi; Kitazawa, Haruki; Rose, Michael T; Nochi, Tomonori; Aso, Hisashi

    2016-06-01

    Microfold (M) cells in the follicle-associated epithelium (FAE) of Peyer's patches contribute to the mucosal immune response by the transcytosis of microorganisms. The mechanism by which M cells take up microorganisms, and the functional proteins by which they do this, are not clear. In order to explore one such protein, we developed a 2H5-F3 monoclonal antibody (2H5-F3 mAb) through its binding to bovine M cells, and identified the antibody reactive molecule as cyclophilin A (Cyp-A). The localization patterns of Cyp-A were very similar to the localization pattern of cytokeratin (CK) 18-positive M cells. Cyp-A was identified at the luminal surface of CK18-positive M cells in bovine jejunal and ileal FAE. The membranous localization of Cyp-A in the bovine intestinal cell line (BIE cells) increased as cells differentiated toward M cells, as determined by flow cytometry analysis. Additionally, BIE cells released Cyp-A to the extracellular space and the differentiation of BIE cells to M cells increased the secretion of Cyp-A, as determined by western blotting. Accordingly, Cyp-A may be localized in M cells in the small intestinal epithelium of cattle. The rise of the membranous localization and secretion of Cyp-A by differentiation toward M cells indicates that Cyp-A has an important role in the function of M cells. While Cyp-A of the M cell membrane may contribute to the uptake of viruses with peptidyl-prolyl cis-trans isomerase activity, in the extracellular space Cyp-A may work as a chemokine and contribute to the distribution of immuno-competent cells.

  14. The oncometabolite R-2-hydroxyglutarate activates NF-κB-dependent tumor-promoting stromal niche for acute myeloid leukemia cells

    PubMed Central

    Chen, Jing-Yi; Lai, You-Syuan; Tsai, Hui-Jen; Kuo, Cheng-Chin; Yen, B. Linju; Yeh, Su-Peng; Sun, H. Sunny; Hung, Wen-Chun

    2016-01-01

    Mutations of isocitrate dehydrogenase 1 (IDH1) and IDH2 in acute myeloid leukemia (AML) cells produce the oncometabolite R-2-hydroxyglutarate (R-2HG) to induce epigenetic alteration and block hematopoietic differentiation. However, the effect of R-2HG released by IDH-mutated AML cells on the bone marrow microenvironment is unclear. Here, we report that R-2HG induces IκB kinase-independent activation of NF-κB in bone marrow stromal cells. R-2HG acts via a reactive oxygen species/extracellular signal-regulated kinase (ERK)-dependent pathway to phosphorylate NF-κB on the Thr254 residue. This phosphorylation enhances the interaction of NF-κB and the peptidyl-prolyl cis-trans isomerase PIN1 and increases the protein stability and transcriptional activity of NF-κB. As a consequence, R-2HG enhances NF-κB-dependent expression of cytokines including IL-6, IL-8 and complement 5a to stimulate proliferation of AML cells. In addition, R-2HG also upregulates vascular endothelial adhesion molecule 1 and CXCR4 in stromal cells to enhance the contact between AML and stromal cells and attenuates chemotherapy-induced apoptosis. More importantly, we validated the R-2HG-activated gene signature in the primary bone marrow stromal cells isolated from IDH-mutated AML patients. Collectively, our results suggest that AML cell-derived R-2HG may be helpful for the establishment of a supportive bone marrow stromal niche to promote AML progression via paracrine stimulation. PMID:27577048

  15. Identification and functional characterization of the putative polysaccharide biosynthesis protein (CapD) of Enterococcus faecium U0317.

    PubMed

    Ali, Liaqat; Spiess, Meike; Wobser, Dominique; Rodriguez, Marta; Blum, Hubert E; Sakιnç, Türkân

    2016-01-01

    Most bacterial species produce capsular polysaccharides that contribute to disease pathogenesis through evasion of the host innate immune system and are also involved in inhibiting leukocyte killing. In the present study, we identified a gene in Enterococcus faecium U0317 with homologies to the polysaccharide biosynthesis protein CapD that is made up of 336 amino acids and putatively catalyzes N-linked glycosylation. A capD deletion mutant was constructed and complemented by homologous recombination that was confirmed by PCR and sequencing. The mutant revealed different growth behavior and morphological changes compared to wild-type by scanning electron microscopy, also the capD mutant showed a strong hydrophobicity and that was reversed in the reconstituted mutant. For further characterization and functional analyses, in-vitro cell culture and in-vivo a mouse infection models were used. Antibodies directed against alpha lipotechoic acid (αLTA) and the peptidyl-prolyl cis-trans isomerase (αPpiC), effectively mediated the opsonophagocytic killing in the capD knock-out mutant, while this activity was not observed in the wild-type and reconstituted mutant. By comparison more than 2-fold decrease was seen in mutant colonization and adherence to both T24 and Caco2 cells. However, a significant higher bacterial colonization was observed in capD mutant during bacteremia in the animal model, while virulence in a mouse UTI (urinary tract infection) model, there were no obvious differences. Further studies are needed to elucidate the function of capsular polysaccharide synthesis gene clusters and its involvement in the disease pathogenesis with the aim to develop targeted therapies to treat multidrug-resistant E. faecium infections.

  16. Integrated proteomic platforms for the comparative characterization of medulloblastoma and pilocytic astrocytoma pediatric brain tumors: a preliminary study.

    PubMed

    Martelli, Claudia; Iavarone, Federica; D'Angelo, Luca; Arba, Morena; Vincenzoni, Federica; Inserra, Ilaria; Delfino, Daniela; Rossetti, Diana Valeria; Caretto, Marta; Massimi, Luca; Tamburrini, Gianpiero; Di Rocco, Concezio; Caldarelli, Massimo; Messana, Irene; Castagnola, Massimo; Sanna, Maria Teresa; Desiderio, Claudia

    2015-06-01

    A top-down/bottom-up integrated proteomic approach based on LC-MS and 2-DE analysis was applied for comparative characterization of medulloblastoma and pilocytic astrocytoma posterior cranial fossa pediatric brain tumor tissues. Although rare, primary brain tumors are the most frequent solid tumors in the pediatric age. Among them the medulloblastoma is the prevalent malignant tumor in childhood while pilocytic astrocytoma is the most common, rarely showing a malignant progression. Due to the limited availability of this kind of sample, the study was applied to pooled tumor tissues for a preliminary investigation. The results showed different proteomic profiles of the two tumors and evidenced interesting differential expression of several proteins and peptides. Top-down proteomics of acid-soluble fractions of brain tumor homogenates ascribed a potential biomarker role of malignancy to β- and α-thymosins and their truncated proteoforms and to C-terminal truncated (des-GG) ubiquitin, resulting exclusively detected or over-expressed in the highly malignant medulloblastoma. The bottom-up proteomics of the acid-soluble fraction identified several proteins, some of them in common with 2-DE analysis of acid-insoluble pellets. Peroxiredoxin-1, peptidyl-prolyl cis-trans isomerase A, triosephosphate isomerase, pyruvate kinase PKM, tubulin beta and alpha chains, heat shock protein HSP-90-beta and different histones characterized the medulloblastoma while the Ig kappa chain C region, serotransferrin, tubulin beta 2A chain and vimentin the pilocytic astrocytoma. The two proteomic strategies, with their pros and cons, well complemented each other in characterizing the proteome of brain tumor tissues and in disclosing potential disease biomarkers to be validated in a future study on individual samples of both tumor histotypes. PMID:25909245

  17. Disentangling mechanisms involved in collagen pyridinoline cross-linking: The immunophilin FKBP65 is critical for dimerization of lysyl hydroxylase 2.

    PubMed

    Gjaltema, Rutger A F; van der Stoel, Miesje M; Boersema, Miriam; Bank, Ruud A

    2016-06-28

    Collagens are subjected to extensive posttranslational modifications, such as lysine hydroxylation. Bruck syndrome (BS) is a connective tissue disorder characterized at the molecular level by a loss of telopeptide lysine hydroxylation, resulting in reduced collagen pyridinoline cross-linking. BS results from mutations in the genes coding for lysyl hydroxylase (LH) 2 or peptidyl-prolyl cis-trans isomerase (PPIase) FKBP65. Given that the immunophilin FKBP65 does not exhibit LH activity, it is likely that LH2 activity is somehow dependent on FKPB65. In this report, we provide insights regarding the interplay between LH2 and FKBP65. We found that FKBP65 forms complexes with LH2 splice variants LH2A and LH2B but not with LH1 and LH3. Ablating the catalytic activity of FKBP65 or LH2 did not affect complex formation. Both depletion of FKBP65 and inhibition of FKBP65 PPIase activity reduced the dimeric (active) form of LH2 but did not affect the binding of monomeric (inactive) LH2 to procollagen Iα1. Furthermore, we show that LH2A and LH2B cannot form heterodimers with each other but are able to form heterodimers with LH1 and LH3. Collectively, our results indicate that FKBP65 is linked to pyridinoline cross-linking by specifically mediating the dimerization of LH2. Moreover, FKBP65 does not interact with LH1 and LH3, explaining why in BS triple-helical hydroxylysines are not affected. Our results provide a mechanistic link between FKBP65 and the loss of pyridinolines and may hold the key to future treatments for diseases related to collagen cross-linking anomalies, such as fibrosis and cancer.

  18. A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication*

    PubMed Central

    Dujardin, Marie; Madan, Vanesa; Montserret, Roland; Ahuja, Puneet; Huvent, Isabelle; Launay, Helene; Leroy, Arnaud; Bartenschlager, Ralf; Penin, François; Lippens, Guy; Hanoulle, Xavier

    2015-01-01

    Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and its interaction with the human chaperone cyclophilin A are both targets for highly potent and promising antiviral drugs that are in the late stages of clinical development. Despite its high interest in regards to the development of drugs to counteract the worldwide HCV burden, NS5A is still an enigmatic multifunctional protein poorly characterized at the molecular level. NS5A is required for HCV RNA replication and is involved in viral particle formation and regulation of host pathways. Thus far, no enzymatic activity or precise molecular function has been ascribed to NS5A that is composed of a highly structured domain 1 (D1), as well as two intrinsically disordered domains 2 (D2) and 3 (D3), representing half of the protein. Here, we identify a short structural motif in the disordered NS5A-D2 and report its NMR structure. We show that this structural motif, a minimal Pro314–Trp316 turn, is essential for HCV RNA replication, and its disruption alters the subcellular distribution of NS5A. We demonstrate that this Pro-Trp turn is required for proper interaction with the host cyclophilin A and influences its peptidyl-prolyl cis/trans isomerase activity on residue Pro314 of NS5A-D2. This work provides a molecular basis for further understanding of the function of the intrinsically disordered domain 2 of HCV NS5A protein. In addition, our work highlights how very small structural motifs present in intrinsically disordered proteins can exert a specific function. PMID:26085105

  19. A serine/arginine-rich nuclear matrix cyclophilin interacts with the C-terminal domain of RNA polymerase II.

    PubMed Central

    Bourquin, J P; Stagljar, I; Meier, P; Moosmann, P; Silke, J; Baechi, T; Georgiev, O; Schaffner, W

    1997-01-01

    The largest subunit of RNA polymerase II shows a striking difference in the degree of phosphorylation, depending on its functional state: initiating and elongating polymerases are unphosphorylated and highly phosphorylated respectively. Phosphorylation mostly occurs at the C-terminal domain (CTD), which consists of a repetitive heptapeptide structure. Using the yeast two-hybrid system, we have selected for mammalian proteins that interact with the phosphorylated CTD of mammalian RNA polymerase II. A prominent isolate, designated SRcyp/CASP10, specifically interacts with the CTD not only in vivo but also in vitro . It contains a serine/arginine-rich (SR) domain, similar to that found in the SR protein family of pre-mRNA splicing factors, which is required for interaction with the CTD. Most remarkably, the N-terminal region of SRcyp includes a peptidyl-prolyl cis - trans isomerase domain characteristic of immunophilins/cyclophilins (Cyp), a protein family implicated in protein folding, assembly and transport. SRcyp is a nuclear protein with a characteristic distribution in large irregularly shaped nuclear speckles and co-localizes perfectly with the SR domain-containing splicing factor SC35. Recent independent investigations have provided complementary data, such as an association of the phosphorylated form of RNA polymerase II with the nuclear speckles, impaired splicing in a CTD deletion background and inhibition of in vitro splicing by CTD peptides. Taken together, these data indicate that factors directly or indirectly involved in splicing are associated with the elongating RNA polymerases, from where they might translocate to the nascent transcripts to ensure efficient splicing, concomitant with transcription. PMID:9153302

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

    PubMed Central

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

    2009-01-01

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

  1. Differential proteome and gene expression for testis of mice exposed to carbon ion radiation

    NASA Astrophysics Data System (ADS)

    Zhang, Hong; Li, Hongyan

    Objective To investigate the effect and mechanism of high linear energy transfer (LET) carbon ion irradiation (CIR) on reproduction in the testis of male Swiss Webster mice, and assess the risk associated with space environment. Methods Male mice underwent whole-body irradiation with CIR (0.5, 1 and 4Gy), and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF) analysis was used to determine the alteration in protein expression in 2-DE (two-dimensional gel electrophoresis) gels of testes caused by irradiation after 7, 14 days. Results 15 differentially expressed proteins, such as glucose-regulated protein(GRP78), aconitate hydratase-mitochondrial precursor (ACO), pyruvate kinase isozymes M1/M2 (PKM1/M2), glutathione-S-transferaseA3 (GSTA3), glutathione S-transferase Pi 1 (GSTP1), Cu/Zn super-oxide dismutase (SOD1), Peptidyl-prolyl cis-trans isomerase (Pin1) and Heat shock 70 kDa protein 4L (HSPa4L), were identified and these proteins were mainly involved in energy supply, the endoplasmic reticulum, cell proliferation, cell cycle, antioxidant capacity and mitochondrial respiration, which play important roles in the inhibition of testicular function in response to CIR. Furthermore, we confirmed the relationship between transcription of mRNA and the abundance of proteins. Conclusion The findings of the present study demonstrated that these proteins may lead to new insights into the molecular mechanism of CIR toxicity, and suggested that the gene expression response to CIR involves diverse regulatory mechanisms from transcription of mRNA to the formation of functional proteins. These data also may provide a scientific basis for protecting astronauts and space traveler’s health and safety.

