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Sample records for peptidyl prolyl cis-trans

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

    PubMed

    Pliyev, B K; Gurvits, B Y

    1999-07-01

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

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

    PubMed

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

    2016-02-01

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

  3. 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

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

    PubMed Central

    Pemberton, Trevor J

    2006-01-01

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

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

    PubMed

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

    1998-04-28

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

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

    PubMed Central

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

    1995-01-01

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

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

    PubMed

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

    2013-02-18

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

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

    PubMed

    Haokip, Nemneineng; Naorem, Aruna

    2017-01-08

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

  9. 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

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

    PubMed Central

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

    1999-01-01

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

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

    PubMed Central

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

    1998-01-01

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

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

    PubMed Central

    van Ooyen, Jan

    2015-01-01

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

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

    PubMed

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

    2015-11-01

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

  14. 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

  15. 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

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

    PubMed

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

    2017-01-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

    2010-01-01

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

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

    PubMed

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

    2015-12-01

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

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

    1992-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2017-03-01

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2013-08-30

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

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

    PubMed

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

    2015-07-31

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

  6. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2005-01-01

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

  11. 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

  12. 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.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  15. 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.

  16. 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.

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

    PubMed

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

    2015-01-01

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

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

    SciTech Connect

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

    2011-03-18

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

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

    EPA Science Inventory

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2017-04-06

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

  3. 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.

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

    PubMed Central

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

    1995-01-01

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

  5. 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.

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

    NASA Astrophysics Data System (ADS)

    Uspenskiy, Igor; Strodel, Birgit; Stock, Gerhard

    2006-10-01

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

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

    PubMed

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

    2017-03-01

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

  8. 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.

  9. 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.

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

    PubMed

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

    2015-04-30

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

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

    PubMed Central

    Dolinski, K J; Heitman, J

    1999-01-01

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

  12. 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

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

    PubMed

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

    2013-06-03

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

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

    NASA Astrophysics Data System (ADS)

    Joukai, Tatsuya; Kita, Kenji; Yano, Ryuzi

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

  15. 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

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

    NASA Astrophysics Data System (ADS)

    Pham, Phong; Guo, Yin

    2013-04-01

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

  17. 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…

  18. 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...

  19. 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

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

  20. 40 CFR 180.545 - Prallethrin (RS)-2-methyl-4-oxo-3-(2-propynyl)cyclopent-2-enyl (1RS)-cis, trans-chrysanthemate...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

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

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

    Code of Federal Regulations, 2012 CFR

    2012-07-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-04-01

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

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

    PubMed

    Tscherbul, Timur V; Brumer, Paul

    2015-12-14

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-08-01

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

  6. 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.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2009-01-01

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

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

    PubMed

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

    2011-06-28

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

  10. Physiological evidence for the presence of a cis-trans isomerase of unsaturated fatty acids in Methylococcus capsulatus Bath to adapt to the presence of toxic organic compounds.

    PubMed

    Löffler, Claudia; Eberlein, Christian; Mäusezahl, Ines; Kappelmeyer, Uwe; Heipieper, Hermann J

    2010-07-01

    The physiology of the response in the methanotrophic bacterium Methylococcus capsulatus Bath towards thermal and solvent stress was studied. A systematic investigation of the toxic effects of organic compounds (chlorinated phenols and alkanols) on the growth of this bacterium was carried out. The sensitivity to the tested alkanols correlated with their chain length and hydrophobicity; methanol was shown to be an exception to which the cells showed a very high tolerance. This can be explained by the adaptation of these bacteria to growth on C1 compounds. On the other hand, M. capsulatus Bath was very sensitive towards the tested chlorinated phenols. The high toxic effect of phenolic compounds on methanotrophic bacteria might be explained by the occurrence of toxic reactive oxygen species. In addition, a physiological proof of the presence of cis-trans isomerization as a membrane-adaptive response mechanism in M. capsulatus was provided. This is the first report on physiological evidence for the presence of the unique postsynthetic membrane-adaptive response mechanism of the cis-trans isomerization of unsaturated fatty acids in a bacterium that does not belong to the genera Pseudomonas and Vibrio where this mechanism was already reported and described extensively.

  11. 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

  12. 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.

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

    DTIC Science & Technology

    2001-07-01

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

  14. 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.

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

    PubMed Central

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

    1998-01-01

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

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

    PubMed

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

    2015-01-15

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

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

    SciTech Connect

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

    1990-07-01

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

  18. 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.

  19. Compensatory role of the cis-trans-isomerase and cardiolipin synthase in the membrane fluidity of Pseudomonas putida DOT-T1E.

    PubMed

    Bernal, Patricia; Segura, Ana; Ramos, Juan-Luis

    2007-07-01

    In Gram-negative bacteria, cell membrane fluidity is influenced by phospholipid head group composition and linked fatty acids. Exposure of Pseudomonas putida to stressing agents results in short- and long-term modifications in membrane lipids. The main adaptive change observed in response to organic solvents in the short term is the cis- to trans-isomerization of unsaturated fatty acids in a reaction mediated by cis/trans-isomerase (CTI); whereas in the long term an increase in cardiolipin content takes place. Despite the interest of these genes in the context of stress responses, the transcriptional regulation of the cti and cls genes has not been studied before. The cti and cls (cardiolipin synthase) genes in the solvent-tolerant P. putida DOT-T1E strain form monocistronic units and are expressed from sigma-70 promoters. Expression from the cls promoter is sixfold higher in the stationary phase than in the log phase, and expression of the cls gene is not influenced by solvents. The cti gene is expressed at fairly constant levels in the log and stationary phase, but its level of expression is moderately upregulated in response to toluene. We used fluorescence polarization assays to show that mutants deficient in the cti gene exhibit less rigid membranes than the wild-type strain, whereas mutants with a knockout in the cls gene exhibit increased membrane rigidity. A double cti/cls mutant has similar membrane rigidity as the wild-type strain, which points towards a compensatory effect of the mutations with regard to membrane fluidity. However, the cls and cls/cti mutants were more sensitive to solvents than the wild-type and the cti mutant because of the impaired functioning of efflux drug transporters.

  20. 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.

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

    PubMed

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

    2015-09-08

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

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

    PubMed

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

    2007-11-09

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

  3. 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

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

    PubMed

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

    2013-11-18

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

  5. An endoplasmic reticulum-specific cyclophilin.

    PubMed Central

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

    1991-01-01

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

  6. 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.

  7. 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

  8. SPPS of protected peptidyl aminoalkyl amides.

    PubMed

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

    2002-11-01

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

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

    PubMed

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

    2015-05-01

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

  10. 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.

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

    PubMed

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

    1996-02-01

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

  12. 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.

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

    PubMed

    Wang, Xiaohong; Bowman, Joel M

    2013-02-12

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

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

    PubMed

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

    2015-05-01

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

  15. 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

  16. 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

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

    PubMed

    MacDonald, Corey A; Boyd, Russell J

    2015-08-15

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

  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. 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.

  20. Collagen Prolyl Hydroxylases are Essential for Breast Cancer Metastasis

    PubMed Central

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

    2013-01-01

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

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

    PubMed

    Barman, Arghya; Batiste, Bruce; Hamelberg, Donald

    2015-04-14

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

  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. Inhibition of serine proteases by peptidyl fluoromethyl ketones

    SciTech Connect

    Imperiali, B.; Abeles, R.H.

    1986-07-01

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

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

    PubMed

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

    2005-11-17

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

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

    PubMed

    Barman, Arghya; Hamelberg, Donald

    2015-03-01

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

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

    PubMed

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

    2005-01-01

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

  7. 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 ...

  8. 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.

  9. Epigenetic Control of Prolyl and Asparaginyl Hydroxylases in Prostate Cancer

    DTIC Science & Technology

    2011-07-01

    containing proteins (PHD/EGLN/HPH) which utilize iron , oxygen and 2-oxoglutarate as co-factors to enzymatically catalyze hydroxylation on the oxygen-dependent...proteosome [6]. Under hypoxic conditions, HIF prolyl hydroxylase activity is decreased and HIF-1a protein accumulates . HIF-a subunits translocate to the...mechanism might be responsible for their silencing. Unlike genetic mutations that accumulate in cancer, epigenetic modifications are reversible [20]. We

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

    PubMed

    Denny, William A

    2012-04-12

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2012-07-01

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

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

    PubMed

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

    2012-10-18

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

  14. Isoform-specific Inhibition of Cyclophilins

    PubMed Central

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

    2009-01-01

    Cyclophilins belong to the enzyme class of peptidyl prolyl cis/trans isomerases which catalyze the cis/trans isomerization of prolyl bonds in peptides and proteins in different folding states. Cyclophilins have been shown to be involved in a multitude of cellular functions like cell growth, proliferation, and motility. Among the 20 human cyclophilin isoenzymes, the two most abundant members of the cyclophilin family CypA and CypB exhibit specific cellular functions in several inflammatory diseases, cancer development and HCV replication. A small-molecule inhibitor on the basis of aryl 1-indanylketones has now been shown to discriminate between CypA and CypB in vitro. CypA binding of this inhibitor has been characterized by fluorescence anisotropy- and isothermal titration calorimetry-based cyclosporin competition assays. Inhibition of CypA- but not CypB-mediated chemotaxis of mouse CD4+ T cells by the inhibitor provided biological proof of discrimination in vivo. PMID:19480458

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

    PubMed

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

    2014-01-07

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

  16. Evolutionary relationships of the prolyl oligopeptidase family enzymes.

    PubMed

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

    2004-07-01

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

  17. 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.

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

    PubMed

    Menninger, J R; Otto, D P

    1982-05-01

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

  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.

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

    PubMed

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

    2009-06-01

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

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

    PubMed

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

    2017-02-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    Sieber, Matthias; Baumgrass, Ria

    2009-01-01

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

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

    PubMed

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

    2017-01-01

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

  5. The Arabidopsis Cyclophilin Gene Family1

    PubMed Central

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

    2004-01-01

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

  6. 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

  7. PIN1 in breast development and cancer: a clinical perspective.

    PubMed

    Rustighi, Alessandra; Zannini, Alessandro; Campaner, Elena; Ciani, Yari; Piazza, Silvano; Del Sal, Giannino

    2017-02-01

    Mammary gland development, various stages of mammary tumorigenesis and breast cancer progression have the peptidyl-prolyl cis/trans isomerase PIN1 at their centerpiece, in virtue of the ability of this unique enzyme to fine-tune the dynamic crosstalk between multiple molecular pathways. PIN1 exerts its action by inducing conformational and functional changes on key cellular proteins, following proline-directed phosphorylation. Through this post-phosphorylation signal transduction mechanism, PIN1 controls the extent and direction of the cellular response to a variety of inputs, in physiology and disease. This review discusses PIN1's roles in normal mammary development and cancerous progression, as well as the clinical impact of targeting this enzyme in breast cancer patients.

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

    PubMed Central

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

    2004-01-01

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

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

    PubMed Central

    Kumar, Amit; Balbach, Jochen

    2017-01-01

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

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

    PubMed

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

    2009-01-01

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

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

    PubMed

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

    2009-11-20

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

  12. 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

  13. 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

  14. 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.

  15. Prolyl carboxypeptidase mRNA expression in the mouse brain.

    PubMed

    Jeong, Jin Kwon; Diano, Sabrina

    2014-01-13

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

  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. 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

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

    PubMed

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

    2012-02-10

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

  19. Soybean cyclophilin GmCYP1 interacts with an isoflavonoid regulator GmMYB176

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2016-03-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    1998-01-01

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

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

    PubMed

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

    2014-09-01

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

  4. Unveiling Prolyl Oligopeptidase Ligand Migration by Comprehensive Computational Techniques

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  6. Pin1 down-regulates transforming growth factor-beta (TGF-beta) signaling by inducing degradation of Smad proteins.

    PubMed

    Nakano, Ayako; Koinuma, Daizo; Miyazawa, Keiji; Uchida, Takafumi; Saitoh, Masao; Kawabata, Masahiro; Hanai, Jun-ichi; Akiyama, Hirotada; Abe, Masahiro; Miyazono, Kohei; Matsumoto, Toshio; Imamura, Takeshi

    2009-03-06

    Transforming growth factor-beta (TGF-beta) is crucial in numerous cellular processes, such as proliferation, differentiation, migration, and apoptosis. TGF-beta signaling is transduced by intracellular Smad proteins that are regulated by the ubiquitin-proteasome system. Smad ubiquitin regulatory factor 2 (Smurf2) prevents TGF-beta and bone morphogenetic protein signaling by interacting with Smads and inducing their ubiquitin-mediated degradation. Here we identified Pin1, a peptidylprolyl cis-trans isomerase, as a novel protein binding Smads. Pin1 interacted with Smad2 and Smad3 but not Smad4; this interaction was enhanced by the phosphorylation of (S/T)P motifs in the Smad linker region. (S/T)P motif phosphorylation also enhanced the interaction of Smad2/3 with Smurf2. Pin1 reduced Smad2/3 protein levels in a manner dependent on its peptidyl-prolyl cis-trans isomerase activity. Knockdown of Pin1 increased the protein levels of endogenous Smad2/3. In addition, Pin1 both enhanced the interaction of Smurf2 with Smads and enhanced Smad ubiquitination. Pin1 inhibited TGF-beta-induced transcription and gene expression, suggesting that Pin1 negatively regulates TGF-beta signaling by down-regulating Smad2/3 protein levels via induction of Smurf2-mediated ubiquitin-proteasomal degradation.

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

    PubMed

    Thirupathi, Ponnaboina; Lee, Keun-Hyeung

    2013-12-15

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

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

    SciTech Connect

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

    1991-02-26

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

  10. 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.

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

    PubMed

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

    2011-02-25

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

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

    PubMed

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

    2006-02-28

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

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

    PubMed Central

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

    2014-01-01

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

  14. 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.

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

    PubMed

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

    2006-10-01

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

  16. 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.

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

    PubMed

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

    2003-10-01

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

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

    PubMed

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

    2002-05-06

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

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

    PubMed

    Karimi, R; Ehrenberg, M

    1996-03-01

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

  20. 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.

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

    PubMed

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

    2000-12-01

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

  2. 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.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

    Majamaa, K; Sasaki, T; Uitto, J

    1987-10-01

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

  5. 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.

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

    PubMed

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

    1997-05-01

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

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

    PubMed

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

    2010-10-01

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

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

    PubMed

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

    2017-01-27

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

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

    PubMed

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

    2015-09-01

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

  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.

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

    PubMed

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

    2016-11-17

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

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

    PubMed

    Thirumoorthy, Krishnan; Nandi, Nilashis

    2007-08-23

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

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

    PubMed

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

    2017-05-15

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

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

    PubMed

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

    1991-01-04

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

  15. 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.

  16. 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

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

    SciTech Connect

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

    2006-12-08

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

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

    PubMed Central

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

    2006-01-01

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

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

    PubMed

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

    2000-06-26

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

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

    PubMed

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

    2016-08-09

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

  1. Understanding the role of PIN1 in hepatocellular carcinoma

    PubMed Central

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

    2016-01-01

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

  2. FK506 binding protein mutational analysis. Defining the surface residue contributions to stability of the calcineurin co-complex.

    PubMed

    Futer, O; DeCenzo, M T; Aldape, R A; Livingston, D J

    1995-08-11

    The 12- and 13-kDa FK506 binding proteins (FKBP12 and FKBP13) are cis-trans peptidyl-prolyl isomerases that bind the macrolides FK506 (Tacrolimus) and rapamycin (Sirolimus). The FKBP12.FK506 complex is immunosuppressive, acting as an inhibitor of the protein phosphatase calcineurin. We have examined the role of the key surface residues of FKBP12 and FKBP13 in calcineurin interactions by generating substitutions at these residues by site-directed mutagenesis. All mutants are active catalysts of the prolyl isomerase reaction, and bind FK506 or rapamycin with high affinity. Mutations at FKBP12 residues Asp-37, Arg-42, His-87, and Ile-90 decrease calcineurin affinity of the mutant FKBP12.FK506 complex by as much as 2600-fold in the case of I90K. Replacement of three FKBP13 surface residues (Gln-50, Ala-95, and Lys-98) with the corresponding homologous FKBP12 residues (Arg-42, His-87, and Ile-90) generates an FKBP13 variant that is equivalent to FKBP12 in its affinity for FK506, rapamycin, and calcineurin. These results confirm the role of two loop regions of FKBP12 (residues 40-44 and 84-91) as part of the effector face that interacts with calcineurin.

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed

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

    2013-07-01

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

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

    PubMed

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

    2000-03-01

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

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

    PubMed

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

    2014-11-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2004-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    1986-01-01

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

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

    PubMed

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

    2002-08-01

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

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

    NASA Technical Reports Server (NTRS)

    Bonner, W. A.

    1972-01-01

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

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

    PubMed

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

    2016-05-01

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

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

    PubMed Central

    Toh, Seok-Ming; Mankin, Alexander S.

    2017-01-01

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

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

    PubMed

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

    2002-02-01

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

  16. Prolyl isomerase Pin1 regulates axon guidance by stabilizing CRMP2A selectively in distal axons

    PubMed Central

    Balastik, Martin; Zhou, Xiao Zhen; Alberich-Jorda, Meritxell; Weissova, Romana; Žiak, Jakub; Pazyra-Murphy, Maria F.; Cosker, Katharina E; Machonova, Olga; Kozmikova, Iryna; Chen, Chun-Hau; Pastorino, Lucia; Asara, John M.; Cole, Adam; Sutherland, Calum; Segal, Rosalind A.; Lu, Kun Ping

    2015-01-01

    SUMMARY Axon guidance relies on precise translation of the gradients of the extracellular signals into local changes of cytoskeletal dynamics, but the molecular mechanisms regulating dose-dependent responses of growth cones are still poorly understood. Here we show that during embryonic development in growing axons low level of Semaphorin3A stimulation is buffered by the prolyl isomerase Pin1. We demonstrate, that Pin1 stabilizes CDK5-phosphorylated CRMP2A, the major isoform of CRMP2 in distal axons. Consequently, Pin1 knockdown or knockout reduces CRMP2A level specifically in distal axons and inhibits axon growth, which can be fully rescued by Pin1 or CRMP2A expression. Moreover, Pin1 knockdown or knockout increases sensitivity to Sema3A-induced growth cone collapse in vitro and in vivo leading to developmental abnormalities in axon guidance. These results identify an important isoform-specific function and regulation of CRMP2A in controlling axon growth, and uncover Pin1-catalyzed prolyl isomerization as a regulatory mechanism in axon guidance. PMID:26489457

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

    PubMed

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

    2006-01-17

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

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

    PubMed Central

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

    2013-01-01

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

  19. 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.

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

    PubMed

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

    1985-08-01

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

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

    PubMed

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

    2005-08-01

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

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

    PubMed

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

    2009-11-24

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

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

    PubMed

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

    2012-04-13

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

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

    EPA Science Inventory

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

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

    PubMed

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

    2008-09-25

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

  6. 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

  7. 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.

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

    PubMed Central

    Howe, Françoise S.; Mellor, Jane

    2014-01-01

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

  9. 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.

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

    EPA Science Inventory

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

  11. 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 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

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

    NASA Astrophysics Data System (ADS)

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

    1993-12-01

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

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

    NASA Astrophysics Data System (ADS)

    Arango, Carlos A.; Brumer, Paul

    2013-02-01

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

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

    PubMed Central

    1992-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Grubbs, G. S.

    2017-01-01

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

  16. 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

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

    PubMed

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

    2012-04-01

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

  18. 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.

  19. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2016-12-01

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

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

    PubMed

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

    2014-04-01

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

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

    PubMed Central

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

    2015-01-01

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

  4. 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

  5. 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

  6. 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.

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

    PubMed Central

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

    2013-01-01

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

  8. 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

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

    PubMed

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

    2016-09-15

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

  10. 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

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

    PubMed Central

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

    2006-01-01

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

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

    PubMed

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

    2015-07-08

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

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

    PubMed

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

    2013-10-10

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

  14. 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.

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2015-04-01

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

  19. 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

  20. 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.

  1. Heterologous expression of a salinity and developmentally regulated rice cyclophilin gene (OsCyp2) in E. coli and S. cerevisiae confers tolerance towards multiple abiotic stresses.

    PubMed

    Kumari, Sumita; Singh, Prabhjeet; Singla-Pareek, Sneh L; Pareek, Ashwani

    2009-06-01

    Cyclophilin 2 (OsCyp2) is a cytosolic member of immunophilin family from rice. We have isolated its full length cDNA (1,056 bp) with an open reading frame of 519 bp encoding a polypeptide of 172 amino acids and an estimated pI of 8.61. Peptidyl prolyl cis-trans isomerase activity of the protein was determined using N-succinyl-ala-ala-pro-phe-p-nitroanilidine as peptide substrate. It has a catalytic efficiency (K (cat)/K (m)) of 4.5 x 10(6)/(mol/l)/s, which is comparable to known cyclophilins from plants. Its activity is specifically inhibited by cyclosporin A, a macrolide drug inhibitor of cyclophilins. Transcript analysis showed it to be a developmentally and differentially regulated gene; showing changes in abundance at seedling, tillering and heading stage under non-stress and salinity stress conditions. Expression of OsCyp2 enhances the ability of Escherichia coli to survive under diverse abiotic stresses viz. salinity, high temperature, osmotic stress (mannitol) and oxidative stress (H(2)O(2)). OsCyp2 was able to complement the yeast mutant lacking native Cyp2 and also improved the growth of wild type yeast under above-mentioned stress conditions. Based on these results, we propose that OsCyp2 may serve as a 'suitable candidate' for raising transgenic plants for enhanced multiple abiotic stress tolerance.

  2. 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

  3. Endoplasmic reticulum stress regulates rat mandibular cartilage thinning under compressive mechanical stress.

    PubMed

    Li, Huang; Zhang, Xiang-Yu; Wu, Tuo-Jiang; Cheng, Wei; Liu, Xin; Jiang, Ting-Ting; Wen, Juan; Li, Jie; Ma, Qiao-Ling; Hua, Zi-Chun

    2013-06-21

    Compressive mechanical stress-induced cartilage thinning has been characterized as a key step in the progression of temporomandibular joint diseases, such as osteoarthritis. However, the regulatory mechanisms underlying this loss have not been thoroughly studied. Here, we used an established animal model for loading compressive mechanical stress to induce cartilage thinning in vivo. The mechanically stressed mandibular chondrocytes were then isolated to screen potential candidates using a proteomics approach. A total of 28 proteins were identified that were directly or indirectly associated with endoplasmic reticulum stress, including protein disulfide-isomerase, calreticulin, translationally controlled tumor protein, and peptidyl-prolyl cis/trans-isomerase protein. The altered expression of these candidates was validated at both the mRNA and protein levels. The induction of endoplasmic reticulum stress by mechanical stress loading was confirmed by the activation of endoplasmic reticulum stress markers, the elevation of the cytoplasmic Ca(2+) level, and the expansion of endoplasmic reticulum membranes. More importantly, the use of a selective inhibitor to block endoplasmic reticulum stress in vivo reduced the apoptosis observed at the early stages of mechanical stress loading and inhibited the proliferation observed at the later stages of mechanical stress loading. Accordingly, the use of the inhibitor significantly restored cartilage thinning. Taken together, these results demonstrated that endoplasmic reticulum stress is significantly activated in mechanical stress-induced mandibular cartilage thinning and, more importantly, that endoplasmic reticulum stress inhibition alleviates this loss, suggesting a novel pharmaceutical strategy for the treatment of mechanical stress-induced temporomandibular joint diseases.

  4. 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.

  5. Discovering Conformational Sub-States Relevant to Protein Function

    PubMed Central

    Ramanathan, Arvind; Savol, Andrej J.; Langmead, Christopher J.; Agarwal, Pratul K.; Chennubhotla, Chakra S.

    2011-01-01

    Background Internal motions enable proteins to explore a range of conformations, even in the vicinity of native state. The role of conformational fluctuations in the designated function of a protein is widely debated. Emerging evidence suggests that sub-groups within the range of conformations (or sub-states) contain properties that may be functionally relevant. However, low populations in these sub-states and the transient nature of conformational transitions between these sub-states present significant challenges for their identification and characterization. Methods and Findings To overcome these challenges we have developed a new computational technique, quasi-anharmonic analysis (QAA). QAA utilizes higher-order statistics of protein motions to identify sub-states in the conformational landscape. Further, the focus on anharmonicity allows identification of conformational fluctuations that enable transitions between sub-states. QAA applied to equilibrium simulations of human ubiquitin and T4 lysozyme reveals functionally relevant sub-states and protein motions involved in molecular recognition. In combination with a reaction pathway sampling method, QAA characterizes conformational sub-states associated with cis/trans peptidyl-prolyl isomerization catalyzed by the enzyme cyclophilin A. In these three proteins, QAA allows identification of conformational sub-states, with critical structural and dynamical features relevant to protein function. Conclusions Overall, QAA provides a novel framework to intuitively understand the biophysical basis of conformational diversity and its relevance to protein function. PMID:21297978

  6. Physiological and comparative genomic analysis of Acidithiobacillus ferrivorans PQ33 provides psychrotolerant fitness evidence for oxidation at low temperature.

    PubMed

    Ccorahua-Santo, Robert; Eca, Anika; Abanto, Michel; Guerra, Gregory; Ramírez, Pablo

    2017-02-21

    Friendly environmental hydrometallurgy at low temperatures is principally promoted by Acidithiobacillus ferrivorans. Until recently, the synergy between cold tolerance and the molecular mechanism of ferrous iron (Fe(2+)) oxidation was unknown. In the present paper, we conducted a physiological and comparative genomics analysis of the new strain A. ferrivorans PQ33 to elucidate the oxidation mechanism at low temperatures, with emphasis placed on trehalose and the Rus operon. PQ33 exhibited a doubling time of 66.6 h in Fe(2+) at pH 1.6 and 63.6 h in CuS at 5 °C. Genomic island (GI) identification and comparative genome analysis were performed with four available genomes of Acidithiobacillus sp. The genome comprised 3,298,172 bp and 56.55% GC content. In contrast to ATCC Acidithiobacillus ferrooxidans strains, the genome of A. ferrivorans PQ33 harbors one GI, which contains a RusB gene. Moreover, five genes of peptidyl-prolyl cis-trans isomerase (PPIases) were observed. Furthermore, comparative analysis of the trehalose operon suggested the presence of a horizontal transfer event. In addition, comparison of rusticyanin proteins revealed that RusB has better intrinsic flexibility than RusA. This comparison suggests psychrotolerant fitness and supports the genetic canalization of A. ferrivorans PQ33 for oxidation at low temperature.