  2. Altered Proteome of Burkholderia pseudomallei Colony Variants Induced by Exposure to Human Lung Epithelial Cells

    PubMed Central

    Al-Maleki, Anis Rageh; Mariappan, Vanitha; Vellasamy, Kumutha Malar; Tay, Sun Tee; Vadivelu, Jamuna

    2015-01-01

    Burkholderia pseudomallei primary diagnostic cultures demonstrate colony morphology variation associated with expression of virulence and adaptation proteins. This study aims to examine the ability of B. pseudomallei colony variants (wild type [WT] and small colony variant [SCV]) to survive and replicate intracellularly in A549 cells and to identify the alterations in the protein expression of these variants, post-exposure to the A549 cells. Intracellular survival and cytotoxicity assays were performed followed by proteomics analysis using two-dimensional gel electrophoresis. B. pseudomallei SCV survive longer than the WT. During post-exposure, among 259 and 260 protein spots of SCV and WT, respectively, 19 were differentially expressed. Among SCV post-exposure up-regulated proteins, glyceraldehyde 3-phosphate dehydrogenase, fructose-bisphosphate aldolase (CbbA) and betaine aldehyde dehydrogenase were associated with adhesion and virulence. Among the down-regulated proteins, enolase (Eno) is implicated in adhesion and virulence. Additionally, post-exposure expression profiles of both variants were compared with pre-exposure. In WT pre- vs post-exposure, 36 proteins were differentially expressed. Of the up-regulated proteins, translocator protein, Eno, nucleoside diphosphate kinase (Ndk), ferritin Dps-family DNA binding protein and peptidyl-prolyl cis-trans isomerase B were implicated in invasion and virulence. In SCV pre- vs post-exposure, 27 proteins were differentially expressed. Among the up-regulated proteins, flagellin, Eno, CbbA, Ndk and phenylacetate-coenzyme A ligase have similarly been implicated in adhesion, invasion. Protein profiles differences post-exposure provide insights into association between morphotypic and phenotypic characteristics of colony variants, strengthening the role of B. pseudomallei morphotypes in pathogenesis of melioidosis. PMID:25996927

  3. Conserved Hydration Sites in Pin1 Reveal a Distinctive Water Recognition Motif in Proteins.

    PubMed

    Barman, Arghya; Smitherman, Crystal; Souffrant, Michael; Gadda, Giovanni; Hamelberg, Donald

    2016-01-25

    Structurally conserved water molecules are important for biomolecular stability, flexibility, and function. X-ray crystallographic studies of Pin1 have resolved a number of water molecules around the enzyme, including two highly conserved water molecules within the protein. The functional role of these localized water molecules remains unknown and unexplored. Pin1 catalyzes cis/trans isomerizations of peptidyl prolyl bonds that are preceded by a phosphorylated serine or threonine residue. Pin1 is involved in many subcellular signaling processes and is a potential therapeutic target for the treatment of several life threatening diseases. Here, we investigate the significance of these structurally conserved water molecules in the catalytic domain of Pin1 using molecular dynamics (MD) simulations, free energy calculations, analysis of X-ray crystal structures, and circular dichroism (CD) experiments. MD simulations and free energy calculations suggest the tighter binding water molecule plays a crucial role in maintaining the integrity and stability of a critical hydrogen-bonding network in the active site. The second water molecule is exchangeable with bulk solvent and is found in a distinctive helix-turn-coil motif. Structural bioinformatics analysis of nonredundant X-ray crystallographic protein structures in the Protein Data Bank (PDB) suggest this motif is present in several other proteins and can act as a water site, akin to the calcium EF hand. CD experiments suggest the isolated motif is in a distorted PII conformation and requires the protein environment to fully form the α-helix-turn-coil motif. This study provides valuable insights into the role of hydration in the structural integrity of Pin1 that can be exploited in protein engineering and drug design. PMID:26651388

  4. Isolation and Characterization of NaCl-Sensitive Mutants of Caulobacter crescentus

    PubMed Central

    Zuleta, Luiz Fernando G.; Italiani, Valéria C. S.; Marques, Marilis V.

    2003-01-01

    An attempt to characterize Caulobacter crescentus genes important for the response to high concentrations of NaCl was initiated by the isolation of mutants defective in survival in the presence of 85 mM NaCl. A transposon Tn5 library was screened, and five strains which contained different genes disrupted by the transposon were isolated. Three of the mutants had the Tn5 in genes involved in lipopolysaccharide biosynthesis, one had the Tn5 in the nhaA gene, which encodes a Na+/H+ antiporter, and one had the Tn5 in the ppiD gene, which encodes a peptidyl-prolyl cis-trans isomerase. All the mutant strains showed severe growth arrest in the presence of 85 mM NaCl, but only the nhaA mutant showed decreased viability under these conditions. All the mutants except the nhaA mutant showed a slightly reduced viability in the presence of 40 mM KCl, but all the strains showed a more severe reduction in viability in the presence of 150 mM sucrose, suggesting that they are defective in responding to osmotic shock. The promoter regions of each disrupted gene were cloned in lacZ reporter vectors, and the pattern of expression in response to NaCl and sucrose was determined; this showed that both agents induced ppiD and nhaA gene expression but did not induce the other genes. Furthermore, the ppiD gene was not induced by heat shock, indicating that it does not belong to the σ32 regulon, as opposed to what was observed for its Escherichia coli homolog. PMID:12788696

  5. Genome wide identification of the immunophilin gene family in Leptosphaeria maculans: a causal agent of Blackleg disease in Oilseed Rape (Brassica napus).

    PubMed

    Singh, Khushwant; Zouhar, Miloslav; Mazakova, Jana; Rysanek, Pavel

    2014-10-01

    Abstract Phoma stem canker (blackleg) is a disease of world-wide importance on oilseed rape (Brassica napus) and can cause serious losses for crops globally. The disease is caused by dothideomycetous fungus, Leptosphaeria maculans, which is highly virulent/aggressive. Cyclophilins (CYPs) and FK506-binding proteins (FKBPs) are ubiquitous proteins belonging to the peptidyl-prolyl cis/trans isomerase (PPIase) family. They are collectively referred to as immunophilins (IMMs). In the present study, IMM genes, CYP and FKBP in haploid strain v23.1.3 of L. maculans genome, were identified and classified. Twelve CYPs and five FKBPs were determined in total. Domain architecture analysis revealed the presence of a conserved cyclophilin-like domain (CLD) in the case of CYPs and FKBP_C in the case of FKBPs. Interestingly, IMMs in L. maculans also subgrouped into single domain (SD) and multidomain (MD) proteins. They were primarily found to be localized in cytoplasm, nuclei, and mitochondria. Homologous and orthologous gene pairs were also determined by comparison with the model organism Saccharomyces cerevisiae. Remarkably, IMMs of L. maculans contain shorter introns in comparison to exons. Moreover, CYPs, in contrast with FKBPs, contain few exons. However, two CYPs were determined as being intronless. The expression profile of IMMs in both mycelium and infected primary leaves of B. napus demonstrated their potential role during infection. Secondary structure analysis revealed the presence of atypical eight β strands and two α helices fold architecture. Gene ontology analysis of IMMs predicted their significant role in protein folding and PPIase activity. Taken together, our findings for the first time present new prospects of this highly conserved gene family in phytopathogenic fungus.

  6. FipB, an Essential Virulence Factor of Francisella tularensis subsp. tularensis, Has Dual Roles in Disulfide Bond Formation

    PubMed Central

    Qin, Aiping; Zhang, Yan; Clark, Melinda E.; Rabideau, Meaghan M.; Millan Barea, Luis R.

    2014-01-01

    FipB, an essential virulence factor of Francisella tularensis, is a lipoprotein with two conserved domains that have similarity to disulfide bond formation A (DsbA) proteins and the amino-terminal dimerization domain of macrophage infectivity potentiator (Mip) proteins, which are proteins with peptidyl-prolyl cis/trans isomerase activity. This combination of conserved domains is unusual, so we further characterized the enzymatic activity and the importance of the Mip domain and lipid modification in virulence. Unlike typical DsbA proteins, which are oxidases, FipB exhibited both oxidase and isomerase activities. FipA, which also shares similarity with Mip proteins, potentiated the isomerase activity of FipB in an in vitro assay and within the bacteria, as measured by increased copper sensitivity. To determine the importance of the Mip domain and lipid modification of FipB, mutants producing FipB proteins that lacked either the Mip domain or the critical cysteine necessary for lipid modification were constructed. Both strains replicated within host cells and retained virulence in mice, though there was some attenuation. FipB formed surface-exposed dimers that were sensitive to dithiothreitol (DTT), dependent on the Mip domain and on at least one cysteine in the active site of the DsbA-like domain. However, these dimers were not essential for virulence, because the Mip deletion mutant, which failed to form dimers, was still able to replicate intracellularly and retained virulence in mice. Thus, the Mip domains of FipB and FipA impart additional isomerase functionality to FipB, but only the DsbA-like domain and oxidase activity are essential for its critical virulence functions. PMID:25092026

  7. Altered Proteome of Burkholderia pseudomallei Colony Variants Induced by Exposure to Human Lung Epithelial Cells.

    PubMed

    Al-Maleki, Anis Rageh; Mariappan, Vanitha; Vellasamy, Kumutha Malar; Tay, Sun Tee; Vadivelu, Jamuna

    2015-01-01

    Burkholderia pseudomallei primary diagnostic cultures demonstrate colony morphology variation associated with expression of virulence and adaptation proteins. This study aims to examine the ability of B. pseudomallei colony variants (wild type [WT] and small colony variant [SCV]) to survive and replicate intracellularly in A549 cells and to identify the alterations in the protein expression of these variants, post-exposure to the A549 cells. Intracellular survival and cytotoxicity assays were performed followed by proteomics analysis using two-dimensional gel electrophoresis. B. pseudomallei SCV survive longer than the WT. During post-exposure, among 259 and 260 protein spots of SCV and WT, respectively, 19 were differentially expressed. Among SCV post-exposure up-regulated proteins, glyceraldehyde 3-phosphate dehydrogenase, fructose-bisphosphate aldolase (CbbA) and betaine aldehyde dehydrogenase were associated with adhesion and virulence. Among the down-regulated proteins, enolase (Eno) is implicated in adhesion and virulence. Additionally, post-exposure expression profiles of both variants were compared with pre-exposure. In WT pre- vs post-exposure, 36 proteins were differentially expressed. Of the up-regulated proteins, translocator protein, Eno, nucleoside diphosphate kinase (Ndk), ferritin Dps-family DNA binding protein and peptidyl-prolyl cis-trans isomerase B were implicated in invasion and virulence. In SCV pre- vs post-exposure, 27 proteins were differentially expressed. Among the up-regulated proteins, flagellin, Eno, CbbA, Ndk and phenylacetate-coenzyme A ligase have similarly been implicated in adhesion, invasion. Protein profiles differences post-exposure provide insights into association between morphotypic and phenotypic characteristics of colony variants, strengthening the role of B. pseudomallei morphotypes in pathogenesis of melioidosis. PMID:25996927

  8. Dephosphorylation of Photosystem II Reaction Center Proteins in Plant Photosynthetic Membranes as an Immediate Response to Abrupt Elevation of Temperature1

    PubMed Central

    Rokka, Anne; Aro, Eva-Mari; Herrmann, Reinhold G.; Andersson, Bertil; Vener, Alexander V.

    2000-01-01

    Kinetic studies of protein dephosphorylation in photosynthetic thylakoid membranes revealed specifically accelerated dephosphorylation of photosystem II (PSII) core proteins at elevated temperatures. Raising the temperature from 22°C to 42°C resulted in a more than 10-fold increase in the dephosphorylation rates of the PSII reaction center proteins D1 and D2 and of the chlorophyll a binding protein CP43 in isolated spinach (Spinacia oleracea) thylakoids. In contrast the dephosphorylation rates of the light harvesting protein complex and the 9-kD protein of the PSII (PsbH) were accelerated only 2- to 3-fold. The use of a phospho-threonine antibody to measure in vivo phosphorylation levels in spinach leaves revealed a more than 20-fold acceleration in D1, D2, and CP43 dephosphorylation induced by abrupt elevation of temperature, but no increase in light harvesting protein complex dephosphorylation. This rapid dephosphorylation is catalyzed by a PSII-specific, intrinsic membrane protein phosphatase. Phosphatase assays, using intact thylakoids, solubilized membranes, and the isolated enzyme, revealed that the temperature-induced lateral migration of PSII to the stroma-exposed thylakoids only partially contributed to the rapid increase in the dephosphorylation rate. Significant activation of the phosphatase coincided with the temperature-induced release of TLP40 from the membrane into thylakoid lumen. TLP40 is a peptidyl-prolyl cis-trans isomerase, which acts as a regulatory subunit of the membrane phosphatase. Thus dissociation of TLP40 caused by an abrupt elevation in temperature and activation of the membrane protein phosphatase are suggested to trigger accelerated repair of photodamaged PSII and to operate as possible early signals initiating other heat shock responses in chloroplasts. PMID:10938368

  9. SUMO-conjugating enzyme (Sce) and FK506-binding protein (FKBP) encoding rice (Oryza sativa L.) genes: genome-wide analysis, expression studies and evidence for their involvement in abiotic stress response.