  7. 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.

  8. Immunoglobulin domains in Escherichia coli and other enterobacteria: from pathogenesis to applications in antibody technologies.

    PubMed

    Bodelón, Gustavo; Palomino, Carmen; Fernández, Luis Ángel

    2013-03-01

    The immunoglobulin (Ig) protein domain is widespread in nature having a well-recognized role in proteins of the immune system. In this review, we describe the proteins containing Ig-like domains in Escherichia coli and enterobacteria, reporting their structural and functional properties, protein folding, and diverse biological roles. In addition, we cover the expression of heterologous Ig domains in E. coli owing to its biotechnological application for expression and selection of antibody fragments and full-length IgG molecules. Ig-like domains in E. coli and enterobacteria are frequently found in cell surface proteins and fimbrial organelles playing important functions during host cell adhesion and invasion of pathogenic strains, being structural components of pilus and nonpilus fimbrial systems and members of the intimin/invasin family of outer membrane (OM) adhesins. Ig-like domains are also found in periplasmic chaperones and OM usher proteins assembling fimbriae, in oxidoreductases and hydrolytic enzymes, ATP-binding cassette transporters, sugar-binding and metal-resistance proteins. The folding of most E. coli Ig-like domains is assisted by periplasmic chaperones, peptidyl-prolyl cis/trans isomerases and disulfide bond catalysts that also participate in the folding of antibodies expressed in this bacterium. The technologies for expression and selection of recombinant antibodies in E. coli are described along with their biotechnological potential.

  9. Proteomic Analysis of Oesophagostomum dentatum (Nematoda) during Larval Transition, and the Effects of Hydrolase Inhibitors on Development

    PubMed Central

    Ondrovics, Martina; Silbermayr, Katja; Mitreva, Makedonka; Young, Neil D.; Razzazi-Fazeli, Ebrahim; Gasser, Robin B.; Joachim, Anja

    2013-01-01

    In this study, in vitro drug testing was combined with proteomic and bioinformatic analyses to identify and characterize proteins involved in larval development of Oesophagostomum dentatum, an economically important parasitic nematode. Four hydrolase inhibitors ο-phenanthroline, sodium fluoride, iodoacetamide and 1,2-epoxy-3-(pnitrophenoxy)-propane (EPNP) significantly inhibited (≥90%) larval development. Comparison of the proteomic profiles of the development-inhibited larvae with those of uninhibited control larvae using two-dimensional gel electrophoresis, and subsequent MALDI-TOF mass spectrometric analysis identified a down-regulation of 12 proteins inferred to be involved in various larval developmental processes, including post-embryonic development and growth. Furthermore, three proteins (i.e. intermediate filament protein B, tropomyosin and peptidyl-prolyl cis-trans isomerase) inferred to be involved in the moulting process were down-regulated in moulting- and development-inhibited O. dentatum larvae. This first proteomic map of O. dentatum larvae provides insights in the protein profile of larval development in this parasitic nematode, and significantly improves our understanding of the fundamental biology of its development. The results and the approach used might assist in developing new interventions against parasitic nematodes by blocking or disrupting their key biological pathways. PMID:23717515

  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. 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

  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. Proteomic analysis of stress-related proteins in transgenic broccoli harboring a gene for cytokinin production during postharvest senescence.

    PubMed

    Liu, Mao-Sen; Li, Hui-Chun; Chang, You-Min; Wu, Min-Tze; Chen, Long-Fang Oliver

    2011-09-01

    Our previous study revealed a cytokinin-related retardation of post-harvest floret yellowing in transgenic broccoli (Brassica oleracea var. italica) that harbored the bacterial isopentenyltransferase (ipt) gene. We aimed to investigate the underlining mechanism of this delayed post-harvest senescence. We used 2D electrophoresis and liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry for a proteomics analysis of heads of ipt-transgenic and non-transgenic inbred lines of broccoli at harvest and after four days post-harvest storage. At harvest, we found an accumulation of stress-responsive proteins involved in maintenance of protein folding (putative protein disulfide isomerase, peptidyl-prolyl cis-trans isomerase and chaperonins), scavenging of reactive oxygen species (Mn superoxide dismutase), and stress protection [myrosinase-binding protein, jasmonate inducible protein, dynamin-like protein, NADH dehydrogenase (ubiquinone) Fe-S protein 1 and stress-inducible tetratricopeptide repeat-containing protein]. After four days' post-harvest storage of non-transgenic broccoli florets, the levels of proteins involved in protein folding and carbon fixation were decreased, which indicates cellular degradation and a change in metabolism toward senescence. In addition, staining for antioxidant enzyme activity of non-transgenic plants after post-harvest storage revealed a marked decrease in activity of Fe-superoxide dismutase and ascorbate peroxidase. Thus, the accumulation of stress-responsive proteins and antioxidant enzyme activity in ipt-transgenic broccoli are most likely associated with retardation of post-harvest senescence.

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

    PubMed

    Engdahl, Cecilia; Näslund, Jonas; Lindgren, Lena; Ahlm, Clas; Bucht, Göran

    2012-07-28

    Rift Valley Fever is an infectious viral disease and an emerging problem in many countries of Africa and on the Arabian Peninsula. The causative virus is predominantly transmitted by mosquitoes and high mortality and abortion rates characterize outbreaks in animals while symptoms ranging from mild to life-threatening encephalitis and hemorrhagic fever are noticed among infected humans. For a better prevention and treatment of the infection, an increased knowledge of the infectious process of the virus is required. The focus of this work was to identify protein-protein interactions between the non-structural protein (NSm), encoded by the M-segment of the virus, and host cell proteins. This study was initiated by screening approximately 26 million cDNA clones of a mouse embryonic cDNA library for interactions with the NSm protein using a yeast two-hybrid system. We have identified nine murine proteins that interact with NSm protein of Rift Valley Fever virus, and the putative protein-protein interactions were confirmed by growth selection procedures and β-gal activity measurements. Our results suggest that the cleavage and polyadenylation specificity factor subunit 2 (Cpsf2), the peptidyl-prolyl cis-trans isomerase (cyclophilin)-like 2 protein (Ppil2), and the synaptosome-associated protein of 25 kDa (SNAP-25) are the most promising targets for the NSm protein of the virus during an infection.

  15. 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.

  16. 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.

  17. 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.

  18. Dipentamethylene thiuram monosulfide is a novel inhibitor of Pin1.

    PubMed

    Tatara, Yota; Lin, Yi-Chin; Bamba, Yoshimasa; Mori, Tadashi; Uchida, Takafumi

    2009-07-03

    Pin1 is involved in eukaryotic cell proliferation by changing the structure and function of phosphorylated proteins. PiB, the Pin1 specific inhibitor, blocks cancer cell proliferation. However, low solubility of PiB in DMSO has limited studies of its effectiveness. We screened for additional Pin1 inhibitors and identified the DMSO-soluble compound dipentamethylene thiuram monosulfide (DTM) that inhibits Pin1 activity with an EC50 value of 4.1 microM. Molecular modeling and enzyme kinetic analysis indicated that DTM competitively inhibits Pin1 activity, with a K(i) value of 0.05 microM. The K(D) value of DTM with Pin1 was determined to be 0.06 microM by SPR technology. Moreover, DTM specifically inhibited peptidyl-prolyl cis/trans isomerase activity in HeLa cells. FACS analysis showed that DTM induced G0 arrest of the HCT116 cells. Our results suggest that DTM has the potential to guide the development of novel antifungal and/or anticancer drugs.

  19. The essential role of FKBP38 in regulating phosphatase of regenerating liver 3 (PRL-3) protein stability.

    PubMed

    Choi, Myung-Suk; Min, Sang-Hyun; Jung, Haiyoung; Lee, Ju Dong; Lee, Tae Ho; Lee, Heung Kyu; Yoo, Ook-Joon

    2011-03-11

    The phosphatase of regenerating liver-3 (PRL-3) is a member of protein tyrosine phosphatases and whose deregulation is implicated in tumorigenesis and metastasis of many cancers. However, the underlying mechanism by which PRL-3 is regulated is not known. In this study, we identified the peptidyl prolyl cis/trans isomerase FK506-binding protein 38 (FKBP38) as an interacting protein of PRL-3 using a yeast two-hybrid system. FKBP38 specifically binds to PRL-3 in vivo, and that the N-terminal region of FKBP38 is crucial for binding with PRL-3. FKBP38 overexpression reduces endogenous PRL-3 expression levels, whereas the depletion of FKBP38 by siRNA increases the level of PRL-3 protein. Moreover, FKBP38 promotes degradation of endogenous PRL-3 protein via protein-proteasome pathway. Furthermore, FKBP38 suppresses PRL-3-mediated p53 activity and cell proliferation. These results demonstrate that FKBP38 is a novel regulator of the oncogenic protein PRL-3 abundance and that alteration in the stability of PRL-3 can have a dramatic impact on cell proliferation. Thus, FKBP38 may play a critical role in tumorigenesis.

  20. 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

  1. Cyclophilin-40 has a cellular role in the aryl hydrocarbon receptor signaling.

    PubMed

    Luu, Tony C; Bhattacharya, Pompeya; Chan, William K

    2008-09-22

    Cyclophilin-40 (CyP40) promotes the formation of the gel shift complex that contains the aryl hydrocarbon receptor (AhR), AhR nuclear translocator (Arnt) and dioxin response element (DRE) using baculovirus expressed proteins. Here we reported that CyP40 plays a role in the AhR signaling. When the CyP40 content in MCF-7 cells is reduced, up-regulation of cyp1a1 and cyp1b1 by 3-methylchloranthrene (3MC) is also reduced, suggesting that CyP40 is essential for maximal AhR function. The CyP40 region containing amino acids 186-215, but not the peptidyl-prolyl cis-trans isomerase and tetratricopeptide repeat domains, is essential for forming the AhR/Arnt/DRE complex. CyP40 is found in the cell nucleus after 3MC treatment and appears to promote the DRE binding form of the AhR/Arnt heterodimer.

  2. Application of cDNA array for studying the gene expression profile of mature appressoria of Magnaporthe grisea *

    PubMed Central

    Jin, Qing-chao; Dong, Hai-tao; Peng, You-liang; Chen, Bao-shan; Shao, Jing; Deng, Ye; Dai, Cheng-en; Fang, Yong-qi; Lou, Yi-chun; Li, You-zhi; Li, De-bao

    2007-01-01

    Appressorium is an infection structure of the phytopathogenic fungus Magnaporthe grisea. Analysis of gene expression profiles of appressorium development provides insight into the molecular basis of pathogenicity and control of this fungal plant disease. A cDNA array representing 2927 unique genes based on a large EST (expressed sequence tag) database of M. grisea strain Y34 was constructed and used to profile the gene expression patterns at mycelium and appressorium maturation stages. Compared with mycelia, 55 up-regulated and 22 down-regulated genes were identified in mature appressoria. Among 77 genes, 16 genes showed no similarity to the genome sequences of M. grisea. A novel homologue of peptidyl-prolyl cis-trans isomerase was found to be expressed at low-level in mature appressoria of M. grisea. The results indicated that the genes such as pyruvate carboxylase, phospholipid metabolism-related protein and glyceraldehyde 3-phosphate dehydrogenase involved in gluconeogenesis, lipid metabolism and glycolysis, showed differential expression in mature appressoria. Furthermore, genes such as PTH11, beta subunit of G protein and SGT1 involved in cell signalling, were expressed differentially in mature appressoria. Northern blot analysis was used to confirm the cDNA array results. PMID:17266183

  3. 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

  4. 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.

  5. Down-regulation of Pin1 in Temporal Lobe Epilepsy Patients and Mouse Model.

    PubMed

    Tang, Lan; Zhang, Yanke; Chen, Guojun; Xiong, Yan; Wang, Xuefeng; Zhu, Binglin

    2017-02-27

    Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) is a unique PPIase belonging to the parvulin family, and it isomerizes peptide bond between phospho-(Ser/Thr) and Pro. Pin1 has been linked to the pathogenesis of various human diseases; however, its exact biological functions remain unclear. The aim of the present study is to explore the expression pattern of Pin1 in patients with refractory epilepsy and in a chronic pilocarpine-induced epileptic mouse model. Using Western blot, immunofluorescence and immunoprecipitation analysis, we found that Pin1 protein was mainly distributed in neurons, demonstrated by colocalization with the dendritic marker, MAP2. However, the expression of Pin1 decreased remarkably in epileptic patients and experimental mice. Furthermore, the reciprocal coimmunoprecipitation analysis showed that Pin1 interacted with NR2A and NR2B-containing NMDA receptors not AMPA receptors in epileptic mouse models. Our results are the first to indicate that the expression of Pin1 in epileptic brain tissue could play important roles in epilepsy.

  6. Comparative proteomic analysis of paclitaxel resistance-related proteins in human breast cancer cell lines

    PubMed Central

    Fujioka, Hiroya; Sakai, Akiko; Tanaka, Satoru; Kimura, Kosei; Miyamoto, Akiko; Iwamoto, Mitsuhiko; Uchiyama, Kazuhisa

    2017-01-01

    Paclitaxel is widely used to treat various cancers; however, resistance to this drug is a major obstacle to breast cancer chemotherapy. To identify the proteins involved in paclitaxel resistance, the present study compared the proteomes of MCF-7 human breast cancer cells and its paclitaxel-resistant subclone MCF-7/PTX. Using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry, 11 upregulated and 12 downregulated proteins were identified in MCF-7/PTX cells compared with the parental cell line. These 23 proteins were functionally classified as stress-induced chaperones, metabolic enzymes and cytoskeletal proteins. The anti-apoptotic proteins, stress-70 protein, 78-kD glucose-regulated protein, peptidyl-prolyl cis-trans isomerase A (PPIA) and heterogeneous nuclear ribonucleoprotein H3, were also upregulated in MCF-7/PTX cells. Notably, knockdown of the stress-response chaperone PPIA using small interfering RNA in MCF-7/PTX cells restored their sensitivity to paclitaxel. These findings indicated that PPIA may have an important role in paclitaxel resistance in MCF-7/PTX cells. PMID:28123557

  7. Responses of Mytilus galloprovincialis to bacterial challenges by metabolomics and proteomics.

    PubMed

    Ji, Chenglong; Wu, Huifeng; Wei, Lei; Zhao, Jianmin; Wang, Qing; Lu, Hongjian

    2013-08-01

    Pathogens can cause diseases and lead to massive mortalities of aquaculture animals and substantial economic loss. In this work, we studied the responses induced by Micrococcus luteus and Vibrio anguillarum in gill of mussel Mytilus galloprovincialis at protein and metabolite levels. Metabolic biomarkers (e.g., amino acids, betaine, ATP) suggested that both M. luteus and V. anguillarum induced disturbances in energy metabolism and osmotic regulation. The unique and some more remarkably altered metabolic biomarkers (threonine, alanine, aspartate, taurine, succinate) demonstrated that V. anguillarum could cause more severe disturbances in osmotic regulation and energy metabolism. Proteomic biomarkers (e.g., goose-type lysozyme 2, matrilin, ependymin-related protein, peptidyl-prolyl cis-trans isomerases) indicated that M. luteus caused immune stress, and disturbances in signaling pathways and protein synthesis. However, V. anguillarum mainly induced oxidative stress and disturbance in energy metabolism in mussel gills indicated by altered procollagen-proline dioxygenase, protein disulfide isomerase, nucleoside diphosphate kinases, electron transfer flavoprotein and glutathione S-transferase. This work confirmed that an integration of proteomics and metabolomics could provide an insightful view into the effects of pathogens to the marine mussel M. galloprovincialis.

  8. Conformational state of a 25-mer peptide from the cyclophilin-binding loop of the HIV type 1 capsid protein.

    PubMed Central

    Reimer, U; Drewello, M; Jakob, M; Fischer, G; Schutkowski, M

    1997-01-01

    Recently a 25-residue part of Gag polyprotein from HIV type 1 (HIV-1) was reported to bind to the cytosolic 18 kDa cyclophilin (Cyp18) with an IC50 value of 180 microM. This peptide corresponds to the Cyp18-binding domain of HIV-1 Gag. A replacement of Gly with Ala in the cyclophilin-binding loop of HIV-1 Gag polyprotein results in the prevention of the packaging of Cyp18 into virions. We found only two conformers of this peptide among 16 possible expected conformers, owing to cis/trans isomerization of four peptidyl-prolyl bonds. Although this finding implicates the existence of a stabilizing structure, we were not able to detect secondary structure formation by 1H-NMR and CD spectroscopy. We characterized the peptide as a substrate for Cyp18 by two-dimensional exchange 1H-NMR spectroscopy. Surprisingly, we found similar binding characteristics for a peptide corresponding to 25-mer peptide containing the above-mentioned Gly to Ala substitution. PMID:9337866

  9. 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

  10. A novel multi-functional chloroplast protein: identification of a 40 kDa immunophilin-like protein located in the thylakoid lumen.

    PubMed Central

    Fulgosi, H; Vener, A V; Altschmied, L; Herrmann, R G; Andersson, B

    1998-01-01

    We describe the identification of the first immunophilin associated with the photosynthetic membrane of chloroplasts. This complex 40 kDa immunophilin, designated TLP40 (thylakoid lumen PPIase), located in the lumen of the thylakoids, was found to play a dual role in photosynthesis involving both biogenesis and intraorganelle signalling. It originates in a single-copy nuclear gene, is made as a precursor of 49.2 kDa with a bipartite lumenal targeting transit peptide, and is characterized by a structure including a cyclophilin-like C-terminal segment of 20 kDa, a predicted N-terminal leucine zipper and a potential phosphatase-binding domain. It can exist in different oligomeric conformations and attach to the inner membrane surface. It is confined predominantly to the non-appressed thylakoid regions, the site of protein integration into the photosynthetic membrane. The isolated protein possesses peptidyl-prolyl cis-trans isomerase protein folding activity characteristic of immunophilins, but is not inhibited by cyclosporin A. TLP40 also exerts an effect on dephosphorylation of several key proteins of photosystem II, probably as a constituent of a transmembrane signal transduction chain. This first evidence for a direct role of immunophilins in a photoautotrophic process suggests that light-mediated protein phosphorylation in photosynthetic membranes and the role of the thylakoid lumen are substantially more complex than anticipated. PMID:9501079

  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. Catalytic and ligand binding properties of the FK506 binding protein FKBP12: effects of the single amino acid substitution of Tyr82 to Leu.

    PubMed Central

    Bossard, M J; Bergsma, D J; Brandt, M; Livi, G P; Eng, W K; Johnson, R K; Levy, M A

    1994-01-01

    The binding of FK506 and rapamycin to their cytosolic receptor FKBP12 is an intermediate step in the paths leading to their potent immunosuppressive properties. One of the amino acids defining the hydrophobic binding cleft for the macrocycles is Tyr82, which is thought to form a hydrogen bond with the amide oxygens of the common pipecolyl structural element within the two macrolides. To understand better the influence of this amino acid residue in catalytic activity (cis-trans peptidyl prolyl isomerization) and ligand binding properties, a Tyr82 to Leu site-specific modification of FKBP12 was prepared, purified and characterized. Kinetic experiments have demonstrated that the Tyr82 to Leu modification has a greater effect on catalytic properties than on ligand binding affinities, a result which indicates that these inhibitors may not be binding as true transition-state analogues. In an additional test for cellular function, expression of both wild-type and mutant human FKBP12 in a strain of Saccharomyces cerevisiae rendered resistant to rapamycin by deletion of the gene encoding a cytosolic rapamycin binding protein (RPB1), the yeast homologue of FKBP12, restored wild-type drug sensitivity. PMID:7507662

  13. Proteomic changes in female rat hippocampus following exposure to a terrified sound stress.

    PubMed

    Yang, Juan; Hu, Lili; Song, Tusheng; Liu, Yong; Wu, Qiuhua; Zhao, Lingyu; Liu, Liying; Zhao, Xiaoge; Zhang, Dianzeng; Huang, Chen

    2014-06-01

    Stress plays a profound role in the onset of affective disorders, including an elevation in risk factors for depression and anxiety. Women are twice as vulnerable to stress as men because of greater sensitivity to a substance produced during times of anxiety. To better define the abnormal proteins implicated in cognitive deficits and other stress-induced dysfunction, female rats were exposed to terrified sound stress, and two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) were utilized to determine the differential protein expression in the hippocampus in sound-stressed female rats compared with controls. Quantitative differences were found in 44 protein spots which were differentially expressed between the stressed and control groups (fold change of >2; p < 0.01). Eighteen protein spots were downregulated, and 26 protein spots were upregulated in the stressed group. The seven most differentially expressed proteins were identified and validated as follows: dihydropyrimidinase-related protein 2 (DRP-2), creatine kinase B type, dynamin-1 protein, alpha-internexin, glial fibrillary acidic protein beta, gamma-enolase, and peptidyl-prolyl cis-trans isomerase A. Changes in protein levels were detected in the hippocampus of female rats subjected to terrified sound stress. The findings herein may open new opportunities for further investigations on the modulation induced in the hippocampus by stress at the molecular level, especially with respect to females stress.

  14. 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.

  15. Hydroxytyrosol rich extract from olive leaves modulates cell cycle progression in MCF-7 human breast cancer cells.

    PubMed

    Bouallagui, Zouhaier; Han, Junkuy; Isoda, Hiroko; Sayadi, Sami

    2011-01-01

    Throughout the history, olive (Olea europea L.) leaves have been heavily exploited for the prevention or the treatment of hypertension, carcinogenesis, diabetes, atherosclerosis and so many other traditional therapeutic uses. These activities are thought to be the output of olive micronutrients especially polyphenols. Hydroxytyrosol and oleuropein are considered as major polyphenolic compounds in olive leaf. In this work, a hydroxytyrosol rich olive leaves extract was investigated for potential anti-tumoral activities. In vitro cytotoxic effects against MCF-7 breast cancer cells were examined using MTT and neutral red tests. The anti-tumor activities were further investigated by flow cytometry and western blotting. Cytotoxicity assays resulted in a dose dependent growth inhibition of MCF-7 cells. This inhibition was due to the cell cycle arrest in the G0/G1 phase. The understanding of the molecular mechanism by which olive leaves extract arrested cell growth showed a down-expression of the peptidyl-prolyl cis-trans isomerase Pin1 which in turn decreased the level of a G1 key protein; Cyclin D1. Additionally, olive leaves extract treatment up-regulated the AP1 transcription factor member, c-jun. Therefore, olive leaves extract will necessitate further deep investigation for a probable use as a cancer preventive food additive.

  16. 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

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

    PubMed

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

    2013-01-15

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

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

    PubMed Central

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

    2014-01-01

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

  19. 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.

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

    PubMed

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

    2008-08-01

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

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

    PubMed

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

    1998-01-02

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

  2. 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

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

    PubMed

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

    2009-11-12

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

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

    PubMed

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

    2016-12-05

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

  5. 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.

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

    PubMed

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

    2014-02-15

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

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

    PubMed

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

    2013-09-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2009-10-01

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

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

    PubMed Central

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

    2017-01-01

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

  12. 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

  13. 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

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

    PubMed Central

    Sanz, Yolanda; Toldrá, Fidel

    2001-01-01

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

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

    PubMed

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

    2011-12-01

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

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

    PubMed

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

    2011-01-01

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

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

    PubMed

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

    2011-09-01

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

  19. 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

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

    PubMed

    Soni, Hitesh

    2014-05-01

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

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

    PubMed Central

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

    2006-01-01

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

  2. Notch Downregulation and Extramedullary Erythrocytosis in Hypoxia-Inducible Factor Prolyl 4-Hydroxylase 2-Deficient Mice.

    PubMed

    Myllymäki, Mikko N M; Määttä, Jenni; Dimova, Elitsa Y; Izzi, Valerio; Väisänen, Timo; Myllyharju, Johanna; Koivunen, Peppi; Serpi, Raisa

    2017-01-15

    Erythrocytosis is driven mainly by erythropoietin, which is regulated by hypoxia-inducible factor (HIF). Mutations in HIF prolyl 4-hydroxylase 2 (HIF-P4H-2) (PHD2/EGLN1), the major downregulator of HIFα subunits, are found in familiar erythrocytosis, and large-spectrum conditional inactivation of HIF-P4H-2 in mice leads to severe erythrocytosis. Although bone marrow is the primary site for erythropoiesis, spleen remains capable of extramedullary erythropoiesis. We studied HIF-P4H-2-deficient (Hif-p4h-2(gt/gt)) mice, which show slightly induced erythropoiesis upon aging despite nonincreased erythropoietin levels, and identified spleen as the site of extramedullary erythropoiesis. Splenic hematopoietic stem cells (HSCs) of these mice exhibited increased erythroid burst-forming unit (BFU-E) growth, and the mice were protected against anemia. HIF-1α and HIF-2α were stabilized in the spleens, while the Notch ligand genes Jag1, Jag2, and Dll1 and target Hes1 became downregulated upon aging HIF-2α dependently. Inhibition of Notch signaling in wild-type spleen HSCs phenocopied the increased BFU-E growth. HIFα stabilization can thus mediate non-erythropoietin-driven splenic erythropoiesis via altered Notch signaling.

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

    PubMed Central

    Thalhammer, Armin; Demetriades, Marina; Chowdhury, Rasheduzzaman; Tian, Ya-Min; Stolze, Ineke; McNeill, Luke A.; Lee, Myung Kyu; Woon, Esther C. Y.; Mackeen, Mukram M.; Kawamura, Akane; Ratcliffe, Peter J.; Mecinović, Jasmin; Schofield, Christopher J.