    PubMed

    Nigam, Neha; Singh, Amanjot; Sahi, Chandan; Chandramouli, Anupama; Grover, Anil

    2008-04-01

    We report an in-depth characterization of two major stress proteins namely SUMO-conjugating enzyme (Sce) and peptidyl prolyl cis-trans isomerase (PPIase) in rice (Oryza sativa L.). Sce mediates addition of SUMO group to various cell proteins, through process referred to as SUMOylation. Rice nuclear genome has two putative genes encoding the Sce protein (OsSce1 and OsSce2). PCR-amplified full-length OsSce1 cDNA functionally complemented the growth defect in yeast cells lacking the equivalent Ubc9 protein (ScDeltaubc9). RT-PCR analysis showed that transcript levels of OsSce1 and OsSce2 in rice seedlings were regulated by temperature stress. OsSce1 protein was localized to the nucleus in onion epidermal cells as evidenced by the transient GFP expression analysis following micro-projectile gun-based shooting of an OsSce1-GFP fusion construct. PPIase proteins assist molecular chaperones in reactions associated with protein folding and protein transport across membrane. There are 23 putative genes encoding for FK506-binding proteins (FKBPs; specific class of PPIase) in rice genome. OsFKBP20 cDNA was isolated as a stress-inducible EST clone. Largest ORF of 561 bases in OsFKBP20 showed characteristic FK506-binding domain at N-terminus and a coiled-coil motif at C-terminus. RNA expression analysis indicated that OsFKBP20 transcript is heat-inducible. OsFKBP20 over-expression in yeast endowed capacity of high temperature tolerance to yeast cells. Yeast two-hybrid analysis showed that OsSce1 protein physically interacts with the OsFKBP20 protein. It is thus proposed that OsSce1 and OsFKBP20 proteins in concert mediate the stress response of rice plants. PMID:18219493

  10. Novel Cycloheximide Derivatives Targeting the Moonlighting Protein Mip Exhibit Specific Antimicrobial Activity Against Legionella pneumophila

    PubMed Central

    Rasch, Janine; Theuerkorn, Martin; Ünal, Can; Heinsohn, Natascha; Tran, Stefan; Fischer, Gunter; Weiwad, Matthias; Steinert, Michael

    2015-01-01

    Macrophage infectivity potentiator (Mip) and Mip-like proteins are virulence factors in a wide range of pathogens including Legionella pneumophila. These proteins belong to the FK506 binding protein (FKBP) family of peptidyl-prolyl-cis/trans-isomerases (PPIases). In L. pneumophila, the PPIase activity of Mip is required for invasion of macrophages, transmigration through an in vitro lung–epithelial barrier, and full virulence in the guinea pig infection model. Additionally, Mip is a moonlighting protein that binds to collagen IV in the extracellular matrix. Here, we describe the development and synthesis of cycloheximide derivatives with adamantyl moieties as novel FKBP ligands, and analyze their effect on the viability of L. pneumophila and other bacteria. All compounds efficiently inhibited PPIase activity of the prototypic human FKBP12 as well as Mip with IC50-values as low as 180 nM and 1.7 μM, respectively. Five of these derivatives inhibited the growth of L. pneumophila at concentrations of 30–40 μM, but exhibited no effect on other tested bacterial species indicating a specific spectrum of antibacterial activity. The derivatives carrying a 3,5-dimethyladamantan-1-[yl]acetamide substitution (MT_30.32), and a 3-ethyladamantan-1-[yl]acetamide substitution (MT_30.51) had the strongest effects in PPIase- and liquid growth assays. MT_30.32 and MT_30.51 were also inhibitory in macrophage infection studies without being cytotoxic. Accordingly, by applying a combinatorial approach, we were able to generate novel, hybrid inhibitors consisting of cycloheximide and adamantane, two known FKBP inhibitors that interact with different parts of the PPIase domain, respectively. Interestingly, despite the proven Mip-inhibitory activity, the viability of a Mip-deficient strain was affected to the same degree as its wild type. Hence, we also propose that cycloheximide derivatives with adamantyl moieties are potent PPIase inhibitors with multiple targets in L

  11. Genome wide identification of the immunophilin gene family in Leptosphaeria maculans: a causal agent of Blackleg disease in Oilseed Rape (Brassica napus).

    PubMed

    Singh, Khushwant; Zouhar, Miloslav; Mazakova, Jana; Rysanek, Pavel

    2014-10-01

    Abstract Phoma stem canker (blackleg) is a disease of world-wide importance on oilseed rape (Brassica napus) and can cause serious losses for crops globally. The disease is caused by dothideomycetous fungus, Leptosphaeria maculans, which is highly virulent/aggressive. Cyclophilins (CYPs) and FK506-binding proteins (FKBPs) are ubiquitous proteins belonging to the peptidyl-prolyl cis/trans isomerase (PPIase) family. They are collectively referred to as immunophilins (IMMs). In the present study, IMM genes, CYP and FKBP in haploid strain v23.1.3 of L. maculans genome, were identified and classified. Twelve CYPs and five FKBPs were determined in total. Domain architecture analysis revealed the presence of a conserved cyclophilin-like domain (CLD) in the case of CYPs and FKBP_C in the case of FKBPs. Interestingly, IMMs in L. maculans also subgrouped into single domain (SD) and multidomain (MD) proteins. They were primarily found to be localized in cytoplasm, nuclei, and mitochondria. Homologous and orthologous gene pairs were also determined by comparison with the model organism Saccharomyces cerevisiae. Remarkably, IMMs of L. maculans contain shorter introns in comparison to exons. Moreover, CYPs, in contrast with FKBPs, contain few exons. However, two CYPs were determined as being intronless. The expression profile of IMMs in both mycelium and infected primary leaves of B. napus demonstrated their potential role during infection. Secondary structure analysis revealed the presence of atypical eight β strands and two α helices fold architecture. Gene ontology analysis of IMMs predicted their significant role in protein folding and PPIase activity. Taken together, our findings for the first time present new prospects of this highly conserved gene family in phytopathogenic fungus. PMID:25259854

  12. Proteome analysis of the Albugo candida–Brassica juncea pathosystem reveals that the timing of the expression of defence-related genes is a crucial determinant of pathogenesis

    PubMed Central

    Kaur, Parwinder; Jost, Ricarda; Sivasithamparam, Krishnapillai; Barbetti, Martin John

    2011-01-01

    White rust, caused by Albugo candida, is a serious pathogen of Brassica juncea (Indian mustard) and poses a potential hazard to the presently developing canola-quality B. juncea industry worldwide. A comparative proteomic study was undertaken to explore the molecular mechanisms that underlie the defence responses of Brassica juncea to white rust disease caused by the biotrophic oomycete Albugo candida. Nineteen proteins showed reproducible differences in abundance between a susceptible (RH 819) and a resistant variety (CBJ 001) of B. juncea following inoculation with A. candida. The identities of all 19 proteins were successfully established through Q-TOF MS/MS. Five of these proteins were only detected in the resistant variety and showed significant differences in their abundance at various times following pathogen inoculation in comparison to mock-inoculated plants. Among these was a thaumatin-like protein (PR-5), a protein not previously associated with the resistance of B. juncea towards A. candida. One protein, peptidyl-prolyl cis/trans isomerase (PPIase) isoform CYP20-3, was only detected in the susceptible variety and increased in abundance in response to the pathogen. PPIases have recently been discovered to play an important role in pathogenesis by suppressing the host cell's immune response. For a subset of seven proteins examined in more detail, an increase in transcript abundance always preceded their induction at the proteome level. These findings are discussed within the context of the A. candida–Brassica juncea pathosystem, especially in relation to host resistance to this pathogen. PMID:21193577

  13. Studies of the toxicological potential of tripeptides (L-valyl-L-prolyl-L-proline and L-isoleucyl-L-prolyl-L-proline): IX. Evaluation of the mutagenic potential of synthesized L-valyl-L-prolyl-L-proline in the Salmonella-Escherichia coli/microsome, incorporation assay.

    PubMed

    Bernard, Bruce K; Nakamura, Yasunori; Aihara, Kotaro; Mennear, John H

    2005-01-01

    The objective of this study was to assess the mutagenic potential of a synthesized tripeptide, L-valyl-L-prolyl-L-proline (VPP), to induce mutational changes in Salmonella typhimurium LT2 strains TA1535, TA1537, TA98, and TA100, and Escherichia coli strain WP2uvrA in the classical Ames test protocol. Bacteria were exposed to plate concentrations of VPP of 0, 156.2, 312.5, 625, 1250, 2500, and 5,000 microg/plate in distilled water, in the presence and absence of Aroclor 1254-induced rat liver homogenate preparation (S9). Positive-control agents included sodium azide (TA100 and TA1535); 2-aminoanthracene (TA98, TA100, TA1535, TA1537, and WP2uvrA); 9-aminoacridine (TA1537); 2-nitrofluorene (TA98); and N-ethyl-N'-nitro-N-nitrosoguanidine (WP2uvrA) in DMSO. Incubations were conducted at 37 degrees C for about 48 h then revertant colonies were counted. All positive-control agents were consistently and unequivocally positive, but there was no evidence that VPP induced increases in the incidences of revertant colonies in any bacterial strain with and without metabolic activation. These findings were replicated in a second, confirmatory test performed with and without S9. The results of the experiments revealed no treatment-associated changes in the incidence of revertant colonies in any bacterial strain tested. These results support a conclusion that, under the experimental conditions described, there is no evidence that VPP possesses mutagenic potential. PMID:16419582

  14. The prolyl isomerase domain of PpiD from Escherichia coli shows a parvulin fold but is devoid of catalytic activity

    PubMed Central

    Weininger, Ulrich; Jakob, Roman P; Kovermann, Michael; Balbach, Jochen; Schmid, Franz X

    2010-01-01

    PpiD is a periplasmic folding helper protein of Escherichia coli. It consists of an N-terminal helix that anchors PpiD in the inner membrane near the SecYEG translocon, followed by three periplasmic domains. The second domain (residues 264–357) shows homology to parvulin-like prolyl isomerases. This domain is a well folded, stable protein and follows a simple two-state folding mechanism. In its solution structure, as determined by NMR spectroscopy, it resembles most closely the first parvulin domain of the SurA protein, which resides in the periplasm of E. coli as well. A previously reported prolyl isomerase activity of PpiD could not be reproduced when using improved protease-free peptide assays or assays with refolding proteins as substrates. The parvulin domain of PpiD interacts, however, with a proline-containing tetrapeptide, and the binding site, as identified by NMR resonance shift analysis, colocalized with the catalytic sites of other parvulins. In its structure, the parvulin domain of PpiD resembles most closely the inactive first parvulin domain of SurA, which is part of the chaperone unit of this protein and presumably involved in substrate recognition. PMID:19866485

  15. Substrate- and pH-dependent contribution of oxyanion binding site to the catalysis of prolyl oligopeptidase, a paradigm of the serine oligopeptidase family.

    PubMed Central

    Szeltner, Z.; Renner, V.; Polgár, L.

    2000-01-01

    Prolyl oligopeptidase, an enzyme implicated in memory disorders, is a member of a new serine peptidase family. Crystallographic studies (Fülöp et al., 1998) revealed a novel oxyanion binding site containing a tyrosine residue, Tyr473. To study the importance of Tyr473 OH, we have produced prolyl oligopeptidase and its Tyr473Phe variant in Escherichia coli. The specificity rate constant, k(cat)/Km, for the modified enzyme decreased by a factor of 8-40 with highly specific substrates, Z-Gly-Pro-Nap, and a fluorogenic octapeptide. With these compounds, the decline in k(cat) was partly compensated for by reduction in Km, a difference from the extensively studied subtilisin. With the less specific suc-Gly-Pro-Nap, the Km value, which approximates Ks, was not significantly changed, resulting in greater diminution (approximately 500-fold) in k(cat)/Km. The second-order rate constant for the reaction with Z-Pro-prolinal, a slow tight-binding transition-state analogue inhibitor, and the Ki values for a slow substrate and two product-like inhibitors were not significantly affected by the Tyr473 OH group. The mechanism of transition-state stabilization was markedly dependent upon the nature of substrate and varied with pH as the enzyme interconverted between its two catalytically competent forms. PMID:10716187

  16. Hearts of Hypoxia-inducible Factor Prolyl 4-Hydroxylase-2 Hypomorphic Mice Show Protection against Acute Ischemia-Reperfusion Injury*

    PubMed Central

    Hyvärinen, Jaana; Hassinen, Ilmo E.; Sormunen, Raija; Mäki, Joni M.; Kivirikko, Kari I.; Koivunen, Peppi; Myllyharju, Johanna

    2010-01-01

    Hypoxia-inducible factor (HIF) has a pivotal role in oxygen homeostasis and cardioprotection mediated by ischemic preconditioning. Its stability is regulated by HIF prolyl 4-hydroxylases (HIF-P4Hs), the inhibition of which is regarded as a promising strategy for treating diseases such as anemia and ischemia. We generated a viable Hif-p4h-2 hypomorph mouse line (Hif-p4h-2gt/gt) that expresses decreased amounts of wild-type Hif-p4h-2 mRNA: 8% in the heart; 15% in the skeletal muscle; 34–47% in the kidney, spleen, lung, and bladder; 60% in the brain; and 85% in the liver. These mice have no polycythemia and show no signs of the dilated cardiomyopathy or hyperactive angiogenesis observed in mice with broad spectrum conditional Hif-p4h-2 inactivation. We focused here on the effects of chronic Hif-p4h-2 deficiency in the heart. Hif-1 and Hif-2 were stabilized, and the mRNA levels of glucose transporter-1, several enzymes of glycolysis, pyruvate dehydrogenase kinase 1, angiopoietin-2, and adrenomedullin were increased in the Hif-p4h-2gt/gt hearts. When isolated Hif-p4h-2gt/gt hearts were subjected to ischemia-reperfusion, the recovery of mechanical function and coronary flow rate was significantly better than in wild type, while cumulative release of lactate dehydrogenase reflecting the infarct size was reduced. The preischemic amount of lactate was increased, and the ischemic versus preischemic [CrP]/[Cr] and [ATP] remained at higher levels in Hif-p4h-2gt/gt hearts, indicating enhanced glycolysis and an improved cellular energy state. Our data suggest that chronic stabilization of Hif-1α and Hif-2α by genetic knockdown of Hif-p4h-2 promotes cardioprotection by induction of many genes involved in glucose metabolism, cardiac function, and blood pressure. PMID:20185832

  17. Mass spectrometric characterization of a prolyl hydroxylase inhibitor GSK1278863, its bishydroxylated metabolite, and its implementation into routine doping controls.

    PubMed

    Thevis, Mario; Milosovich, Susan; Licea-Perez, Hermes; Knecht, Dana; Cavalier, Tom; Schänzer, Wilhelm

    2016-08-01

    Drug candidates, which have the potential of enhancing athletic performance represent a risk of being misused in elite sport. Therefore, there is a need for early consideration by anti-doping authorities and implementation into sports drug testing programmes. The hypoxia-inducible factor (HIF) or prolyl hydroxylase inhibitor (PHI) GSK1278863 represents an advanced candidate of an emerging class of therapeutics that possess substantial potential for abuse in sport due to their capability to stimulate the biogenesis of erythrocytes and, consequently, the individual's oxygen transport capacity. A thorough characterization of such analytes by technologies predominantly used for doping control purposes and the subsequent implementation of the active drug and/or its main urinary metabolite(s) are vital for comprehensive, preventive, and efficient anti-doping work. In the present study, the HIF PHI drug candidate GSK1278863 (comprising a 6-hydroxypyrimidine-2,4-dione nucleus) and its bishydroxylated metabolite M2 (GSK2391220A) were studied regarding their mass spectrometric behaviour under electrospray ionization (ESI-MS/MS) conditions. Synthesized reference materials were used to elucidate dissociation pathways by means of quadrupole/time-of-flight high resolution/high accuracy tandem mass spectrometry, and their detection from spiked urine and elimination study urine samples under routine doping control conditions was established using liquid chromatography-electrospray ionization-tandem mass spectrometry with direct injection. Dissociation pathways to diagnostic product ions of GSK1278863 (e.g. m/z 291, 223, and 122) were proposed as substantiated by determined elemental compositions and MS(n) experiments as well as comparison to spectra of the bishydroxylated analogue M2. An analytical assay based on direct urine injection using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous determination of GSK1278863 in

  18. Distribution of peptidyl-glycine alpha-amidating monooxygenase immunoreactivity in the brain, pituitary and islet organ of the anglerfish (Lophius americanus).