    2015-01-01

    Inhibition of the hypoxia-inducible factor (HIF) prolyl-hydroxylases (PHD or EGLN enzymes) is of interest for the treatment of anemia and ischemia-related diseases. Most PHD inhibitors work by binding to the single ferrous ion and competing with 2-oxoglutarate (2OG) co-substrate for binding at the PHD active site. Non-specific iron chelators also inhibit the PHDs, both in vitro and in cells. We report the identification of dual action PHD inhibitors, which bind to the active site iron and also induce the binding of a second iron ion at the active site. Following analysis of small-molecule iron complexes and application of non-denaturing protein mass spectrometry to assess PHD2·iron·inhibitor stoichimetry, selected diacylhydrazines were identified as PHD2 inhibitors that induce the binding of a second iron ion. Some compounds were shown to inhibit the HIF hydroxylases in human hepatoma and renal carcinoma cell lines. PMID:23151668

  4. Hyperplasia of pulmonary neuroepithelial bodies (NEB) in lungs of prolyl hydroxylase -1(PHD-1) deficient mice.

    PubMed

    Pan, Jie; Yeger, Herman; Ratcliffe, Peter; Bishop, Tammie; Cutz, Ernest

    2012-01-01

    Pulmonary NEB, widely distributed within the airway mucosa of mammalian lungs, are presumed hypoxia sensitive airway O(2) sensors responding to changes in airway gas concentration. NEB cell hyperplasia has been reported after exposure to chronic hypoxia and in a variety of paediatric and adult lung disorders. Prolyl hydroxylases (PHD 1-3) regulate the stability of hypoxia-inducible factors (HIF's) in an O(2)-dependent manner and function as intrinsic oxygen sensors. To determine a possible role of PHD-1in NEB cells we have quantitated NEB's in lungs of neonatal (P2) and adult (2 months) PHD-1-deficient mice and compared them to wild type (WT) control mice. Lung tissues fixed in formalin and embedded in paraffin were processed for immunoperoxidase method and frozen sections for multilabel immunoflourescence using antibodies for NEB markers synaptophysin, synaptic vesicle protein 2 and the peptide CGRP. The frequency and size of NEB in lungs of PHD-1 deficient neonatal mice (P2) and at 2 months was increased significantly compared to WT controls (p < 0.01). The present data suggests an important role for PHD enzymes in NEB cell biology deserving further studies. Since the PHD-1 deficient mouse appears to be the first animal model showing NEB cell hyperplasia it may be useful for studies of NEB physiology and pathobiology.

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

    PubMed

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

    2017-02-01

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

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

    PubMed

    Simonović, Miljan; Steitz, Thomas A

    2008-11-01

    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.

  7. 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.

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

    PubMed

    Arafet, Kemel; Ferrer, Silvia; Moliner, Vicent

    2015-06-02

    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.

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

    PubMed

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

    2014-07-01

    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.

  10. Alternative mRNA splicing generates multiple forms of peptidyl-glycine alpha-amidating monooxygenase in rat atrium.

    PubMed Central

    Stoffers, D A; Green, C B; Eipper, B A

    1989-01-01

    Peptidyl-glycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the conversion of a variety of glycine-extended peptides into biologically active alpha-amidated product peptides in a reaction dependent on copper, ascorbate, and molecular oxygen. We have isolated and sequenced cDNAs representing the two major classes of PAM mRNA in the adult rat heart atrium. The two types of cDNA, rPAM-1 and rPAM-2, are identical except for the deletion of a 315-base-pair segment within the protein coding region in rPAM-2, suggesting that rPAM-1 and rPAM-2 arise by alternative splicing. Northern analysis using a cDNA probe derived from within the 315-base-pair region deleted in rPAM-2 visualized the larger of the PAM mRNAs in adult rat atrium and not the smaller, indicating that the presence or absence of this 315-nucleotide segment is a major feature distinguishing the two size forms of PAM mRNA. The 105 amino acid segment that distinguishes the two forms of atrial PAM contains a consensus N-glycosylation site and a paired basic amino acid site of potential importance in endoproteolytic processing. Comparison of the nucleotide sequences of rat, frog, and bovine PAM cDNAs reveals an extremely well conserved segment in the 3' untranslated region. The high degree of conservation in amino acid sequence throughout the catalytic, intragranular, and cytoplasmic domains of rat atrium, bovine pituitary, and frog skin PAM suggests that both the catalytic and noncatalytic domains of the protein subserve important functions. Images PMID:2911604

  11. Molecular docking and 3D-quantitative structure activity relationship analyses of peptidyl vinyl sulfones: Plasmodium Falciparum cysteine proteases inhibitors

    NASA Astrophysics Data System (ADS)

    Teixeira, Cátia; Gomes, José R. B.; Couesnon, Thierry; Gomes, Paula

    2011-08-01

    Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) based on three-dimensional quantitative structure-activity relationship (3D-QSAR) studies were conducted on a series (39 molecules) of peptidyl vinyl sulfone derivatives as potential Plasmodium Falciparum cysteine proteases inhibitors. Two different methods of alignment were employed: (i) a receptor-docked alignment derived from the structure-based docking algorithm GOLD and (ii) a ligand-based alignment using the structure of one of the ligands derived from a crystal structure from the PDB databank. The best predictions were obtained for the receptor-docked alignment with a CoMFA standard model ( q 2 = 0.696 and r 2 = 0.980) and with CoMSIA combined electrostatic, and hydrophobic fields ( q 2 = 0.711 and r 2 = 0.992). Both models were validated by a test set of nine compounds and gave satisfactory predictive r 2 pred values of 0.76 and 0.74, respectively. CoMFA and CoMSIA contour maps were used to identify critical regions where any change in the steric, electrostatic, and hydrophobic fields may affect the inhibitory activity, and to highlight the key structural features required for biological activity. Moreover, the results obtained from 3D-QSAR analyses were superimposed on the Plasmodium Falciparum cysteine proteases active site and the main interactions were studied. The present work provides extremely useful guidelines for future structural modifications of this class of compounds towards the development of superior antimalarials.

  12. Identification of prolyl carboxypeptidase as an alternative enzyme for processing of renal angiotensin II using mass spectrometry

    PubMed Central

    Grobe, Nadja; Weir, Nathan M.; Leiva, Orly; Ong, Frank S.; Bernstein, Kenneth E.; Schmaier, Alvin H.; Morris, Mariana

    2013-01-01

    Angiotensin-converting enzyme 2 (ACE2) catalyzes conversion of ANG II to ANG-(1–7). The present study uses newly established proteomic approaches and genetic mouse models to examine the contribution of alternative renal peptidases to ACE2-independent formation of ANG-(1–7). In situ and in vitro mass spectrometric characterization showed that substrate concentration and pH control renal ANG II processing. At pH ≥6, ANG-(1–7) formation was significantly reduced in ACE2 knockout (KO) mice. However, at pH <6, formation of ANG-(1–7) in ACE2 KO mice was similar to that in wild-type (WT) mice, suggesting alternative peptidases for renal ANG II processing. Furthermore, the dual prolyl carboxypeptidase (PCP)-prolyl endopeptidase (PEP) inhibitor Z-prolyl-prolinal reduced ANG-(1–7) formation in ACE2 KO mice, while the ACE2 inhibitor MLN-4760 had no effect. Unlike the ACE2 KO mice, ANG-(1–7) formation from ANG II in PEP KO mice was not different from that in WT mice at any tested pH. However, at pH 5, this reaction was significantly reduced in kidneys and urine of PCP-depleted mice. In conclusion, results suggest that ACE2 metabolizes ANG II in the kidney at neutral and basic pH, while PCP catalyzes the same reaction at acidic pH. This is the first report demonstrating that renal ANG-(1–7) formation from ANG II is independent of ACE2. Elucidation of ACE2-independent ANG-(1–7) production pathways may have clinically important implications in patients with metabolic and renal disease. PMID:23392115

  13. The effectiveness of inhibitors of soluble prolyl hydroxylase against the enzyme in the cisternae of isolated bone microsomes.

    PubMed

    Tschank, G; Hanauske-Abel, H M; Peterkofsky, B

    1988-03-01

    Inhibitors of purified, soluble prolyl hydroxylase (K. Majamaa et al. (1984) Eur. J. Biochem. 138, 239-245; K. Majamaa et al. (1986) J. Biol. Chem. 261, 7819-7823) were tested against isolated chick embryo bone microsomes containing intracisternal prolyl hydroxylase and its radiolabeled, unhydroxylated procollagen substrate. Two groups of inhibitors were used which consisted of pyridine-2-carboxylate and 1,2-dihydroxybenzene (catechol) derivatives. The 2,4- and 2,5-pyridine dicarboxylic acids, which are potent inhibitors of the soluble enzyme (Ki values 2 and 0.8 microM, respectively), were effective in the same concentration range against intracisternal prolyl hydroxylase, although their relative affinities were reversed. Inhibition by pyridine-2,4-dicarboxylate in the microsomal system was reversed by increasing the concentration of 2-oxoglutarate. Pyridine-2,4-dicarboxylic acid did not inhibit the uptake of 2-[14C]oxoglutarate into microsomes, so it appears likely that the inhibitor must traverse the microsomal membrane and act directly at the enzyme level. Pyridine-2-carboxylic acid was ineffective in the microsomal system at 1 mM whereas it is a relatively potent inhibitor of the soluble enzyme with a Ki of 25 microM. This finding suggests that the second carboxyl group of the pyridine carboxylate derivatives may be required for their transport into the microsomal lumen. In the soluble system, 3,4-dihydroxybenzoic acid and 1,2-dihydroxybenzene had been found to be competitive inhibitors with relatively low Ki values of 5 and 25 microM, respectively. In the microsomal system, half-maximal inhibition was obtained at approximately 50-100 microM and inhibition was not reversed by increasing the concentrations of either 2-oxoglutarate or ascorbate, alone or together. These results imply that in situ these compounds do not inhibit prolyl hydroxylase directly. Thus, the microsomal system can assess the accessibility of the intracisternal enzyme to potential

  14. Effect of prolyl hydroxylase domain 2 haplodeficiency on liver progenitor cell characteristics in early mouse hepatocarcinogenesis

    PubMed Central

    Bogaerts, Eliene; Paridaens, Annelies; Verhelst, Xavier; Carmeliet, Peter; Geerts, Anja; Van Vlierberghe, Hans; Devisscher, Lindsey

    2016-01-01

    Activation of the hypoxia-inducible factor (HIF)-pathway in hepatocellular carcinoma (HCC) induces therapy resistant tumours, characterized by increased liver progenitor cell (LPCs) characteristics and poor prognosis. We previously reported corresponding results in mice with HCC in which hypoxia was mimicked by prolyl hydroxylase domain (PHD) inhibition. Here, we aimed at investigating whether induction of LPC characteristics occurs during the onset of hepatocarcinogenesis and if this is associated with activation of Notch signalling. Dietheylnitrosamine (DEN) was used to induce hepatic tumours in PHD2 haplodeficient (PHD2+/-) mice which were euthanized at 5, 10, 15 and 17 weeks following DEN during neoplastic transformation, before tumour formation. Neoplasia and mRNA expression of LPC and Notch markers were evaluated by histology and qPCR on isolated livers. PHD2 haplodeficiency resulted in enhanced expression of HIF target genes after 17 weeks of DEN compared to wild type (WT) littermates but had no effect on the onset of neoplastic transformation. The mRNA expression of Afp and Epcam was increased at all time points following DEN whereas CK19, Prom1 and Notch3 were increased after 17 weeks of DEN, without difference between PHD2+/- and WT mice. MDR1 mRNA expression was increased in all DEN treated mice compared to saline control with increased expression in PHD2+/- compared to WT from 15 weeks. These results indicate that the effects of PHD2 haplodeficiency on the expression of LPC and Notch markers manifest during tumour nodule formation and not early on during neoplastic transformation. PMID:28337100

  15. Slow-binding inhibitors of prolyl oligopeptidase with different functional groups at the P1 site.

    PubMed

    Venäläinen, Jarkko I; Juvonen, Risto O; Garcia-Horsman, J Arturo; Wallén, Erik A A; Christiaans, Johannes A M; Jarho, Elina M; Gynther, Jukka; Männistö, Pekka T

    2004-09-15

    POP (prolyl oligopeptidase) specifically hydrolyses a number of small proline-containing peptides at the carboxy end of the proline residue and POP inhibitors have been shown to have cognition-enhancing properties. It has been noted that certain functional groups at the P1 site of the inhibitor, which correspond to the substrate residue on the N-terminal side of the bond to be cleaved, increase the inhibitory potency. However, detailed mechanistic and kinetic analysis of the inhibition has not been studied. In the present study, we examined the effect of different functional groups at the P1 site of the parent inhibitor isophthalic acid bis-(L-prolylpyrrolidine) amide on the binding kinetics to POP. Addition of CHO, CN or COCH(2)OH groups to the P1 site increased the inhibitory potency by two orders of magnitude (K(i)=11.8-0.1 nM) and caused a clear slow-binding inhibition. The inhibitor containing a CHO group had the lowest association rate constant, k(on)=(2.43+/-0.12) x 10(5) M(-1) x s(-1), whereas the inhibitor with a CN group exhibited the fastest binding, k(on)=(12.0+/-0.08)x10(5) M(-1) x s(-1). In addition, the dissociation rate was found to be crucially dependent on the type of the functional group. Compounds with COCH(2)OH and CHO groups had much longer half-lives of dissociation (over 5 h) compared with the compound with the CN group (25 min), although the K(i) values of the compounds were relatively similar. A possibility to optimize the duration of inhibition by changing the functional group at the P1 site is important when planning therapeutically useful POP inhibitors.