    PubMed

    McDonald, J K; Klein, K; Noe, B D

    1995-04-01

    Peptidyl-glycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is an enzyme that catalyzes conversion of glycine-extended peptides to alpha-amidated bioactive peptides. Two peptides that are processed at their carboxyl-termini by this enzyme are neuropeptide Y and anglerfish peptide Y, both of which possess a C-terminal glycine that is used as a substrate for amidation. Results from previous reports have demonstrated that neuropeptide Y-like and anglerfish peptide Y-like immunoreactivities are present in the brain of anglerfish (Lophius americanus). Furthermore, neuropeptide Y-like peptides, namely anglerfish peptide Y and anglerfish peptide YG (the homologues of pancreatic polypeptide) are present in the islet organ of this species. Neuropeptide Y has also been localized in the anterior, intermediate and posterior lobes of the pituitary gland in a variety of species. In order to learn more about the distribution of the enzyme responsible for alpha amidation of these peptides in the brain and pituitary and to specifically investigate the relationship of this enzyme to peptide synthesizing endocrine cells of the anglerfish islet, we performed an immunohistochemical study using several antisera generated against different peptide sequences of the enzyme. PAM antisera labeled cells in the islet organ, pituitary and brain, and fibers in the brain and pituitary gland. The PAM staining pattern in the brain was remarkably similar to the distribution of neuropeptide Y immunoreactivity reported previously. Clusters of cells adjacent to vessels in the anterior pituitary displayed punctate PAM immunoreactivity while varicose fibers were observed in the pituitary stalk and neurohypophysis. Endocrine cells of the islet organ were differentially labeled with different PAM antisera. Comparison of the staining patterns of insulin, glucagon, and anglerfish peptide Y in the islet organ to PAM immunoreactivity suggests a distribution of forms of PAM enzyme in insulin and

  19. Enzyme-catalyzed formation of beta-peptides: beta-peptidyl aminopeptidases BapA and DmpA acting as beta-peptide-synthesizing enzymes.

    PubMed

    Heck, Tobias; Kohler, Hans-Peter E; Limbach, Michael; Flögel, Oliver; Seebach, Dieter; Geueke, Birgit

    2007-09-01

    In recent studies, we discovered that the three beta-peptidyl aminopeptidases, BapA from Sphingosinicella xenopeptidilytica 3-2W4, BapA from S. microcystinivorans Y2, and DmpA from Ochrobactrum anthropi LMG7991, possess the unique feature of cleaving N-terminal beta-amino acid residues from beta- and alpha/beta-peptides. Herein, we investigated the use of the same three enzymes for the reverse reaction catalyzing the oligomerization of beta-amino acids and the synthesis of mixed peptides with N-terminal beta-amino acid residues. As substrates, we employed the beta-homoamino acid derivatives H-beta hGly-pNA, H-beta3 hAla-pNA, H-(R)-beta3 hAla-pNA, H-beta3 hPhe-pNA, H-(R)-beta3 hPhe-pNA, and H-beta3 hLeu-pNA. All three enzymes were capable of coupling the six beta-amino acids to oligomers with chain lengths of up to eight amino acid residues. With the enzyme DmpA as the catalyst, we observed very high conversion rates, which correspond to dimer yields of up to 76%. The beta-dipeptide H-beta3 hAla-beta3 hLeu-OH and the beta/alpha-dipeptide H-beta hGly-His-OH (carnosine) were formed with almost 50% conversion, when a five-fold excess of beta3-homoleucine or histidine was incubated with H-beta3 hAla-pNA and H-beta hGly-pNA, respectively, in the presence of the enzyme BapA from S. microcystinivorans Y2. BapA from S. xenopeptidilytica 3-2W4 turned out to be a versatile catalyst capable of coupling various beta-amino acid residues to the free N-termini of beta- and alpha-amino acids and even to an alpha-tripeptide. Thus, these aminopeptidases might be useful to introduce a beta-amino acid residue as an N-terminal protecting group into a 'natural' alpha-peptide, thereby stabilizing the peptide against degradation by other proteolytic enzymes. PMID:17886858

  20. Enzyme-catalyzed formation of beta-peptides: beta-peptidyl aminopeptidases BapA and DmpA acting as beta-peptide-synthesizing enzymes.

    PubMed

    Heck, Tobias; Kohler, Hans-Peter E; Limbach, Michael; Flögel, Oliver; Seebach, Dieter; Geueke, Birgit

    2007-09-01

    In recent studies, we discovered that the three beta-peptidyl aminopeptidases, BapA from Sphingosinicella xenopeptidilytica 3-2W4, BapA from S. microcystinivorans Y2, and DmpA from Ochrobactrum anthropi LMG7991, possess the unique feature of cleaving N-terminal beta-amino acid residues from beta- and alpha/beta-peptides. Herein, we investigated the use of the same three enzymes for the reverse reaction catalyzing the oligomerization of beta-amino acids and the synthesis of mixed peptides with N-terminal beta-amino acid residues. As substrates, we employed the beta-homoamino acid derivatives H-beta hGly-pNA, H-beta3 hAla-pNA, H-(R)-beta3 hAla-pNA, H-beta3 hPhe-pNA, H-(R)-beta3 hPhe-pNA, and H-beta3 hLeu-pNA. All three enzymes were capable of coupling the six beta-amino acids to oligomers with chain lengths of up to eight amino acid residues. With the enzyme DmpA as the catalyst, we observed very high conversion rates, which correspond to dimer yields of up to 76%. The beta-dipeptide H-beta3 hAla-beta3 hLeu-OH and the beta/alpha-dipeptide H-beta hGly-His-OH (carnosine) were formed with almost 50% conversion, when a five-fold excess of beta3-homoleucine or histidine was incubated with H-beta3 hAla-pNA and H-beta hGly-pNA, respectively, in the presence of the enzyme BapA from S. microcystinivorans Y2. BapA from S. xenopeptidilytica 3-2W4 turned out to be a versatile catalyst capable of coupling various beta-amino acid residues to the free N-termini of beta- and alpha-amino acids and even to an alpha-tripeptide. Thus, these aminopeptidases might be useful to introduce a beta-amino acid residue as an N-terminal protecting group into a 'natural' alpha-peptide, thereby stabilizing the peptide against degradation by other proteolytic enzymes.

  1. Isolated erythrocytosis: study of 67 patients and identification of three novel germ-line mutations in the prolyl hydroxylase domain protein 2 (PHD2) gene

    PubMed Central

    Albiero, Elena; Ruggeri, Marco; Fortuna, Stefania; Finotto, Silvia; Bernardi, Martina; Madeo, Domenico; Rodeghiero, Francesco

    2012-01-01

    The oxygen sensing pathway modulates erythropoietin expression. In normal cells, intracellular oxygen tensions are directly sensed by prolyl hydroxylase domain (PHD)-containing proteins. PHD2 isozyme has a key role in tagging hypoxia-inducible factor (HIF)-α subunits for polyubiquitination and proteasomal degradation. Erythrocytosis-associated PHD2 mutations reduce hydroxylation of HIF-α. The investigation of 67 patients with isolated erythrocytosis, either sporadic or familial, allowed the identification of three novel mutations in the catalytic domain of the PHD2 protein. All new mutations are germ-line, heterozygous and missense, and code for a predicted full length mutant PHD2 protein. Identification of the disease-causing genes will be of critical importance for a better classification of familial and acquired erythrocytosis, offering additional insight into the erythropoietin regulating oxygen sensing pathway. PMID:21828119

  2. Potent and Selective Triazole-Based Inhibitors of the Hypoxia-Inducible Factor Prolyl-Hydroxylases with Activity in the Murine Brain

    PubMed Central

    Chan, Mun Chiang; Atasoylu, Onur; Hodson, Emma; Tumber, Anthony; Leung, Ivanhoe K. H.; Chowdhury, Rasheduzzaman; Gómez-Pérez, Verónica; Demetriades, Marina; Rydzik, Anna M.; Holt-Martyn, James; Tian, Ya-Min; Bishop, Tammie; Claridge, Timothy D. W.; Kawamura, Akane; Pugh, Christopher W.; Ratcliffe, Peter J.; Schofield, Christopher J.

    2015-01-01

    As part of the cellular adaptation to limiting oxygen availability in animals, the expression of a large set of genes is activated by the upregulation of the hypoxia-inducible transcription factors (HIFs). Therapeutic activation of the natural human hypoxic response can be achieved by the inhibition of the hypoxia sensors for the HIF system, i.e. the HIF prolyl-hydroxylases (PHDs). Here, we report studies on tricyclic triazole-containing compounds as potent and selective PHD inhibitors which compete with the 2-oxoglutarate co-substrate. One compound (IOX4) induces HIFα in cells and in wildtype mice with marked induction in the brain tissue, revealing that it is useful for studies aimed at validating the upregulation of HIF for treatment of cerebral diseases including stroke. PMID:26147748

  3. Inhibitory efficacy of cyclo(L-leucyl-L-prolyl) from mangrove rhizosphere bacterium-Bacillus amyloliquefaciens (MMS-50) toward cariogenic properties of Streptococcus mutans.

    PubMed

    Gowrishankar, Shanmugaraj; Poornima, Balan; Pandian, Shunmugiah Karutha

    2014-05-01

    Since Streptococcus mutans is the principal etiologic agent causing dental caries, by encompassing an array of unique virulence traits, emerging treatment strategies that specifically target the virulence of this pathogen may be promising as alternative approaches compared to conventional antibiotic therapy. In this perspective, we investigated chloroform extract of cell-free culture supernatant from mangrove rhizosphere bacterium Bacillus amyloliquefaciens (MMS-50) in terms of anticariogenic properties of S. mutans, without suppressing its viability. Crude chloroform extract of MMS-50 was subjected to column and high performance liquid chromatographic techniques to obtain the active fraction (AF), and MMS-50 AF was used for all further assays. GC-MS and FT-IR were carried out to identify the major components present in MMS-50 AF. Comparative gene expression analysis of some biofilm-forming and virulence genes (vicR, comDE, gtfC, and gbpB) was done by real-time PCR. Cyclo(L-leucyl-L-prolyl) was found to be the chief compound in MMS-50 AF responsible for bioactivity. The minimum and maximum inhibitory concentrations of MMS-50 AF against S. mutans were found to be 100 and 250 μg/mL, respectively. Anti-virulence assays performed using below-sub-MIC levels of MMS-50 AF (30 μg/mL) resulted in significant reduction in adherence (68%), acid production, acid tolerance, glucan synthesis (32%), biofilm formation (53.5%) and cell surface hydrophobicity, all devoid of affecting its viability. The micrographs of CLSM and SEM further confirmed the antibiofilm and anti-virulence efficacies of MMS-50 AF. Expression data showed significant reduction in expression of all studied virulence genes. Thus, the current study unveils the anticariogenic potential of cyclo(L-leucyl-L-prolyl) from B. amyloliquefaciens, as well as its suitability as a novel and alternative anticariogenic agent against dental caries.

  4. Inhibitory efficacy of cyclo(L-leucyl-L-prolyl) from mangrove rhizosphere bacterium-Bacillus amyloliquefaciens (MMS-50) toward cariogenic properties of Streptococcus mutans.

    PubMed

    Gowrishankar, Shanmugaraj; Poornima, Balan; Pandian, Shunmugiah Karutha

    2014-05-01

    Since Streptococcus mutans is the principal etiologic agent causing dental caries, by encompassing an array of unique virulence traits, emerging treatment strategies that specifically target the virulence of this pathogen may be promising as alternative approaches compared to conventional antibiotic therapy. In this perspective, we investigated chloroform extract of cell-free culture supernatant from mangrove rhizosphere bacterium Bacillus amyloliquefaciens (MMS-50) in terms of anticariogenic properties of S. mutans, without suppressing its viability. Crude chloroform extract of MMS-50 was subjected to column and high performance liquid chromatographic techniques to obtain the active fraction (AF), and MMS-50 AF was used for all further assays. GC-MS and FT-IR were carried out to identify the major components present in MMS-50 AF. Comparative gene expression analysis of some biofilm-forming and virulence genes (vicR, comDE, gtfC, and gbpB) was done by real-time PCR. Cyclo(L-leucyl-L-prolyl) was found to be the chief compound in MMS-50 AF responsible for bioactivity. The minimum and maximum inhibitory concentrations of MMS-50 AF against S. mutans were found to be 100 and 250 μg/mL, respectively. Anti-virulence assays performed using below-sub-MIC levels of MMS-50 AF (30 μg/mL) resulted in significant reduction in adherence (68%), acid production, acid tolerance, glucan synthesis (32%), biofilm formation (53.5%) and cell surface hydrophobicity, all devoid of affecting its viability. The micrographs of CLSM and SEM further confirmed the antibiofilm and anti-virulence efficacies of MMS-50 AF. Expression data showed significant reduction in expression of all studied virulence genes. Thus, the current study unveils the anticariogenic potential of cyclo(L-leucyl-L-prolyl) from B. amyloliquefaciens, as well as its suitability as a novel and alternative anticariogenic agent against dental caries. PMID:24698790

  5. The Skp1 protein from Toxoplasma is modified by a cytoplasmic prolyl 4-hydroxylase associated with oxygen sensing in the social amoeba Dictyostelium.

    PubMed

    Xu, Yuechi; Brown, Kevin M; Wang, Zhuo A; van der Wel, Hanke; Teygong, Crystal; Zhang, Dongmei; Blader, Ira J; West, Christopher M

    2012-07-20

    In diverse types of organisms, cellular hypoxic responses are mediated by prolyl 4-hydroxylases that use O(2) and α-ketoglutarate as substrates to hydroxylate conserved proline residues in target proteins. Whereas in metazoans these enzymes control the stability of the HIFα family of transcription factor subunits, the Dictyostelium enzyme (DdPhyA) contributes to O(2) regulation of development by a divergent mechanism involving hydroxylation and subsequent glycosylation of DdSkp1, an adaptor subunit in E3(SCF) ubiquitin ligases. Sequences related to DdPhyA, DdSkp1, and the glycosyltransferases that cap Skp1 hydroxyproline occur also in the genomes of Toxoplasma and other protists, suggesting that this O(2) sensing mechanism may be widespread. Here we show by disruption of the TgphyA locus that this enzyme is required for Skp1 glycosylation in Toxoplasma and that disrupted parasites grow slowly at physiological O(2) levels. Conservation of cellular function was tested by expression of TgPhyA in DdphyA-null cells. Simple gene replacement did not rescue Skp1 glycosylation, whereas overexpression not only corrected Skp1 modification but also restored the O(2) requirement to a level comparable to that of overexpressed DdPhyA. Bacterially expressed TgPhyA protein can prolyl hydroxylate both Toxoplasma and Dictyostelium Skp1s. Kinetic analyses showed that TgPhyA has similar properties to DdPhyA, including a superimposable dependence on the concentration of its co-substrate α-ketoglutarate. Remarkably, however, TgPhyA had a significantly higher apparent affinity for O(2). The findings suggest that Skp1 hydroxylation by PhyA is a conserved process among protists and that this biochemical pathway may indirectly sense O(2) by detecting the levels of O(2)-regulated metabolites such as α-ketoglutarate.