  16. 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

  17. Nitric oxide mediates prolyl hydroxylase 3 expression in mesangial cells and in glomerulonephritis.

    PubMed

    Aglan, Ahmed; Longen, Sebastian; Dehne, Nathalie; Köhler, Yvette; Hassan, Mohamed; Beck, Martina; Tredup, Claudia; Boosen, Meike; Hsieh, Tzung-Harn Louise; Schaefer, Liliana; Beck, Karl-Friedrich; Pfeilschifter, Josef

    2017-03-01

    Renal mesangial cells are regarded as main players in glomerular inflammatory diseases. To investigate a possible crosstalk between inflammatory and hypoxia-driven signaling processes, we stimulated cultured mouse mesangial cells with different inflammatory agents and analyzed the expression of prolyl hydroxylase domain containing proteins (PHDs), the main regulators of hypoxia-inducible factor (HIF) stability. Administration of IL-1β (1 nM) and TNF-α (1 nM), a combination further referred to as cytokine mix (CM), resulted in a fivefold increase in PHD3 but not PHD1 and PHD2 mRNA expression compared to untreated controls. In contrast, a combination of IL-1β, TNF-α with lipopolysaccharide (10 μg/ml), and interferon-γ (20 ng/ml) designated as CM+ showed a high (60-fold) induction of PHD3 and a moderate (twofold) induction of PHD2 mRNA expression. Interestingly, CM+ but not CM induced the expression of inducible NO synthase and endogenously produced NO was responsible for the immense induction of PHD3 in mesangial cells treated with CM+. We found that CM+ affected PHD3 expression mainly via the NO/HIF axis, whereas PHD3 regulation by CM occurred in a NF-κB-dependent manner. In turn, silencing of PHD3 expression resulted in a decrease in the mRNA expression of ICAM-1, MIP-2, MCP-1, and CXCL-10, which are under control of NF-κB. In a rat model of mesangio-proliferative glomerulonephritis, PHD3 mRNA and protein expression was markedly induced and this effect was nearly abolished when rats were treated with the iNOS-specific inhibitor L-NIL, thus confirming our findings also in vivo.

  18. 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.

  19. Minute Time Scale Prolyl Isomerization Governs Antibody Recognition of an Intrinsically Disordered Immunodominant Epitope*

    PubMed Central

    Fassolari, Marisol; Chemes, Lucia B.; Gallo, Mariana; Smal, Clara; Sánchez, Ignacio E.; de Prat-Gay, Gonzalo

    2013-01-01

    Conformational rearrangements in antibody·antigen recognition are essential events where kinetic discrimination of isomers expands the universe of combinations. We investigated the interaction mechanism of a monoclonal antibody, M1, raised against E7 from human papillomavirus, a prototypic viral oncoprotein and a model intrinsically disordered protein. The mapped 12-amino acid immunodominant epitope lies within a “hinge” region between the N-terminal intrinsically disordered and the C-terminal globular domains. Kinetic experiments show that despite being within an intrinsically disordered region, the hinge E7 epitope has at least two populations separated by a high energy barrier. Nuclear magnetic resonance traced the origin of this barrier to a very slow (t½ ∼4 min) trans-cis prolyl isomerization event involving changes in secondary structure. The less populated (10%) cis isomer is the binding-competent species, thus requiring the 90% of molecules in the trans configuration to isomerize before binding. The association rate for the cis isomer approaches 6 × 107 m−1 s−1, a ceiling for antigen-antibody interactions. Mutagenesis experiments showed that Pro-41 in E7Ep was required for both binding and isomerization. After a slow postbinding unimolecular rearrangement, a consolidated complex with KD = 1.2 × 10−7 m is reached. Our results suggest that presentation of this viral epitope by the antigen-presenting cells would have to be “locked” in the cis conformation, in opposition to the most populated trans isomer, in order to select the specific antibody clone that goes through affinity and kinetic maturation. PMID:23504368

  20. Prolyl oligopeptidase attenuates hepatic stellate cell activation through induction of Smad7 and PPAR-γ

    PubMed Central

    Zhou, Da; Wang, Jing; He, Ling-Nan; Li, Bing-Hang; Ding, Yong-Nian; Chen, Yuan-Wen; Fan, Jian-Gao

    2017-01-01

    Prolyl oligopeptidase (POP) is a serine endopeptidase widely distributed in vivo with high activity in the liver. However, its biological functions in the liver have remained largely elusive. A previous study by our group has shown that POP produced N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) and thereby exerted an anti-fibrogenic effect on hepatic stellate cells (HSCs) in vitro. It was therefore hypothesized that POP may affect the activation state of HSCs and has an important role in liver fibrosis. The HSC-T6 immortalized rat liver stellate cell line was treated with the POP inhibitor S17092 or transfected with recombinant lentivirus to overexpress POP. Cell proliferation and apoptosis were determined using a Cell Counting Kit-8 and flow cytometry, respectively. The activation status of HSCs was determined by examination of the expression of α-smooth muscle actin (α-SMA), collagen I, monocyte chemoattractant protein-1 (MCP-1), transforming growth factor (TGF)-β-Smad signaling and peroxisome proliferator activated receptor-γ (PPAR-γ). Inhibition by S17092 decreased, whereas lentiviral expression increased the activity of POP and cell proliferation, while neither of the treatments affected cell apoptosis. Of note, S17092 significantly increased, whereas POP overexpression decreased the expression of α-SMA and MCP-1 without affecting the expression of collagen I and TGF-β1. Furthermore, S17092 caused a reduction, whereas POP overexpression caused an upregulation of Smad7 protein and PPAR-γ, but not phosphorylated-Smad2/3 expression. In conclusion, POP attenuated the activation of HSCs through inhibition of TGF-β signaling and induction of PPAR-γ, which may have therapeutic potential in liver fibrosis. PMID:28352366

  1. The role of prolyl hydroxylase domain protein (PHD) during rosiglitazone-induced adipocyte differentiation.

    PubMed

    Kim, Juyoung; Kwak, Hyun Jeong; Cha, Ji-Young; Jeong, Yun-Seung; Rhee, Sang Dahl; Cheon, Hyae Gyeong

    2014-01-31

    Rosiglitazone, a well known insulin sensitizer, stimulates adipocyte differentiation via the activation of peroxisome proliferator-activated receptor γ (PPARγ). Previous two-dimensional proteomics studies using C3H10T1/2 murine mesenchymal pluripotent stem cells revealed that prolyl hydroxylase domain protein (PHD) levels significantly increased during rosiglitazone-induced adipocyte differentiation (RIAD). In this study, we investigated the functional role played by PHD during RIAD. Three PHD isoforms (PHD1, 2, and 3) were found to be up-regulated in C3H10T1/2 cells during RIAD, whereas PHD knockdown and treatment with PHD inhibitors (dimethyloxalyl glycine or ethyl-3,4-dihydroxybenzoate) blocked RIAD. PHD inhibition was found to be associated with increases in the levels of anti-adipogenic proteins such as GATA-3, KLF-2, and transcriptional coactivator with PDZ binding motif (TAZ), with their reduced ubiquitination, suggesting that PHDs evoke the ubiquitination/proteasomal degradation of anti-adipogenic proteins. On the other hand, MG-132 (a proteasomal inhibitor) prevented the degradation of anti-adipogenic proteins and retarded RIAD. PPARγ antagonists (bisphenol A diglycidyl ether or GW9662) blunted the effects of rosiglitazone on PHD regulation. Furthermore, putative PPARγ binding sites were identified in the promoter region of PHDs by ChIP-PCR, implying that rosiglitazone may induce PHD up-regulation directly by PPARγ activation. Consistent with in vitro results, oral administration of rosiglitazone to ob/ob mice for 2 weeks increased adipose PHD levels and decreased anti-adipogenic protein levels by increasing their ubiquitination. These results suggest that rosiglitazone increases PHD expression in a PPARγ-dependent manner and that this leads to the commitment of anti-adipogenic proteins to the ubiquitination-proteasomal pathway and to the subsequent induction of adipocyte differentiation.

  2. A Cu-Catalyzed, pH-Neutral, Aerobic, Room Temperature Construction of High Enantiopurity Peptidyl Ketones from Peptidic S-Acylthiosalicylamides

    PubMed Central

    Liebeskind, Lanny S.; Yang, Hao; Li, Hao

    2009-01-01

    Described herein is a Cu-catalyzed transformation of peptidic thiol esters and boronic acids into peptidyl ketones that takes place at room temperature in DMF or in DMF/H2O open to air and uses only catalytic quantities of a Cu carboxylate to mediate the reaction. This aerobic transformation occurs only at a thiol ester capable of coordinating to Cu through its S-appendage and is hampered neither by racemization of the reactants or products, nor by the presence of disulfides or of unprotected phenols, alcohols, or indoles. PMID:19145620

  3. 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.

  4. Gene Transfer of Prolyl Hydroxylase Domain 2 Inhibits Hypoxia-inducible Angiogenesis in a Model of Choroidal Neovascularization.

    PubMed

    Takei, Anna; Ekström, Malena; Mammadzada, Parviz; Aronsson, Monica; Yu, Ma; Kvanta, Anders; André, Helder

    2017-02-10

    Cellular responses to hypoxia are mediated by the hypoxia-inducible factors (HIF). In normoxia, HIF-α proteins are regulated by a family of dioxygenases, through prolyl and asparagyl hydroxylation, culminating in proteasomal degradation and transcriptional inactivation. In hypoxia, the dioxygenases become inactive and allow formation of HIF transcription factor, responsible for upregulation of hypoxia genes. In ocular neoangiogenic diseases, such as neovascular age-related macular degeneration (nAMD), hypoxia seems pivotal. Here, we investigate the effects of HIF regulatory proteins on the hypoxia pathway in retinal pigment epithelium (RPE) cells, critically involved in nAMD pathogenesis. Our data indicates that, in ARPE-19 cells, prolyl hydroxylase domain (PHD)2 is the most potent negative-regulator of the HIF pathway. The negative effects of PHD2 on the hypoxia pathway were associated with decreased HIF-1α protein levels, and concomitant decrease in angiogenic factors. ARPE-19 cells stably expressing PHD2 impaired angiogenesis in vitro by wound healing, tubulogenesis, and sprouting assays, as well as in vivo by iris-induced angiogenesis. Gene transfer of PHD2 in vivo resulted in mitigation of HIF-mediated angiogenesis in a mouse model of nAMD. These results may have implications for the clinical treatment of nAMD patients, particularly regarding the use of gene therapy to negatively regulate neoangiogenesis.

  5. 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.

  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. Gene Transfer of Prolyl Hydroxylase Domain 2 Inhibits Hypoxia-inducible Angiogenesis in a Model of Choroidal Neovascularization

    PubMed Central

    Takei, Anna; Ekström, Malena; Mammadzada, Parviz; Aronsson, Monica; Yu, Ma; Kvanta, Anders; André, Helder

    2017-01-01

    Cellular responses to hypoxia are mediated by the hypoxia-inducible factors (HIF). In normoxia, HIF-α proteins are regulated by a family of dioxygenases, through prolyl and asparagyl hydroxylation, culminating in proteasomal degradation and transcriptional inactivation. In hypoxia, the dioxygenases become inactive and allow formation of HIF transcription factor, responsible for upregulation of hypoxia genes. In ocular neoangiogenic diseases, such as neovascular age-related macular degeneration (nAMD), hypoxia seems pivotal. Here, we investigate the effects of HIF regulatory proteins on the hypoxia pathway in retinal pigment epithelium (RPE) cells, critically involved in nAMD pathogenesis. Our data indicates that, in ARPE-19 cells, prolyl hydroxylase domain (PHD)2 is the most potent negative-regulator of the HIF pathway. The negative effects of PHD2 on the hypoxia pathway were associated with decreased HIF-1α protein levels, and concomitant decrease in angiogenic factors. ARPE-19 cells stably expressing PHD2 impaired angiogenesis in vitro by wound healing, tubulogenesis, and sprouting assays, as well as in vivo by iris-induced angiogenesis. Gene transfer of PHD2 in vivo resulted in mitigation of HIF-mediated angiogenesis in a mouse model of nAMD. These results may have implications for the clinical treatment of nAMD patients, particularly regarding the use of gene therapy to negatively regulate neoangiogenesis. PMID:28186209

  8. 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

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

    PubMed

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

    2016-05-01

    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.

  10. 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

  11. 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.

  12. 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

  13. The three-dimensional structure of the RNA-binding domain of ribosomal protein L2; a protein at the peptidyl transferase center of the ribosome.