  6. Changes in the Chondrocyte and Extracellular Matrix Proteome during Post-natal Mouse Cartilage Development*

    PubMed Central

    Wilson, Richard; Norris, Emma L.; Brachvogel, Bent; Angelucci, Constanza; Zivkovic, Snezana; Gordon, Lavinia; Bernardo, Bianca C.; Stermann, Jacek; Sekiguchi, Kiyotoshi; Gorman, Jeffrey J.; Bateman, John F.

    2012-01-01

    cartilage development. Although the multifunctional chaperone BiP was not differentially expressed, enzymes and chaperones required specifically for collagen biosynthesis, such as the prolyl 3-hydroxylase 1, cartilage-associated protein, and peptidyl prolyl cis-trans isomerase B complex, were down-regulated during maturation. Conversely, the lumenal proteins calumenin, reticulocalbin-1, and reticulocalbin-2 were significantly increased, signifying a shift toward calcium binding functions. This first proteomic analysis of cartilage development in vivo reveals the breadth of protein expression changes during chondrocyte maturation and ECM remodeling in the mouse femoral head. PMID:21989018

  7. HIF-1α is required for hematopoietic stem cell mobilization and 4-prolyl hydroxylase inhibitors enhance mobilization by stabilizing HIF-1α.

    PubMed

    Forristal, C E; Nowlan, B; Jacobsen, R N; Barbier, V; Walkinshaw, G; Walkley, C R; Winkler, I G; Levesque, J P

    2015-06-01

    Many patients with hematological neoplasms fail to mobilize sufficient numbers of hematopoietic stem cells (HSCs) in response to granulocyte colony-stimulating factor (G-CSF) precluding subsequent autologous HSC transplantation. Plerixafor, a specific antagonist of the chemokine receptor CXCR4, can rescue some but not all patients who failed to mobilize with G-CSF alone. These refractory poor mobilizers cannot currently benefit from autologous transplantation. To discover alternative targetable pathways to enhance HSC mobilization, we studied the role of hypoxia-inducible factor-1α (HIF-1α) and the effect of HIF-1α pharmacological stabilization on HSC mobilization in mice. We demonstrate in mice with HSC-specific conditional deletion of the Hif1a gene that the oxygen-labile transcription factor HIF-1α is essential for HSC mobilization in response to G-CSF and Plerixafor. Conversely, pharmacological stabilization of HIF-1α with the 4-prolyl hydroxylase inhibitor FG-4497 synergizes with G-CSF and Plerixafor increasing mobilization of reconstituting HSCs 20-fold compared with G-CSF plus Plerixafor, currently the most potent mobilizing combination used in the clinic.

  8. HIF-1α is required for hematopoietic stem cell mobilization and 4-prolyl hydroxylase inhibitors enhance mobilization by stabilizing HIF-1α

    PubMed Central

    Forristal, C E; Nowlan, B; Jacobsen, R N; Barbier, V; Walkinshaw, G; Walkley, C R; Winkler, I G; Levesque, J P

    2015-01-01

    Many patients with hematological neoplasms fail to mobilize sufficient numbers of hematopoietic stem cells (HSCs) in response to granulocyte colony-stimulating factor (G-CSF) precluding subsequent autologous HSC transplantation. Plerixafor, a specific antagonist of the chemokine receptor CXCR4, can rescue some but not all patients who failed to mobilize with G-CSF alone. These refractory poor mobilizers cannot currently benefit from autologous transplantation. To discover alternative targetable pathways to enhance HSC mobilization, we studied the role of hypoxia-inducible factor-1α (HIF-1α) and the effect of HIF-1α pharmacological stabilization on HSC mobilization in mice. We demonstrate in mice with HSC-specific conditional deletion of the Hif1a gene that the oxygen-labile transcription factor HIF-1α is essential for HSC mobilization in response to G-CSF and Plerixafor. Conversely, pharmacological stabilization of HIF-1α with the 4-prolyl hydroxylase inhibitor FG-4497 synergizes with G-CSF and Plerixafor increasing mobilization of reconstituting HSCs 20-fold compared with G-CSF plus Plerixafor, currently the most potent mobilizing combination used in the clinic. PMID:25578474

  9. Transport of a Prolyl Endopeptidase Inhibitory Peptide across the Blood-Brain Barrier Demonstrated Using the hCMEC/D3 Cell Line Transcytosis Assay.

    PubMed

    Hayes, Maria; Moen, Lars Fredrik; Auty, Mark A E; Lea, Tor Erling

    2016-01-13

    The blood-brain barrier (BBB) remains a significant hurdle for treatment of central nervous system (CNS) and mental health disorders. A prolyl endopeptidase (PEP) inhibitory peptide with the amino acid sequence proline-proline-leucine (PPL) was chemically synthesized labeled with 5-FAM and assessed using a transcytosis assay for its ability to cross the BBB. Transport of this peptide across the BBB was determined using an in vitro model of the human BBB, which utilizes the human cerebral microvascular endothelial cell line (hCMEC/D3). Uptake and transport of 5-FAM-PPL across the hCMEC/D3 cell model was determined using confocal microscopy and mass spectrometry. This is an important parameter in determining whether peptides may reach the target organ (i.e., the brain and central nervous system).This work assessed, for the first time, the ability of a food-derived PEP inhibitory peptide to cross the BBB without the use of animal models.

  10. Overexpression of Prolyl-4-Hydroxylase-α1 Stabilizes but Increases Shear Stress-Induced Atherosclerotic Plaque in Apolipoprotein E-Deficient Mice

    PubMed Central

    Liu, Xin-xin; Li, Meng-meng; Zhang, Yu; Chen, Liang; Wang, Lin; Di, Ming-xue

    2016-01-01

    The rupture and erosion of atherosclerotic plaque can induce coronary thrombosis. Prolyl-4-hydroxylase (P4H) plays a central role in the synthesis of all known types of collagens, which are the most abundant constituent of the extracellular matrix in atherosclerotic plaque. The pathogenesis of atherosclerosis is thought to be in part caused by shear stress. In this study, we aimed to investigate a relationship between P4Hα1 and shear stress-induced atherosclerotic plaque. Carotid arteries of ApoE−/− mice were exposed to low and oscillatory shear stress conditions by the placement of a shear stress cast for 2 weeks; we divided 60 male ApoE−/− mice into three groups for treatments with saline (mock) (n = 20), empty lentivirus (lenti-EGFP) (n = 20), and lentivirus-P4Hα1 (lenti-P4Hα1) (n = 20). Our results reveal that after 2 weeks of lenti-P4Hα1 treatment both low and oscillatory shear stress-induced plaques increased collagen and the thickness of fibrous cap and decreased macrophage accumulation but no change in lipid accumulation. We also observed that overexpression of P4Ha1 increased plaque size. Our study suggests that P4Hα1 overexpression might be a potential therapeutic target in stabilizing vulnerable plaques.

  11. Pharmacokinetics, pharmacodynamics and safety of single, oral doses of GSK1278863, a novel HIF-prolyl hydroxylase inhibitor, in healthy Japanese and Caucasian subjects.

    PubMed

    Hara, Katsutoshi; Takahashi, Naoki; Wakamatsu, Akira; Caltabiano, Stephen

    2015-12-01

    This study was performed to evaluate the pharmacokinetics (PK), pharmacodynamics (PD) and safety of GSK1278863, a novel prolyl hydroxylase inhibitor, following a single oral administration of GSK1278863 from 10 to 100 mg or placebo in Japanese (n = 19), and 10, 25 and 100 mg in Caucasians (n = 14). Dose-proportional increases were observed in AUCinf of GSK1278863 in both ethnic groups, with a 1.3-1.5-fold higher exposure seen in Japanese relative to Caucasians for all doses. This difference in exposure can be mainly explained by the observed differences in body weights between the two groups. Statistically significant increases in erythropoietin (EPO), vascular endothelial growth factor (VEGF) and reticulocyte counts were observed in Japanese subjects after the 50 and 100 mg dose as compared to placebo. In Caucasians, similar to Japanese, EPO and VEGF levels were observed to be increased in response to the 100 mg dose. Drug-related adverse events, including headache and abdominal pain were reported in 3 Japanese subjects, while headache was reported in 3 Caucasians. In conclusion, GSK1278863 was well tolerated, with dose-proportional increases in exposure observed in both groups. There was no evidence of ethnic differences between Japanese and Caucasian with regard to PK or PD.

  12. Chiral separation of new cathinone- and amphetamine-related designer drugs by gas chromatography-mass spectrometry using trifluoroacetyl-l-prolyl chloride as chiral derivatization reagent.

    PubMed

    Mohr, Stefan; Weiß, Jennifer A; Spreitz, Josef; Schmid, Martin G

    2012-12-21

    Since cathinone derivatives gained high popularity on the recreational drugs market within the past 5 years the development of analytical methods for the achiral and chiral determination of this substance class is of great interest. Not at least because it is obvious that the pharmacological potency differs between both enantiomers. Cathinones are structurally closely related to amphetamines, which have similar stimulating effects and are somehow better investigated. The goal of this research was to perform indirect enantioseparation of novel psychoactive cathinone and amphetamine derivatives. Trifluoroacetyl-l-prolyl chloride was served as chiral derivatization agent (CDA). Chromatographic separation was performed using a commercially available HP5-MS capillary column with a length of 30 m. Helium was used as carrier gas with a constant flow of 1.0 ml/min. Under optimum conditions 14 amphetamine derivatives were successfully resolved into their enantiomers and detected with the single quadrupol detector. Racemic methcathinone derivatives analyzed with the same method showed different peak areas for each of the produced diastereomeric isomers, even if they are structurally closely related to the amphetamines. Derivatization experiments with the single isomers of methcathinone led to both diastereomers whereas the S(-) enantiomer seemed to racemize more likely. Based on comparative experiments with R-(-)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (MTPA) as CDA, racemization due to the keto-enol-tautomerism of the cathinone derivatives seemed to be responsible for this phenomenon. Nevertheless, 18 cathinone derivatives were successfully enantioseparated and an approach of quantitative evaluation is demonstrated.

  13. Indirect chiral separation of new recreational drugs by gas chromatography-mass spectrometry using trifluoroacetyl-L-prolyl chloride as chiral derivatization reagent.

    PubMed

    Weiß, Jennifer A; Mohr, Stefan; Schmid, Martin G

    2015-03-01

    New recreational drugs such as amphetamine-, cathinone, and benzofury derivatives gained high popularity on the drug market in recent years. They can be purchased via the Internet from different providers and online portals. Most of these compounds are chiral, which makes the development of chiral separation methods necessary. Besides this, it is useful to find out if the compounds were sold as racemic mixtures. Also, it is important to check whether the new psychoactive compounds contain further ingredients or impurities. The aim of this research was the continuation of the application of a method for indirect chiral separation of 24 new psychoactive compounds recently purchased via the Internet. After derivatization with the chiral derivatization reagent trifluoroacetyl-L-prolyl chloride, chromatographic separation of diastereomers was achieved using a 30 m HP5-MS capillary column. As carrier gas, helium was used with a constant flow of 1.0 ml/min. Three different column temperature programs were tested. Under optimum conditions 13 out of 24 compounds were successfully resolved into their enantiomers obtaining Rs values up to 7.0. The use of a single quadrupole mass spectrometer as the detector allowed the identification of the compounds in multicomponent samples.

  14. Lactoferrin from bovine colostrum regulates prolyl hydroxylase 2 activity and prevents prion protein-mediated neuronal cell damage via cellular prion protein.

    PubMed

    Park, Y-G; Moon, J-H; Park, S-Y

    2014-08-22

    Prion disorders are associated with the conversion of normal cellular prion protein (PrPc) to the abnormal scrapie isoform of prion protein (PrPsc). Recent studies have shown that expression of normal PrPc is regulated by hypoxia-inducible factor-1 alpha (HIF-1α), and that lactoferrin increases full-length PrPc on the cell surface. Lactoferrin is an 80-kDa iron-binding glycoprotein with various biological activities, including iron-chelating ability. HIF-1α and the associated ubiquitin-proteasome pathway are regulated by HIF prolyl-hydroxylases 2 (PHD2). We hypothesized that lactoferrin regulates PHD2 expression and enzymatic activity, and the PHD2 regulation promotes HIF-1α stability and prevention of neuronal cell death mediated by prion protein (PrP) residues (106-126). Lactoferrin prevented PrP (106-126)-induced neurotoxicity by the induction of PrPc expression via promoting HIF-1α stability in neuronal cells. Our results demonstrated that lactoferrin prevented PrP (106-126)-induced neurotoxicity via the up-regulation of HIF-1α stability determined by PHD2 expression and enzymatic activity. These findings suggest that possible therapies such as PHD2 inhibition, or promotion of lactoferrin secretion, may have clinical benefits in neurodegenerative diseases, including prion disease. PMID:24875174

  15. Genetic interactions between the ESS1 prolyl-isomerase and the RSP5 ubiquitin ligase reveal opposing effects on RNA polymerase II function.

    PubMed

    Wu, X; Chang, A; Sudol, M; Hanes, S D

    2001-12-01

    Transcription of protein-coding genes by RNA polymerase II (pol II) is a highly coordinated process that requires the stepwise association of distinct protein complexes with the C-terminal domain (CTD) of Rpbl, the largest subunit of RNA pol II. Interaction of these complexes with the CTD might be subject to regulation by proteins such as Ess1 and Rsp5. Ess1, a prolyl-isomerase, binds the CTD and is thought to play a positive role in pol II transcription by generating conformational isomers of the CTD. Rsp5, a ubiquitin ligase, binds the CTD and is thought to play a negative role in transcription by mediating Rpbl ubiquitination and degradation. In this paper, we demonstrate that ESS1 and RSP5 interact genetically and that these interactions occur via RPBI. We show that over-expression of RSP5 enhances the growth defect of ess1ts cells and this effect is reversed by introducing extra copies of RPB1. Over-expression of RSP5 also mimics the sensitivity of ess1ts mutant cells to the toxicity of plasmids carrying dominant-negative CTD mutations, whereas mutations in RSP5 suppress this effect. Using a modified two-hybrid assay, we also demonstrate that Essl and Rsp5 compete directly for binding to the CTD. The results suggest a model in which Essl and Rsp5 act opposingly on pol II function to control the level of pol II available for transcription.

  16. Pichia pastoris production of a prolyl 4-hydroxylase derived from Chondrosia reniformis sponge: A new biotechnological tool for the recombinant production of marine collagen.

    PubMed

    Pozzolini, Marina; Scarfì, Sonia; Mussino, Francesca; Salis, Annalisa; Damonte, Gianluca; Benatti, Umberto; Giovine, Marco

    2015-08-20

    Prolyl 4-hydroxylase (P4H) is a α2β2 tetramer catalyzing the post-translational hydroxylation of prolines in collagen. Its recombinant production is mainly pursued to realize biotechnological tools able to generate animal contaminant-free hydroxylated collagen. One promising candidate for biomedical applications is the collagen extracted from the marine sponge Chondrosia reniformis, because of its biocompatibility and because is devoid of the health risks associated with bovine and porcine collagens. Here we report on the production and selection, by enzymatic and biomolecular analyses, of a triple transformed Pichia pastoris strain expressing a stable P4H tetramer derived from C. reniformis sponge and a hydroxylated non fibrillar procollagen polypeptide from the same animal. The percentage of recombinant procollagen hydroxylated prolines inside the transformed yeast was of 36.3% analyzed by mass spectrometry indicating that the recombinant enzyme is active on its natural substrate inside the yeast cell host. Furthermore, the recombinant sponge P4H has the ability to hydroxylate its natural substrate in both X and Y positions in the Xaa-Yaa-Gly collagenous triplets. In conclusion this Pichia system seems ideal for high-level production of hydroxylated sponge- or marine-derived collagen polypeptides as well as of conotoxins or other marine proteins of high pharmacological interest needing this particular post-translational modification.

  17. Enhancement of ACE and prolyl oligopeptidase inhibitory potency of protein hydrolysates from sardine and tuna by-products by simulated gastrointestinal digestion.

    PubMed

    Martínez-Alvarez, Oscar; Batista, Irineu; Ramos, Cristina; Montero, Pilar

    2016-04-01

    This work was focused on the study of the bioactive potential of three fish protein hydrolysates, one of them prepared from industrial sardine by-products (head and viscera) and the others from tuna by-products (head, and muscle and viscera). These protein hydrolysates exhibited moderate ability to inhibit Angiotensin Converting Enzyme or ACE (IC50 between 0.24-1.16 mg dry weight per ml) and prolyl oligopeptidase or PO (IC50 between 3.30-9.57 mg ml(-1)), those obtained from tuna by-products being the most effective. Overall, ACE- and PO-inhibiting activities were enhanced by sequential nanofiltration through 3 and 1 kDa MWCO membranes (IC50 between 0.02-0.16 mg ml(-1) (ACE) and 1.10-4.21 mg ml(-1) (PO)). The inhibitory properties of the hydrolysates were greatly improved by in vitro gastric digestion, and were barely affected by further intestinal digestion. The digested tuna hydrolysates, mainly that from heads, proved to be the best source of PO- and ACE- inhibiting molecules (IC50 = 0.16 mg ml(-1) (ACE) and 1.04 mg ml(-1) (PO)) and could be potential new ingredients in food with interest in the prevention or treatment of cardiovascular and neurological diseases. PMID:27045751

  18. Alternative splicing transcription of Megalobrama amblycephala HIF prolyl hydroxylase PHD3 and up-regulation of PHD3 by HIF-1α.

    PubMed

    Chen, Nan; Huang, Cui-Hong; Chen, Bo-Xiang; Liu, Hong; Wang, Wei-Min; Gul, Yasmeen; Wang, Huan-Ling

    2016-01-15

    PHD3 is a hydroxylase that hydroxylates prolyl residues on hypoxia-inducible factors (HIFs) in mammals. In this study, the full-length cDNA and promoter sequences of Megalobrama amblycephala PHD3 gene were isolated by a modified RACE method. PHD3 cDNA was 1622 bp in length, including an ORF of 717 bp encoding 238 amino acid residues. The semi-quantitative PCR results suggested that PHD3 was highly expressed in liver in the normal condition, while after hypoxia treatment this gene was significantly increased in all analyzed tissues. PHD3 was detected only in the initial stages of M. amblycephala embryo development. In addition, the presence of another alternatively processed PHD3 transcript, designated PHD3Δ1 was observed in the process of analyzing the expression of PHD3. Both PHD3 and PHD3Δ1 were up-regulated under hypoxia, and had five the hypoxia response elements (HREs) by in silico scanning on the promoter. Further luciferase assay indicated that all HREs significantly responded to hypoxia. Taken together, these results suggest that PHD3 plays important roles in hypoxia response and early embryo development of M. amblycephala. PMID:26697748

  19. Therapeutic targeting of oxygen-sensing prolyl hydroxylases abrogates ATF4-dependent neuronal death and improves outcomes after brain hemorrhage in several rodent models.

    PubMed

    Karuppagounder, Saravanan S; Alim, Ishraq; Khim, Soah J; Bourassa, Megan W; Sleiman, Sama F; John, Roseleen; Thinnes, Cyrille C; Yeh, Tzu-Lan; Demetriades, Marina; Neitemeier, Sandra; Cruz, Dana; Gazaryan, Irina; Killilea, David W; Morgenstern, Lewis; Xi, Guohua; Keep, Richard F; Schallert, Timothy; Tappero, Ryan V; Zhong, Jian; Cho, Sunghee; Maxfield, Frederick R; Holman, Theodore R; Culmsee, Carsten; Fong, Guo-Hua; Su, Yijing; Ming, Guo-li; Song, Hongjun; Cave, John W; Schofield, Christopher J; Colbourne, Frederick; Coppola, Giovanni; Ratan, Rajiv R

    2016-03-01

    Disability or death due to intracerebral hemorrhage (ICH) is attributed to blood lysis, liberation of iron, and consequent oxidative stress. Iron chelators bind to free iron and prevent neuronal death induced by oxidative stress and disability due to ICH, but the mechanisms for this effect remain unclear. We show that the hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) family of iron-dependent, oxygen-sensing enzymes are effectors of iron chelation. Molecular reduction of the three HIF-PHD enzyme isoforms in the mouse striatum improved functional recovery after ICH. A low-molecular-weight hydroxyquinoline inhibitor of the HIF-PHD enzymes, adaptaquin, reduced neuronal death and behavioral deficits after ICH in several rodent models without affecting total iron or zinc distribution in the brain. Unexpectedly, protection from oxidative death in vitro or from ICH in vivo by adaptaquin was associated with suppression of activity of the prodeath factor ATF4 rather than activation of an HIF-dependent prosurvival pathway. Together, these findings demonstrate that brain-specific inactivation of the HIF-PHD metalloenzymes with the blood-brain barrier-permeable inhibitor adaptaquin can improve functional outcomes after ICH in several rodent models.

  20. Pichia pastoris production of a prolyl 4-hydroxylase derived from Chondrosia reniformis sponge: A new biotechnological tool for the recombinant production of marine collagen.

    PubMed

    Pozzolini, Marina; Scarfì, Sonia; Mussino, Francesca; Salis, Annalisa; Damonte, Gianluca; Benatti, Umberto; Giovine, Marco

    2015-08-20

    Prolyl 4-hydroxylase (P4H) is a α2β2 tetramer catalyzing the post-translational hydroxylation of prolines in collagen. Its recombinant production is mainly pursued to realize biotechnological tools able to generate animal contaminant-free hydroxylated collagen. One promising candidate for biomedical applications is the collagen extracted from the marine sponge Chondrosia reniformis, because of its biocompatibility and because is devoid of the health risks associated with bovine and porcine collagens. Here we report on the production and selection, by enzymatic and biomolecular analyses, of a triple transformed Pichia pastoris strain expressing a stable P4H tetramer derived from C. reniformis sponge and a hydroxylated non fibrillar procollagen polypeptide from the same animal. The percentage of recombinant procollagen hydroxylated prolines inside the transformed yeast was of 36.3% analyzed by mass spectrometry indicating that the recombinant enzyme is active on its natural substrate inside the yeast cell host. Furthermore, the recombinant sponge P4H has the ability to hydroxylate its natural substrate in both X and Y positions in the Xaa-Yaa-Gly collagenous triplets. In conclusion this Pichia system seems ideal for high-level production of hydroxylated sponge- or marine-derived collagen polypeptides as well as of conotoxins or other marine proteins of high pharmacological interest needing this particular post-translational modification. PMID:26022422

  1. Transport of a Prolyl Endopeptidase Inhibitory Peptide across the Blood-Brain Barrier Demonstrated Using the hCMEC/D3 Cell Line Transcytosis Assay.

    PubMed

    Hayes, Maria; Moen, Lars Fredrik; Auty, Mark A E; Lea, Tor Erling

    2016-01-13

    The blood-brain barrier (BBB) remains a significant hurdle for treatment of central nervous system (CNS) and mental health disorders. A prolyl endopeptidase (PEP) inhibitory peptide with the amino acid sequence proline-proline-leucine (PPL) was chemically synthesized labeled with 5-FAM and assessed using a transcytosis assay for its ability to cross the BBB. Transport of this peptide across the BBB was determined using an in vitro model of the human BBB, which utilizes the human cerebral microvascular endothelial cell line (hCMEC/D3). Uptake and transport of 5-FAM-PPL across the hCMEC/D3 cell model was determined using confocal microscopy and mass spectrometry. This is an important parameter in determining whether peptides may reach the target organ (i.e., the brain and central nervous system).This work assessed, for the first time, the ability of a food-derived PEP inhibitory peptide to cross the BBB without the use of animal models. PMID:26716467

  2. A simple HPLC method for the comprehensive analysis of cis/trans (Z/E) geometrical isomers of carotenoids for nutritional studies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Geometrical isomers of carotenoids behave differently in aspects like stability towards oxidants, bioavailability, vitamin A activity and specificity for enzymes. The availability of HPLC methods for their detailed profiling is therefore advisable to expand our knowledge on their metabolism and biol...

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

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... shall be limited to a maximum of 2.0% active ingredient. Contamination of food or food contact surfaces... cleaning compound and rinsed with potable water after use of the product. (4) To assure safe use of...

  4. 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

    ... shall be limited to a maximum of 2.0% active ingredient. Contamination of food or food contact surfaces... cleaning compound and rinsed with potable water after use of the product. (4) To assure safe use of...

  5. Estimation of procyanidin/prodelphinidin and cis/trans flavanol ratios of condensed tannin fractions by 1H-13C HSQC NMR spectroscopy: Correlation with thiolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Integration of cross-peak contours of H/C-2’,6’ signals from prodelphinidin (PD) and of H/C-6’ signals from procyanidin (PC) units in 1H-13C HSQC nuclear magnetic resonance (NMR) spectra of condensed tannins yielded nuclei-adjusted PC/PD estimates that were highly correlated with PC/PD ratios obtain...

  6. 40 CFR 180.545 - Prallethrin (RS)-2-methyl-4-oxo-3-(2-propynyl)cyclopent-2-enyl (1RS)-cis, trans-chrysanthemate...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Prallethrin (RS)-2-methyl-4-oxo-3-(2... Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND... served. (3) Application shall be limited to space, general surface, and spot and/or crack and...

  7. A critical evaluation of the s-cis-trans isomerism of 2-acetylpyrrole and its N-methyl derivative through infrared and NMR spectroscopies and theoretical calculations.

    PubMed

    Ducati, Lucas C; Braga, Carolyne B; Rittner, Roberto; Tormena, Cláudio F

    2013-12-01

    Literature data are controversial regarding the conformational equilibria of 2-acetylpyrrole (AP) and its N-methyl derivative (AMP). Now, a detailed study through infrared spectroscopy and theoretical calculations has shown that previous data were erroneously interpreted, since only a N,O-cis conformer is present in solution and that it is the stable conformer in the isolated state (ΔE(trans-cis) = 5.05 kcal mol(-1), for AP; ΔE(trans-cis) = 7.14 kcal mol(-1), for AMP). Carbonyl and NH absorption data in different solvents, supported by theoretical results taking into account the solvent effects [at IEFPCM-B3LYP/6-311++G(3df,3p) level of theory] clearly demonstrated that only the N,O-cis conformer is present in solution. However, a doublet was observed for AP, in CCl4, which can be attributed to this conformer and the lowest wavenumber component to the cis dimer form, stabilized through intermolecular hydrogen bonds (NH · · · OC). The overall preference for the N,O-cis conformer, in AP and AMP, as interpreted by the NBO analysis, indicated that the hyperconjugative effect is the main contribution to stabilize this rotamer, overcoming the possible steric repulsion. (13)C NMR experiments at low temperature in two different solvents (CS2/CDCl2 and acetone-d6) confirmed the occurrence of a single conformer since no separated signals were observed.

  8. A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold.

    PubMed

    Rendón-Ramírez, Adela; Shukla, Manish; Oda, Masataka; Chakraborty, Sandeep; Minda, Renu; Dandekar, Abhaya M; Ásgeirsson, Bjarni; Goñi, Félix M; Rao, Basuthkar J

    2013-01-01

    Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

  9. Regulation of lung maturation by prolyl hydroxylase domain inhibition in the lung of the normally grown and placentally restricted fetus in late gestation.

    PubMed

    McGillick, Erin V; Orgeig, Sandra; Morrison, Janna L

    2016-06-01

    Intrauterine growth restriction induced by placental restriction (PR) in sheep leads to chronic hypoxemia and reduced surfactant maturation. The underlying molecular mechanism involves altered regulation of hypoxia signaling by increased prolyl hydroxylase domain (PHD) expression. Here, we evaluated the effect of intratracheal administration of the PHD inhibitor dimethyloxalylglycine (DMOG) on functional, molecular, and structural determinants of lung maturation in the control and PR sheep fetus. There was no effect of DMOG on fetal blood pressure or fetal breathing movements. DMOG reduced lung expression of genes regulating hypoxia signaling (HIF-3α, ACE1), antioxidant defense (CAT), lung liquid reabsorption (SCNN1-A, ATP1-A1, AQP-1, AQP-5), and surfactant maturation (SFTP-A, SFTP-B, SFTP-C, PCYT1A, LPCAT, ABCA3, LAMP3) in control fetuses. There were very few effects of DMOG on gene expression in the PR fetal lung (reduced lung expression of angiogenic factor ADM, water channel AQP-5, and increased expression of glucose transporter SLC2A1). DMOG administration in controls reduced total lung lavage phosphatidylcholine to the same degree as in PR fetuses. These changes appear to be regulated at the molecular level as there was no effect of DMOG on the percent tissue, air space, or numerical density of SFTP-B positive cells in the control and PR lung. Hence, DMOG administration mimics the effects of PR in reducing surfactant maturation in the lung of control fetuses. The limited responsiveness of the PR fetal lung suggests a potential biochemical limit or reduced plasticity to respond to changes in regulation of hypoxia signaling following exposure to chronic hypoxemia in utero. PMID:26936783

  10. The Crystal Structure of an Algal Prolyl 4-Hydroxylase Complexed with a Proline-rich Peptide Reveals a Novel Buried Tripeptide Binding Motif*

    PubMed Central

    Koski, M. Kristian; Hieta, Reija; Hirsilä, Maija; Rönkä, Anna; Myllyharju, Johanna; Wierenga, Rik K.