    PubMed Central

    Nakagawa, A; Nakashima, T; Taniguchi, M; Hosaka, H; Kimura, M; Tanaka, I

    1999-01-01

    Ribosomal protein L2 is the largest protein component in the ribosome. It is located at or near the peptidyl transferase center and has been a prime candidate for the peptidyl transferase activity. It binds directly to 23S rRNA and plays a crucial role in its assembly. The three-dimensional structure of the RNA-binding domain of L2 from Bacillus stearothermophilus has been determined at 2.3 A resolution by X-ray crystallography using the selenomethionyl MAD method. The RNA-binding domain of L2 consists of two recurring motifs of approximately 70 residues each. The N-terminal domain (positions 60-130) is homologous to the OB-fold, and the C-terminal domain (positions 131-201) is homologous to the SH3-like barrel. Residues Arg86 and Arg155, which have been identified by mutation experiments to be involved in the 23S rRNA binding, are located at the gate of the interface region between the two domains. The molecular architecture suggests how this important protein has evolved from the ancient nucleic acid-binding proteins to create a 23S rRNA-binding domain in the very remote past. PMID:10075918

  14. Fractionation factors and activation energies for exchange of the low barrier hydrogen bonding proton in peptidyl trifluoromethyl ketone complexes of chymotrypsin.

    PubMed

    Lin, J; Westler, W M; Cleland, W W; Markley, J L; Frey, P A

    1998-12-08

    NMR investigations have been carried out of complexes between bovine chymotrypsin Aalpha and a series of four peptidyl trifluoromethyl ketones, listed here in order of increasing affinity for chymotrypsin: N-Acetyl-L-Phe-CF3, N-Acetyl-Gly-L-Phe-CF3, N-Acetyl-L-Val-L-Phe-CF3, and N-Acetyl-L-Leu-L-Phe-CF3. The D/H fractionation factors (phi) for the hydrogen in the H-bond between His 57 and Asp 102 (His 57-Hdelta1) in these four complexes at 5 degreesC were in the range phi = 0.32-0.43, expected for a low-barrier hydrogen bond. For this series of complexes, measurements also were made of the chemical shifts of His 57-Hepsilon1 (delta2,2-dimethylsilapentane-5-sulfonic acid 8.97-9. 18), the exchange rate of the His 57-Hdelta1 proton with bulk water protons (284-12.4 s-1), and the activation enthalpies for this hydrogen exchange (14.7-19.4 kcal.mol-1). It was found that the previously noted correlations between the inhibition constants (Ki 170-1.2 microM) and the chemical shifts of His 57-Hdelta1 (delta2, 2-dimethylsilapentane-5-sulfonic acid 18.61-18.95) for this series of peptidyl trifluoromethyl ketones with chymotrypsin [Lin, J., Cassidy, C. S. & Frey, P. A. (1998) Biochemistry 37, 11940-11948] could be extended to include the fractionation factors, hydrogen exchange rates, and hydrogen exchange activation enthalpies. The results support the proposal of low barrier hydrogen bond-facilitated general base catalysis in the addition of Ser 195 to the peptidyl carbonyl group of substrates in the mechanism of chymotrypsin-catalyzed peptide hydrolysis. Trends in the enthalpies for hydrogen exchange and the fractionation factors are consistent with a strong, double-minimum or single-well potential hydrogen bond in the strongest complexes. The lifetimes of His 57-Hdelta1, which is solvent shielded in these complexes, track the strength of the hydrogen bond. Because these lifetimes are orders of magnitude shorter than those of the complexes themselves, the enzyme must have a

  15. Computational analysis of prolyl hydroxylase domain-containing protein 2 (PHD2) mutations promoting polycythemia insurgence in humans

    PubMed Central

    Minervini, Giovanni; Quaglia, Federica; Tosatto, Silvio CE

    2016-01-01

    Idiopathic erythrocytosis is a rare disease characterized by an increase in red blood cell mass due to mutations in proteins of the oxygen-sensing pathway, such as prolyl hydroxylase 2 (PHD2). Here, we present a bioinformatics investigation of the pathological effect of twelve PHD2 mutations related to polycythemia insurgence. We show that few mutations impair the PHD2 catalytic site, while most localize to non-enzymatic regions. We also found that most mutations do not overlap the substrate recognition site, suggesting a novel PHD2 binding interface. After a structural analysis of both binding partners, we suggest that this novel interface is responsible for PHD2 interaction with the LIMD1 tumor suppressor. PMID:26754054

  16. Manganese (II) induces chemical hypoxia by inhibiting HIF-prolyl hydroxylase: Implication in manganese-induced pulmonary inflammation

    SciTech Connect

    Han, Jeongoh; Lee, Jong-Suk; Choi, Daekyu; Lee, Youna; Hong, Sungchae; Choi, Jungyun; Han, Songyi; Ko, Yujin; Kim, Jung-Ae; Mi Kim, Young; Jung, Yunjin

    2009-03-15

    Manganese (II), a transition metal, causes pulmonary inflammation upon environmental or occupational inhalation in excess. We investigated a potential molecular mechanism underlying manganese-induced pulmonary inflammation. Manganese (II) delayed HIF-1{alpha} protein disappearance, which occurred by inhibiting HIF-prolyl hydroxylase (HPH), the key enzyme for HIF-1{alpha} hydroxylation and subsequent von Hippel-Lindau(VHL)-dependent HIF-1{alpha} degradation. HPH inhibition by manganese (II) was neutralized significantly by elevated dose of iron. Consistent with this, the induction of cellular HIF-1{alpha} protein by manganese (II) was abolished by pretreatment with iron. Manganese (II) induced the HIF-1 target gene involved in pulmonary inflammation, vascular endothelial growth factor (VEGF), in lung carcinoma cell lines. The induction of VEGF was dependent on HIF-1. Manganese-induced VEGF promoted tube formation of HUVEC. Taken together, these data suggest that HIF-1 may be a potential mediator of manganese-induced pulmonary inflammation.

  17. Computational analysis of prolyl hydroxylase domain-containing protein 2 (PHD2) mutations promoting polycythemia insurgence in humans.

    PubMed

    Minervini, Giovanni; Quaglia, Federica; Tosatto, Silvio C E

    2016-01-12

    Idiopathic erythrocytosis is a rare disease characterized by an increase in red blood cell mass due to mutations in proteins of the oxygen-sensing pathway, such as prolyl hydroxylase 2 (PHD2). Here, we present a bioinformatics investigation of the pathological effect of twelve PHD2 mutations related to polycythemia insurgence. We show that few mutations impair the PHD2 catalytic site, while most localize to non-enzymatic regions. We also found that most mutations do not overlap the substrate recognition site, suggesting a novel PHD2 binding interface. After a structural analysis of both binding partners, we suggest that this novel interface is responsible for PHD2 interaction with the LIMD1 tumor suppressor.

  18. X-Prolyl-Dipeptidyl Aminopeptidase of Lactobacillus delbrueckii subsp. bulgaricus: Characterization of the Enzyme and Isolation of Deficient Mutants

    PubMed Central

    Atlan, Danièle; Laloi, Patrick; Portalier, Raymond

    1990-01-01

    Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397 is able to hydrolyze X-proline-para-nitroanilides and X-proline-β-naphthylamides (X for alanyl- or glycyl-). A single metal-independent cytoplasmic enzyme with a molecular weight estimated to be 82,000 is responsible for these activities and was named X-prolyl-dipeptidyl aminopeptidase (X-Pro-DPAP). Isolation and analysis of mutants totally deficient for X-Pro-DPAP activity showed that a total lack of this enzyme induces (i) a decrease in the growth rate; (ii) an increase in cell wall proteinase activity; (iii) the loss of three cell wall proteins with respective molecular masses of 16, 40, and 52 kilodaltons; and (iv) enhancement of a cell wall protein with a molecular mass of 150 kilodaltons. The involvement of X-Pro-DPAP in casein catabolism is discussed. Images PMID:16348239

  19. 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.

  20. Hypoxia-inducible factor prolyl hydroxylases as targets for neuroprotection by "antioxidant" metal chelators: From ferroptosis to stroke.

    PubMed

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

    2013-09-01

    Neurologic conditions including stroke, Alzheimer disease, Parkinson disease, and Huntington 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 (HIF)-1α mediates a broad, evolutionarily conserved, endogenous adaptive program to hypoxia, and manipulation of components of the HIF pathway is neuroprotective in a number of human neurological diseases and experimental models. In this review, we discuss molecular components of one aspect of hypoxic adaptation in detail and provide perspective on which targets within this pathway seem 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.

  1. Crystal Structures of Trypanosoma brucei Oligopeptidase B Broaden the Paradigm of Catalytic Regulation in Prolyl Oligopeptidase Family Enzymes

    PubMed Central

    Morty, Rory E.; Fülöp, Vilmos

    2013-01-01

    Oligopeptidase B cleaves after basic amino acids in peptides up to 30 residues. As a virulence factor in bacteria and trypanosomatid pathogens that is absent in higher eukaryotes, this is a promising drug target. Here we present ligand-free open state and inhibitor-bound closed state crystal structures of oligopeptidase B from Trypanosoma brucei, the causative agent of African sleeping sickness. These (and related) structures show the importance of structural dynamics, governed by a fine enthalpic and entropic balance, in substrate size selectivity and catalysis. Peptides over 30 residues cannot fit the enzyme cavity, preventing the complete domain closure required for a key propeller Asp/Glu to fix the catalytic His and Arg in the catalytically competent conformation. This size exclusion mechanism protects larger peptides and proteins from degradation. Similar bacterial prolyl endopeptidase and archael acylaminoacyl peptidase structures demonstrate this mechanism is conserved among oligopeptidase family enzymes across all three domains of life. PMID:24265767

  2. Exploration of the One-Bead One-Compound Methodology for the Design of Prolyl Oligopeptidase Substrates

    PubMed Central

    Comellas, Gemma; Kaczmarska, Zusanna; Tarragó, Teresa; Teixidó, Meritxell; Giralt, Ernest

    2009-01-01

    Here we describe the design, synthesis and evaluation of the first solid-phase substrates for prolyl oligopeptidase (POP), a cytosolic serine peptidase associated with schizophrenia, bipolar affective disorder and related neuropsychiatric disorders. This study seeks to contribute to the future design of a one-bead one-compound (OBOC) peptide library of POP substrates, based on an intramolecular energy transfer substrate. Unexpectedly, the enzymatic evaluation of the substrates attached on solid-phase by means of the HMBA linker were cleaved through the ester bond, thereby suggesting an unknown esterase activity of POP, in addition to its known peptidase activity. By performing multiple activity assays, we have confirmed the esterase activity of this enzyme and its capacity to process the substrates on solid-phase. Finally, we tested a new linker, compatible with both the solid-phase peptide-synthesis used and the enzymatic assay, for application in the future design of an OBOC library. PMID:19593439

  3. Hypoxia-inducible factor prolyl hydroxylase inhibition: robust new target or another big bust for stroke therapeutics?

    PubMed Central

    Karuppagounder, Saravanan S; Ratan, Rajiv R

    2012-01-01

    A major challenge in developing stroke therapeutics that augment adaptive pathways to stress has been to identify targets that can activate compensatory programs without inducing or adding to the stress of injury. In this regard, hypoxia-inducible factor prolyl hydroxylases (HIF PHDs) are central gatekeepers of posttranscriptional and transcriptional adaptation to hypoxia, oxidative stress, and excitotoxicity. Indeed, some of the known salutary effects of putative ‘antioxidant' iron chelators in ischemic and hemorrhagic stroke may derive from their abilities to inhibit this family of iron, 2-oxoglutarate, and oxygen-dependent enzymes. Evidence from a number of laboratories supports the notion that HIF PHD inhibition can improve histological and functional outcomes in ischemic and hemorrhagic stroke models. In this review, we discuss this evidence and highlight important gaps in our understanding that render HIF PHD inhibition a promising but not yet preclinically validated target for protection and repair after stroke. PMID:22415525

  4. 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

  5. Hypoxia-inducible factor prolyl hydroxylase inhibition: robust new target or another big bust for stroke therapeutics?

    PubMed

    Karuppagounder, Saravanan S; Ratan, Rajiv R

    2012-07-01

    A major challenge in developing stroke therapeutics that augment adaptive pathways to stress has been to identify targets that can activate compensatory programs without inducing or adding to the stress of injury. In this regard, hypoxia-inducible factor prolyl hydroxylases (HIF PHDs) are central gatekeepers of posttranscriptional and transcriptional adaptation to hypoxia, oxidative stress, and excitotoxicity. Indeed, some of the known salutary effects of putative 'antioxidant' iron chelators in ischemic and hemorrhagic stroke may derive from their abilities to inhibit this family of iron, 2-oxoglutarate, and oxygen-dependent enzymes. Evidence from a number of laboratories supports the notion that HIF PHD inhibition can improve histological and functional outcomes in ischemic and hemorrhagic stroke models. In this review, we discuss this evidence and highlight important gaps in our understanding that render HIF PHD inhibition a promising but not yet preclinically validated target for protection and repair after stroke.

  6. Crystal structures of Trypanosoma brucei oligopeptidase B broaden the paradigm of catalytic regulation in prolyl oligopeptidase family enzymes.