    2009-01-01

    Plant and algal prolyl 4-hydroxylases (P4Hs) are key enzymes in the synthesis of cell wall components. These monomeric enzymes belong to the 2-oxoglutarate dependent superfamily of enzymes characterized by a conserved jelly-roll framework. This algal P4H has high sequence similarity to the catalytic domain of the vertebrate, tetrameric collagen P4Hs, whereas there are distinct sequence differences with the oxygen-sensing hypoxia-inducible factor P4H subfamily of enzymes. We present here a 1.98-Å crystal structure of the algal Chlamydomonas reinhardtii P4H-1 complexed with Zn2+ and a proline-rich (Ser-Pro)5 substrate. This ternary complex captures the competent mode of binding of the peptide substrate, being bound in a left-handed (poly)l-proline type II conformation in a tunnel shaped by two loops. These two loops are mostly disordered in the absence of the substrate. The importance of these loops for the function is confirmed by extensive mutagenesis, followed up by enzyme kinetic characterizations. These loops cover the central Ser-Pro-Ser tripeptide of the substrate such that the hydroxylation occurs in a highly buried space. This novel mode of binding does not depend on stacking interactions of the proline side chains with aromatic residues. Major conformational changes of the two peptide binding loops are predicted to be a key feature of the catalytic cycle. These conformational changes are probably triggered by the conformational switch of Tyr140, as induced by the hydroxylation of the proline residue. The importance of these findings for understanding the specific binding and hydroxylation of (X-Pro-Gly)n sequences by collagen P4Hs is also discussed. PMID:19553701

  11. Identifying a novel role for X-prolyl aminopeptidase (Xpnpep) 2 in CrVI-induced adverse effects on germ cell nest breakdown and follicle development in rats.

    PubMed

    Banu, Sakhila K; Stanley, Jone A; Sivakumar, Kirthiram K; Arosh, Joe A; Barhoumi, Rola; Burghardt, Robert C

    2015-03-01

    Environmental exposure to endocrine-disrupting chemicals (EDCs) is one cause of premature ovarian failure (POF). Hexavalent chromium (CrVI) is a heavy metal EDC widely used in more than 50 industries, including chrome plating, welding, wood processing, and tanneries. Recent data from U.S. Environmental Protection Agency indicate increased levels of Cr in drinking water from several American cities, which potentially predispose residents to various health problems. Recently, we demonstrated that gestational exposure to CrVI caused POF in F1 offspring. The current study was performed to identify the molecular mechanism behind CrVI-induced POF. Pregnant rats were treated with 25 ppm of potassium dichromate from Gestational Day (GD) 9.5 to GD 14.5 through drinking water, and the fetuses were exposed to CrVI through transplacental transfer. Ovaries were removed from the fetuses or pups on Embryonic Day (ED) 15.5, ED 17.5, Postnatal Day (PND) 1, PND 4, or PND 25, and various analyses were performed. Results showed that gestational exposure to CrVI: 1) increased germ cell/oocyte apoptosis and advanced germ cell nest (GCN) breakdown; 2) increased X-prolyl aminopeptidase (Xpnpep) 2, a POF marker in humans, during GCN breakdown; 3) decreased Xpnpep2 during postnatal follicle development; and 4) increased colocalization of Xpnpep2 with Col3 and Col4. We also found that Xpnpep2 inversely regulated the expression of Col1, Col3, and Col4 in all the developmental stages studied. Thus, CrVI advanced GCN breakdown and increased follicle atresia in F1 female progeny by targeting Xpnpep2.

  12. Proline with or without hydroxyproline influences collagen concentration and regulates prolyl 4-hydroxylase α (I) gene expression in juvenile turbo ( Scophthalmus maximus L.)

    NASA Astrophysics Data System (ADS)

    Zhang, Kaikai; Mai, Kangsen; Xu, Wei; Zhou, Huihui; Liufu, Zhiguo; Zhang, Yanjiao; Peng, Mo; Ai, Qinghui

    2015-06-01

    This study was conducted to investigate the effect of dietary proline (Pro), and Pro and hydroxyproline (Hyp) in combination on the growth performance, total Hyp and collagen concentrations of tissues, and prolyl 4-hydroxylase α(I) (P4H α(I)) gene expression in juvenile turbot feeding high plant protein diets. A diet containing 50% crude protein and 12% crude lipid was formulated as the basal and control, on which other two protein and lipid contents identical experimental diets were formulated by supplementing the basal with either 0.75% Pro (Pro-0.75) or 0.75% Pro and 0.75% Hyp (Pro+Hyp). Four groups of fish in indoor seawater recirculating systems, 35 individuals each, were fed twice a day to apparent satiation for 10 weeks. The results showed that dietary Pro and Hyp supplementation had no significant effect on growth performance and feed utilization of juvenile turbot (P > 0.05). Total Hyp and collagen concentrations in muscle were significantly increased when dietary Pro and Hyp increased (P <0.05), and fish fed diet Pro+Hyp showed significantly higher free Hyp content in plasma than those fed other diets (P <0.05). The expression of P4H a(I) gene in liver and muscle was significantly up regulated in fish fed diet Pro-0.75 in comparison with control (P <0.05); however the gene was significantly down regulated in fish fed diet Pro+Hyp in muscle in comparison with fish fed diet Pro-0.75 (P <0.05). It can be concluded that supplement of crystal L-Pro and L-Hyp to high plant protein diets did not show positive effects on growth performance of juvenile turbot, but enhanced total collagen concentrations in muscle.

  13. The anti-inflammatory peptide Ac-SDKP is released from thymosin-β4 by renal meprin-α and prolyl oligopeptidase.

    PubMed

    Kumar, Nitin; Nakagawa, Pablo; Janic, Branislava; Romero, Cesar A; Worou, Morel E; Monu, Sumit R; Peterson, Edward L; Shaw, Jiajiu; Valeriote, Frederick; Ongeri, Elimelda M; Niyitegeka, Jean-Marie V; Rhaleb, Nour-Eddine; Carretero, Oscar A

    2016-05-15

    N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a natural tetrapeptide with anti-inflammatory and antifibrotic properties. Previously, we have shown that prolyl oligopeptidase (POP) is involved in the Ac-SDKP release from thymosin-β4 (Tβ4). However, POP can only hydrolyze peptides shorter than 30 amino acids, and Tβ4 is 43 amino acids long. This indicates that before POP hydrolysis takes place, Tβ4 is hydrolyzed by another peptidase that releases NH2-terminal intermediate peptide(s) with fewer than 30 amino acids. Our peptidase database search pointed out meprin-α metalloprotease as a potential candidate. Therefore, we hypothesized that, prior to POP hydrolysis, Tβ4 is hydrolyzed by meprin-α. In vitro, we found that the incubation of Tβ4 with both meprin-α and POP released Ac-SDKP, whereas no Ac-SDKP was released when Tβ4 was incubated with either meprin-α or POP alone. Incubation of Tβ4 with rat kidney homogenates significantly released Ac-SDKP, which was blocked by the meprin-α inhibitor actinonin. In addition, kidneys from meprin-α knockout (KO) mice showed significantly lower basal Ac-SDKP amount, compared with wild-type mice. Kidney homogenates from meprin-α KO mice failed to release Ac-SDKP from Tβ4. In vivo, we observed that rats treated with the ACE inhibitor captopril increased plasma concentrations of Ac-SDKP, which was inhibited by the coadministration of actinonin (vehicle, 3.1 ± 0.2 nmol/l; captopril, 15.1 ± 0.7 nmol/l; captopril + actinonin, 6.1 ± 0.3 nmol/l; P < 0.005). Similar results were obtained with urinary Ac-SDKP after actinonin treatment. We conclude that release of Ac-SDKP from Tβ4 is mediated by successive hydrolysis involving meprin-α and POP.

  14. Isolation and characterization of cyclo-(tryptophanyl-prolyl) and chloramphenicol from Streptomyces sp. SUK 25 with antimethicillin-resistant Staphylococcus aureus activity

    PubMed Central

    Alshaibani, Muhanna M; Jalil, Juriyati; Sidik, Nik M; Edrada-Ebel, Ruangelie; Zin, Noraziah M

    2016-01-01

    Background Zingiber spectabile, commonly known as Beehive Ginger, is used as an ethnobotanical plant in many countries as an appetizer or to treat stomachache, toothache, muscle sprain, and as a cure for swelling, sores and cuts. This is the first report of isolation of Streptomyces strain from the root of this plant. Strain Universiti Kebangsaan 25 (SUK 25) has a very high activity to produce secondary metabolites against methicillin-resistant Staphylococcus aureus (MRSA), which is associated with high morbidity and mortality rates due to acquired multidrug resistance genes and causes medication failure in some clinical cases worldwide. Phylogenetic analysis based on the 16S ribosomal RNA gene sequence exhibited that the most closely related strain was Streptomyces omiyaensis NBRC 13449T (99.0% similarity). Aim This study was conducted to carry out the extraction, identification, and biological evaluation of active metabolites isolated from SUK 25 against three MRSA strains, namely, MRSA ATCC 43300, MRSA ATCC 33591, and MRSA ATCC 49476. Materials and methods The production of secondary metabolites by this strain was optimized through Thronton’s media. Isolation, purification, and identification of the bioactive compounds were carried out using reversed-phase high-performance liquid chromatography, high-resolution mass spectrometry, Fourier transform infrared, and one-dimensional and two-dimensional nuclear magnetic resonance. Results During screening procedure, SUK 25 exhibited good antimicrobial potential against several strains of MRSA. The best biological activity was shown from fraction number VII and its subfractions F2 and F3 with minimum inhibitory concentration values at 16 µg/mL and 8 µg/mL, respectively. These two subfractions were identified as diketopiperazine cyclo-(tryptophanyl-prolyl) and chloramphenicol. Conclusion On the basis of obtained results, SUK 25 isolated from Z. spectabile can be regarded as a new valuable source to produce secondary

  15. Cell-free synthesis and assembly of prolyl 4-hydroxylase: the role of the beta-subunit (PDI) in preventing misfolding and aggregation of the alpha-subunit.

    PubMed Central

    John, D C; Grant, M E; Bulleid, N J

    1993-01-01

    Prolyl 4-hydroxylase (P4-H) catalyses a vital post-translational modification in the biosynthesis of collagen. The enzyme consists of two distinct polypeptides forming an alpha 2 beta 2 tetramer (alpha = 64 kDa, beta = 60 kDa), the beta-subunit being identical to the multifunctional enzyme protein disulfide isomerase (PDI). By studying the cell-free synthesis of the rat alpha-subunit of P4-H we have shown that the alpha-subunit can be translocated, glycosylated and the signal peptide cleaved by dog pancreatic microsomal membranes to yield both singly and doubly glycosylated forms. When translations were carried out under conditions which prevent disulfide bond formation, the product synthesized formed aggregates which were associated with the immunoglobulin heavy chain binding protein (BiP). Translations carried out under conditions that promote disulfide bond formation yielded a product that was not associated with BiP but formed a complex with the endogenous beta-subunit (PDI). Complex formation was detected by co-precipitation of the newly synthesized alpha-subunit with antibodies raised against PDI, by sucrose gradient centrifugation and by chemical cross-linking. When microsomal vesicles were depleted of PDI, BiP and other soluble endoplasmic reticulum proteins, no complex formation was observed and the alpha-subunit aggregated even under conditions that promote disulfide bond formation. We have therefore demonstrated that the enzyme P4-H can be assembled at synthesis in a cell-free system and that the solubility of the alpha-subunit is dependent upon its association with PDI. Images PMID:8385607

  16. Molecular Mechanisms of Action and Therapeutic Uses of Pharmacological Inhibitors of HIF–Prolyl 4-Hydroxylases for Treatment of Ischemic Diseases

    PubMed Central

    Selvaraju, Vaithinathan; Parinandi, Narasimham L.; Adluri, Ram Sudheer; Goldman, Joshua W.; Hussain, Naveed; Sanchez, Juan A.

    2014-01-01

    Abstract Significance: In this review, we have discussed the efficacy and effect of small molecules that act as prolyl hydroxylase domain inhibitors (PHDIs). The use of these compounds causes upregulation of the pro-angiogenic factors and hypoxia inducible factor-1α and -2α (HIF-1α and HIF-2α) to enhance angiogenic, glycolytic, erythropoietic, and anti-apoptotic pathways in the treatment of various ischemic diseases responsible for significant morbidity and mortality in humans. Recent Advances: Sprouting of new blood vessels from the existing vasculature and surgical intervention, such as coronary bypass and stent insertion, have been shown to be effective in attenuating ischemia. However, the initial reentry of oxygen leads to the formation of reactive oxygen species that cause oxidative stress and result in ischemia/reperfusion (IR) injury. This apparent “oxygen paradox” must be resolved to combat IR injury. During hypoxia, decreased activity of PHDs initiates the accumulation and activation of HIF-1α, wherein the modulation of both PHD and HIF-1α appears as promising therapeutic targets for the pharmacological treatment of ischemic diseases. Critical Issues: Research on PHDs and HIFs has shown that these molecules can serve as therapeutic targets for ischemic diseases by modulating glycolysis, erythropoiesis, apoptosis, and angiogenesis. Efforts are underway to identify and synthesize safer small-molecule inhibitors of PHDs that can be administered in vivo as therapy against ischemic diseases. Future Directions: This review presents a comprehensive and current account of the existing small-molecule PHDIs and their use in the treatment of ischemic diseases with a focus on the molecular mechanisms of therapeutic action in animal models. Antioxid. Redox Signal. 20, 2631–2665. PMID:23992027

  17. Hypoxia-Inducible Factor α and Hif-prolyl Hydroxylase Characterization and Gene Expression in Short-Time Air-Exposed Mytilus galloprovincialis.

    PubMed

    Giannetto, Alessia; Maisano, Maria; Cappello, Tiziana; Oliva, Sabrina; Parrino, Vincenzo; Natalotto, Antonino; De Marco, Giuseppe; Barberi, Chiara; Romeo, Orazio; Mauceri, Angela; Fasulo, Salvatore

    2015-12-01

    Aquatic organisms experience environmental hypoxia as a result of eutrophication and naturally occurring tidal cycles. Mytilus galloprovincialis, being an anoxic/hypoxic-tolerant bivalve, provides an excellent model to investigate the molecular mechanisms regulating oxygen sensing. Across the animal kingdom, inadequacy in oxygen supply is signalled predominantly by hypoxia-inducible factors (HIF) and Hif-prolyl hydroxylases (PHD). In this study, hif-α 5'-end and partial phd mRNA sequences from M. galloprovincialis were obtained. Phylogenetic and molecular characterization of both HIF-α and PHD putative proteins showed shared key features with the respective orthologues from animals strongly suggesting their crucial involvement in the highly conserved oxygen sensing pathway. Both transcripts displayed a tissue-specific distribution with prominent expression in gills. Quantitative gene expression analysis of hif-α and phd mRNAs from gills of M. galloprovincialis demonstrated that both these key sensors are transcriptionally modulated by oxygen availability during the short-time air exposure and subsequent re-oxygenation treatments proving that they are critical players of oxygen-sensing mechanisms in mussels. Remarkably, hif-α gene expression showed a prompt and transient response suggesting the precocious implication of this transcription factor in the early phase of the adaptive response to hypoxia in Mytilus. HIF-α and PHD proteins were modulated in a time-dependent manner with trends comparable to mRNA expression patterns, thus suggesting a central role of their transcriptional regulation in the hypoxia tolerance strategies in marine bivalves. These results provide molecular information about the effects of oxygen deficiency and identify hypoxia-responsive biomarker genes in mussels applicable in ecotoxicological studies of natural marine areas.

  18. Selective inhibition of hypoxia-inducible factor (HIF) prolyl-hydroxylase 1 mediates neuroprotection against normoxic oxidative death via HIF- and CREB-independent pathways.

    PubMed

    Siddiq, Ambreena; Aminova, Leila R; Troy, Carol M; Suh, Kyungsun; Messer, Zachary; Semenza, Gregg L; Ratan, Rajiv R

    2009-07-01

    Oxidative stress contributes to tissue injury in conditions ranging from cardiovascular disease to stroke, spinal cord injury, neurodegeneration, and perhaps even aging. Yet the efficacy of antioxidants in human disease has been mixed at best. We need a better understanding of the mechanisms by which established antioxidants combat oxidative stress. Iron chelators are well established inhibitors of oxidative death in both neural and non-neural tissues, but their precise mechanism of action remains elusive. The prevailing but not completely substantiated view is that iron chelators prevent oxidative injury by suppressing Fenton chemistry and the formation of highly reactive hydroxyl radicals. Here, we show that iron chelation protects, rather unexpectedly, by inhibiting the hypoxia-inducible factor prolyl 4-hydroxylase isoform 1 (PHD1), an iron and 2-oxoglutarate-dependent dioxygenase. PHD1 and its isoforms 2 and 3 are best known for stabilizing transcriptional regulators involved in hypoxic adaptation, such as HIF-1alpha and cAMP response element-binding protein (CREB). Yet we find that global hypoxia-inducible factor (HIF)-PHD inhibition protects neurons even when HIF-1alpha and CREB are directly suppressed. Moreover, two global HIF-PHD inhibitors continued to be neuroprotective even in the presence of diminished HIF-2alpha levels, which itself increases neuronal susceptibility to oxidative stress. Finally, RNA interference to PHD1 but not isoforms PHD2 or PHD3 prevents oxidative death, independent of HIF activation. Together, these studies suggest that iron chelators can prevent normoxic oxidative neuronal death through selective inhibition of PHD1 but independent of HIF-1alpha and CREB; and that HIF-2alpha, not HIF-1alpha, regulates susceptibility to normoxic oxidative neuronal death. PMID:19587290

  19. Expression of Prolyl Hydroxylases (PHDs) Is Selectively Controlled by HIF-1 and HIF-2 Proteins in Nucleus Pulposus Cells of the Intervertebral Disc

    PubMed Central

    Fujita, Nobuyuki; Markova, Dessislava; Anderson, D. Greg; Chiba, Kazuhiro; Toyama, Yoshiaki; Shapiro, Irving M.; Risbud, Makarand V.

    2012-01-01

    Adaptive response to hypoxia in nucleus pulposus cells of the intervertebral disc is regulated by the hypoxia-inducible factors, HIF-1α and HIF-2α. Moreover, oxygen-dependent turnover of HIF-1α in these cells is controlled by the prolyl-4-hydroxylase domain (PHD) family of proteins. Whether HIF homologues control expression of PHDs and whether PHDs control hypoxia-inducible factor (HIF) turnover and/or activity under hypoxia is not known. Here, we show that in nucleus pulposus cells, hypoxia robustly induces PHD3 expression and, to a lesser extent, of PHD2 and PHD1. Reporter analysis shows that the hypoxic induction of the PHD2 promoter is HIF-1α dependent, whereas PHD3 promoter/enhancer activity is dependent on both HIF-1α and HIF-2α. Lentiviral delivery of HIF-1α, ShHIF-1α, and ShHIF-1β confirmed these observations. Noteworthy, HIF-1α maintains basal expression of PHD1 in hypoxia at the posttranscriptional level. Finally, loss of function studies using lentiviral transduction of ShPHDs clearly shows that even at 1% O2, PHD2 selectively degrades HIF-1α. In contrast, in hypoxia, PHD3 enhances HIF-1α transcriptional activity without affecting protein levels. To correlate these observations with disc disease, a condition characterized by tissue vascularization, we analyzed human tissues. Increased PHD1 mRNA expression but decreased PHD2 and PHD3 expression is observed in degenerate tissues. Interestingly, the hypoxic responsiveness of all the PHDs is maintained in isolated nucleus pulposus cells regardless of the disease state. We propose that PHD2 and PHD3 can be used as a biomarker of tissue oxygenation in the disc and that, as such, it may have important clinical implications. PMID:22451659

  20. Prolyl-4-hydroxylase Domain Protein 2 Controls NF-κB/p65 Transactivation and Enhances the Catabolic Effects of Inflammatory Cytokines on Cells of the Nucleus Pulposus*

    PubMed Central

    Li, Jun; Yuan, Wen; Jiang, Shuai; Ye, Wei; Yang, Hao; Shapiro, Irving M.; Risbud, Makarand V.

    2015-01-01

    Prolyl-4-hydroxylase (PHD) proteins are key in sensing tissue hypoxia. In nucleus pulposus (NP) cells, our previous work demonstrated that PHD isoforms have a differential contribution in controlling hypoxia-inducible factor (HIF)-α degradation and activity. Recently we have shown that a regulatory relationship exists between PHD3 and inflammatory cytokines in NP cells. With respect to PHD2, the most abundant PHD isoform in NP cells, very little is known concerning its function and regulation under inflammatory conditions that characterize intervertebral disc degeneration. Here, we show that PHD2 is a potent regulator of the catabolic activities of TNF-α; silencing of PHD2 significantly decreased TNF-α-induced expression of catabolic markers including SDC4, MMP-3, MMP-13, and ADAMTS5, as well as several inflammatory cytokines and chemokines, while partially restoring aggrecan and collagen II expression. Use of NF-κB reporters with ShPHD2, SiHIF-1α, as well as p65−/−, PHD2−/−, and PHD3−/− cells, shows that PHD2 serves as a co-activator of NF-κB/p65 signaling in HIF-1-independent fashion. Immunoprecipitation of endogenous and exogenously expressed tagged proteins, as well as fluorescence microscopy, indicates that following TNF-α treatment, PHD2 interacts and co-localizes with p65. Conversely, loss of function experiments using lentivirally delivered Sh-p65, Sh-IKKβ, and NF-κB inhibitor confirmed that cytokine-dependent PHD2 expression in NP cells requires NF-κB signaling. These findings clearly demonstrate that PHD2 forms a regulatory circuit with TNF-α via NF-κB and thereby plays an important role in enhancing activity of this cytokine. We propose that during disc degeneration PHD2 may offer a therapeutic target to mitigate the deleterious actions of TNF-α, a key proinflammatory cytokine. PMID:25635047

  1. Solution Structure of Archaeoglobus fulgidis Peptidyl-tRNA Hydrolase(Pth2) Provides Evidence for an Extensive Conserved Family of Pth2 Enzymes in Archaea, Bacteria and Eukaryotes.

    SciTech Connect

    Powers, Robert; Mirkovic, Nebojsa; Goldsmith-Fischman, Sharon; Acton, Thomas; Chiang, Yiwen; Huang, Yuanpeng; Ma, LiChung; Rajan, Paranji K.; Cort, John R.; Kennedy, Michael A.; Liu, Jinfeng; Rost, Burkhard; Honig, Barry; Murray, Diana; Montelione, Gaetano

    2005-11-01

    The solution structure of protein AF2095 from the thermophilic archaea Archaeglobus fulgidis, a 123-residue (13.6 kDa) protein, has been determined by NMR methods. The structure of AF2095 is comprised of four a-helices and a mixed b-sheet consisting of four parallel and anti-parallel b-strands, where the a-helices sandwich the b-sheet. Sequence and structural comparison of AF2095 with proteins from Homo sapiens, Methanocaldococcus jannaschii and Sulfolobus solfataricus, reveals that AF2095 is a peptidyl-tRNA hydrolase (Pth2). This structural comparison also identifies putative catalytic residues and a tRNA interaction region for AF2095. The structure of AF2095 is also similar to the structure of protein TA0108 from archaea Thermoplasma acidophilum, which is deposited in the Protein Database but not functionally annotated. The NMR structure of AF2095 has been further leveraged to obtain good quality structural models for 55 other proteins. Although earlier studies have proposed that the Pth2 protein family is restricted to archeal and eukaryotic organisms, the similarity of the AF2095 structure to human Pth2, the conservation of key active-site residues, and the good quality of the resulting homology models demonstrate a large family of homologous Pth2 proteins that are conserved in eukaryotic, archaeal and bacterial organisms, providing novel insights in the evolution of the Pth and Pth2 enzyme families.

  2. Severe Extracellular Matrix Abnormalities and Chondrodysplasia in Mice Lacking Collagen Prolyl 4-Hydroxylase Isoenzyme II in Combination with a Reduced Amount of Isoenzyme I*

    PubMed Central

    Aro, Ellinoora; Salo, Antti M.; Khatri, Richa; Finnilä, Mikko; Miinalainen, Ilkka; Sormunen, Raija; Pakkanen, Outi; Holster, Tiina; Soininen, Raija; Prein, Carina; Clausen-Schaumann, Hauke; Aszódi, Attila; Tuukkanen, Juha; Kivirikko, Kari I.; Schipani, Ernestina; Myllyharju, Johanna

    2015-01-01

    Collagen prolyl 4-hydroxylases (C-P4H-I, C-P4H-II, and C-P4H-III) catalyze formation of 4-hydroxyproline residues required to form triple-helical collagen molecules. Vertebrate C-P4Hs are α2β2 tetramers differing in their catalytic α subunits. C-P4H-I is the major isoenzyme in most cells, and inactivation of its catalytic subunit (P4ha1−/−) leads to embryonic lethality in mouse, whereas P4ha1+/− mice have no abnormalities. To study the role of C-P4H-II, which predominates in chondrocytes, we generated P4ha2−/− mice. Surprisingly, they had no apparent phenotypic abnormalities. To assess possible functional complementarity, we established P4ha1+/−;P4ha2−/− mice. They were smaller than their littermates, had moderate chondrodysplasia, and developed kyphosis. A transient inner cell death phenotype was detected in their developing growth plates. The columnar arrangement of proliferative chondrocytes was impaired, the amount of 4-hydroxyproline and the Tm of collagen II were reduced, and the extracellular matrix was softer in the growth plates of newborn P4ha1+/−;P4ha2−/− mice. No signs of uncompensated ER stress were detected in the mutant growth plate chondrocytes. Some of these defects were also found in P4ha2−/− mice, although in a much milder form. Our data show that C-P4H-I can to a large extent compensate for the lack of C-P4H-II in proper endochondral bone development, but their combined partial and complete inactivation, respectively, leads to biomechanically impaired extracellular matrix, moderate chondrodysplasia, and kyphosis. Our mouse data suggest that inactivating mutations in human P4HA2 are not likely to lead to skeletal disorders, and a simultaneous decrease in P4HA1 function would most probably be required to generate such a disease phenotype. PMID:26001784

  3. Hyperplasia and hypertrophy of pulmonary neuroepithelial bodies, presumed airway hypoxia sensors, in hypoxia-inducible factor prolyl hydroxylase-deficient mice

    PubMed Central

    Pan, Jie; Bishop, Tammie; Ratcliffe, Peter J; Yeger, Herman; Cutz, Ernest

    2016-01-01

    Pulmonary neuroepithelial bodies (NEBs), presumed polymodal airway sensors, consist of innervated clusters of amine (serotonin) and peptide-producing cells. While NEB responses to acute hypoxia are mediated by a membrane-bound O2 sensor complex, responses to sustained and/or chronic hypoxia involve a prolyl hydroxylase (PHD)–hypoxia-inducible factor-dependent mechanism. We have previously reported hyperplasia of NEBs in the lungs of Phd1−/− mice associated with enhanced serotonin secretion. Here we use a novel multilabel immunofluorescence method to assess NEB distribution, frequency, and size, together with the number and size of NEB cell nuclei, and to colocalize multiple cytoplasmic and nuclear epitopes in the lungs of Phd1−/−, Phd2+/−, and Phd3−/− mice and compare them with wild-type controls. To define the mechanisms of NEB cell hyperplasia, we used antibodies against Mash1 and Prox1 (neurogenic genes involved in NEB cell differentiation/maturation), hypoxia-inducible factor-1alpha, and the cell proliferation marker Ki67. Morphometric analysis of (% total lung area) immunostaining for synaptophysin (% synaptophysin), a cytoplasmic marker of NEB cells, was significantly increased in Phd1−/− and Phd3−/− mice compared to wild-type mice. In addition, NEB size and the number and size of NEB nuclei were also significantly increased, indicating that deficiency of Phds is associated with striking hyperplasia and hypertrophy of NEBs. In Phd2+/− mice, while mean % synaptophysin was comparable to wild-type controls, the NEB size was moderately increased, suggesting an effect even in heterozygotes. NEBs in all Phd-deficient mice showed increased expression of Mash1, Prox1, Ki67, and hypoxia-inducible factor-1alpha, in keeping with enhanced differentiation from precursor cells and a minor component of cell proliferation. Since the loss of PHD activity mimics chronic hypoxia, our data provide critical information on the potential role of PHDs in

  4. NMR studies of conformational states of proteins involved in biosynthesis of iron-sulfur clusters

    NASA Astrophysics Data System (ADS)

    Dai, Ziqi

    Iron-sulfur (Fe-S) clusters are the most ancient and ubiquitous cofactors that exist throughout evolution. The most important biosynthetic system of the cluster in both prokaryotes and eukaryotes is the ISC system. Defects in this system can be lethal and have been associated with a number of human diseases. Previous works show that a number of proteins are involved in the [Fe-S] biosynthetic processes and the structural flexibility may play an important role. For example, it was shown that apo-IscU, the scaffold protein, from Escherichia coli populates two functionally important conformational states, one dynamically disordered (D-state) and the other more structured (S-state) (Kim et al., 2009; Kim et al., 2012c). To further investigate the characteristics and transition of the conformational states of proteins involved in this system, I performed extensive NMR studies. Here, I present the findings based on my studies of two important players of the ISC system, IscU and HscB. In this research, I find that a pepti