    PubMed

    Canning, Peter; Rea, Dean; Morty, Rory E; Fülöp, Vilmos

    2013-01-01

    Oligopeptidase B cleaves after basic amino acids in peptides up to 30 residues. As a virulence factor in bacteria and trypanosomatid pathogens that is absent in higher eukaryotes, this is a promising drug target. Here we present ligand-free open state and inhibitor-bound closed state crystal structures of oligopeptidase B from Trypanosoma brucei, the causative agent of African sleeping sickness. These (and related) structures show the importance of structural dynamics, governed by a fine enthalpic and entropic balance, in substrate size selectivity and catalysis. Peptides over 30 residues cannot fit the enzyme cavity, preventing the complete domain closure required for a key propeller Asp/Glu to fix the catalytic His and Arg in the catalytically competent conformation. This size exclusion mechanism protects larger peptides and proteins from degradation. Similar bacterial prolyl endopeptidase and archael acylaminoacyl peptidase structures demonstrate this mechanism is conserved among oligopeptidase family enzymes across all three domains of life.

  7. 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

  8. Oxygen Sensing in Drosophila: Multiple Isoforms of the Prolyl Hydroxylase Fatiga Have Different Capacity to Regulate HIFα/Sima

    PubMed Central

    Dekanty, Andrés; Wappner, Pablo

    2010-01-01

    Background The Hypoxia Inducible Factor (HIF) mediates cellular adaptations to low oxygen. Prolyl-4-hydroxylases are oxygen sensors that hydroxylate the HIF alpha-subunit, promoting its proteasomal degradation in normoxia. Three HIF-prolyl hydroxylases, encoded by independent genes, PHD1, PHD2, and PHD3, occur in mammals. PHD2, the longest PHD isoform includes a MYND domain, whose biochemical function is unclear. PHD2 and PHD3 genes are induced in hypoxia to shut down HIF dependent transcription upon reoxygenation, while expression of PHD1 is oxygen-independent. The physiologic significance of the diversity of the PHD oxygen sensors is intriguing. Methodology and Principal Findings We have analyzed the Drosophila PHD locus, fatiga, which encodes 3 isoforms, FgaA, FgaB and FgaC that are originated through a combination of alternative initiation of transcription and alternative splicing. FgaA includes a MYND domain and is homologous to PHD2, while FgaB and FgaC are shorter isoforms most similar to PHD3. Through a combination of genetic experiments in vivo and molecular analyses in cell culture, we show that fgaB but not fgaA is induced in hypoxia, in a Sima-dependent manner, through a HIF-Responsive Element localized in the first intron of fgaA. The regulatory capacity of FgaB is stronger than that of FgaA, as complete reversion of fga loss-of-function phenotypes is observed upon transgenic expression of the former, and only partial rescue occurs after expression of the latter. Conclusions and Significance Diversity of PHD isoforms is a conserved feature in evolution. As in mammals, there are hypoxia-inducible and non-inducible Drosophila PHDs, and a fly isoform including a MYND domain co-exists with isoforms lacking this domain. Our results suggest that the isoform devoid of a MYND domain has stronger regulatory capacity than that including this domain. PMID:20811646

  9. 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

  10. 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.

  11. Deoxyribozyme-based, semisynthetic access to stable peptidyl-tRNAs exemplified by tRNAVal carrying a macrolide antibiotic resistance peptide.

    PubMed

    Graber, Dagmar; Trappl, Krista; Steger, Jessica; Geiermann, Anna-Skrollan; Rigger, Lukas; Moroder, Holger; Polacek, Norbert; Micura, Ronald

    2012-01-01

    We present a protocol for the reliable synthesis of non-hydrolyzable 3'-peptidyl-tRNAs that contain all the respective genuine nucleoside modifications. The approach is exemplified by tRNA(Val)-3'-NH-VFLVM-NH(2) and relies on commercially available Escherichia coli tRNA(Val). This tRNA was cleaved site-specifically within the TΨC loop using a 10-23 type DNA enzyme to obtain a 58 nt tRNA 5'-fragment which contained the modifications. After cleavage of the 2',3'-cyclophosphate moiety from the 5'-fragment, it was ligated to the 18 nt RNA-pentapeptide conjugate which had been chemically synthesized. By this methodology, tRNA(Val)-3'-NH-VFLVM-NH(2) is accessible in efficient manner. Furthermore, we point out that the approach is applicable to other types of tRNA.

  12. Solid phase synthesis and binding affinity of peptidyl transferase transition state mimics containing 2′-OH at P-site position A76

    PubMed Central

    Weinger, Joshua S.; Kitchen, David; Scaringe, Stephen A.; Strobel, Scott A.; Muth, Gregory W.

    2004-01-01

    All living cells are dependent on ribosomes to catalyze the peptidyl transfer reaction, by which amino acids are assembled into proteins. The previously studied peptidyl transferase transition state analog CC-dA-phosphate-puromycin (CCdApPmn) has important differences from the transition state, yet current models of the ribosomal active site have been heavily influenced by the properties of this molecule. One significant difference is the substitution of deoxyadenosine for riboadenosine at A76, which mimics the 3′ end of a P-site tRNA. We have developed a solid phase synthetic approach to produce inhibitors that more closely match the transition state, including the critical P-site 2′-OH. Inclusion of the 2′-OH or an even bulkier OCH3 group causes significant changes in binding affinity. We also investigated the effects of changing the A-site amino acid side chain from phenylalanine to alanine. These results indicate that the absence of the 2′-OH is likely to play a significant role in the binding and conformation of CCdApPmn in the ribosomal active site by eliminating steric clash between the 2′-OH and the tetrahedral phosphate oxygen. The conformation of the actual transition state must allow for the presence of the 2′-OH, and transition state mimics that include this critical hydroxyl group must bind in a different conformation from that seen in prior analog structures. These new inhibitors will provide valuable insights into the geometry and mechanism of the ribosomal active site. PMID:14999092

  13. cis-cis-trans-Bis(acetonitrile-κN)dichloridobis(triphenyl­phosphine-κP)ruthenium(II) acetonitrile disolvate

    PubMed Central

    Al-Far, Ahmad M.; Slaughter, LeGrande M.

    2008-01-01

    The title compound, [RuCl2(C2H3N)2(C18H15P)2]·2C2H3N, was obtained upon stirring an acetonitrile/ethanol solution of [RuCl2(PPh3)3]. In the crystal structure, each RuII ion is coordinated by two Cl [Ru—Cl = 2.4308 (7) and 2.4139 (7) Å], two N [Ru—N = 2.016 (2) and 2.003 (2) Å], and two P [Ru—P = 2.3688 (7) and 2.3887 (7) Å] atoms in a distorted octa­hedral geometry. Packing inter­actions include typical C—H⋯π contacts involving phenyl groups as well as weak hydrogen bonds between CH3CN methyl H atoms and Cl or solvent CH3CN N atoms. PMID:21200532

  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. Light-controlled ion channels formed by amphiphilic small molecules regulate ion conduction via cis-trans photoisomerization.

    PubMed

    Liu, Tao; Bao, Chunyan; Wang, Haiyan; Lin, Yao; Jia, Huijuan; Zhu, Linyong

    2013-11-11

    Light-regulated ion channel-transport across lipid bilayers was realized using structurally simple azobenzene-based amphiphilic small molecules. UV or visible irradiation triggers molecular photoisomerization, which induces structural and membrane affinity changes in self-assembled channels, thus resulting in light-regulated ion transmembrane transport.

  16. Studies of the toxicological potential of tripeptides (L-valyl-L-prolyl-L-proline and L-isoleucyl-L-prolyl-L-proline): VI. Effects of Lactobacillus helveticus-fermented milk powder on fertility and reproductive performance of rats.

    PubMed

    Kurosaki, Takahiro; Maeno, Masafumi; Mennear, John H; Bernard, Bruce K

    2005-01-01

    The objective of these studies was to assess the effects of the tripeptides, L-valyl-L-prolyl-L-proline (VPP) and L-isoleucyl-L-prolyl-L-proline (IPP), on reproductive capabilities of male and female rats. The specific goals of the experiments were (1) to determine the effects of orally administered tripeptides on (a) fertility and reproductive behavior in both sexes of rats, (b) embryo-fetal development in pregnant rats, and (c) pre- and postnatal development of rats exposed to tripeptides in utero and during lactation; and (2) to estimate the no-observable-adverse-effect doses of tripeptides in maternal and fetal rats. During the conduct of these classical segment I, II, and III studies, the test material was powdered Lactobacillus helveticus-fermented milk (FM), which contains the tripeptides, VPP and IPP. FM (0, 500, 1000 or 2000 mg/kg body weight [BW]/day--equivalent to 0, 0.8, 1.6, or 3.3 mg/kg BW/day of VPP plus IPP) was administered to males by oral gavage from 4 weeks prior to mating until sacrifice, and to females from 2 weeks prior to mating through day 20 of lactation. Evaluative parameters included monitoring grossly observable clinical signs; food consumption and body weight gains; mating behavior and fertility indices of both sexes; implantation and maintenance of embryos; sex ratio of live pups; fetal viability; incidences of external, visceral or skeletal variations; growth and behavioral development; as well as reproductive capabilities of F1 offspring exposed to FM during gestation and lactation. All animals were subjected to macroscopic examination at termination of their segment of the studies. Clinical signs, body weights, and food consumption were unaffected by administration of FM. During segment I, the test agent had no effect on estrus cycle, mating behavior, fertility index, or reproductive competence of either males or females. The results of segment II experiments revealed no effects of FM on postimplantation survival-loss, sex ratio or

  17. 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

  18. 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

  19. The Zinc Finger of Prolyl Hydroxylase Domain Protein 2 Is Essential for Efficient Hydroxylation of Hypoxia-Inducible Factor α

    PubMed Central

    Arsenault, Patrick R.; Song, Daisheng; Chung, Yu Jin; Khurana, Tejvir S.

    2016-01-01

    Prolyl hydroxylase domain protein 2 (PHD2) (also known as EGLN1) is a key oxygen sensor in mammals that posttranslationally modifies hypoxia-inducible factor α (HIF-α) and targets it for degradation. In addition to its catalytic domain, PHD2 contains an evolutionarily conserved zinc finger domain, which we have previously proposed recruits PHD2 to the HSP90 pathway to promote HIF-α hydroxylation. Here, we provide evidence that this recruitment is critical both in vitro and in vivo. We show that in vitro, the zinc finger can function as an autonomous recruitment domain to facilitate interaction with HIF-α. In vivo, ablation of zinc finger function by a C36S/C42S Egln1 knock-in mutation results in upregulation of the erythropoietin gene, erythrocytosis, and augmented hypoxic ventilatory response, all hallmarks of Egln1 loss of function and HIF stabilization. Hence, the zinc finger ordinarily performs a critical positive regulatory function. Intriguingly, the function of this zinc finger is impaired in high-altitude-adapted Tibetans, suggesting that their adaptation to high altitude may, in part, be due to a loss-of-function EGLN1 allele. Thus, these findings have important implications for understanding both the molecular mechanism of the hypoxic response and human adaptation to high altitude. PMID:27325674

  20. Prolyl endopeptidase is revealed following SILAC analysis to be a novel mediator of human microglial and THP-1 cell neurotoxicity.

    PubMed

    Klegeris, Andis; Li, Jane; Bammler, Theo K; Jin, Jinghua; Zhu, David; Kashima, Daniel T; Pan, Sheng; Hashioka, Sadayuki; Maguire, John; McGeer, Patrick L; Zhang, Jing

    2008-04-15

    Reactive microglial cells may exacerbate the pathology in some neurodegenerative disorders. Supernatants of stimulated human microglial cells, or their surrogate THP-1 cells, are lethal to cultured human neuroblastoma SH-SY5Y cells. To explore this neurotoxicity, we examined the spectrum of proteins generated by THP-1 cells using the technique of stable isotope labeling by amino acids in cell culture (SILAC). Unstimulated cells were grown in medium with light L-[(12)C(6)] arginine while cells stimulated by lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) were grown in medium with heavy L-[(13)C(6)] arginine. Proteins isolated from the media were digested with trypsin, and relative concentrations of generated peptides determined by mass spectrometry. More than 1,500 proteins or putative proteins were identified. Of these, 174 were increased and 189 decreased by more than twofold in the stimulated cell supernatant. We selected one upregulated protein, prolyl endopeptidase (PEP), for further investigation of its potential contribution to neurotoxicity. We first confirmed its upregulation by comparing its enzymatic activity in stimulated and unstimulated cell supernatants. We then evaluated two specific PEP inhibitors, Boc-Asn-Phe-Pro-aldehyde and Z-Pro-Pro-aldehyde-dimethyl acetal, for their potential to reduce toxicity of stimulated THP-1 cell and human microglia supernatants towards SH-SY5Y cells. We found both to be partially protective in a concentration-dependent manner. Inhibition of PEP may be a therapeutic approach to neurodegenerative disorders including Alzheimer and Parkinson diseases.

  1. 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

  2. Ion-exchange chromatography: basic principles and application to the partial purification of soluble mammalian prolyl oligopeptidase.

    PubMed

    Cummins, Philip M; Dowling, Oonagh; O'Connor, Brendan F

    2011-01-01

    Ion-exchange chromatography (IEC) allows for the separation of ionizable molecules on the basis of differences in charge properties. Its large sample-handling capacity, broad applicability (particularly to proteins and enzymes), moderate cost, powerful resolving ability, and ease of scale-up and automation have led to it becoming one of the most versatile and widely used of all liquid chromatography (LC) techniques. In this chapter, we review the basic principles of IEC, as well as the broader criteria for selecting IEC conditions. By way of further illustration, we outline protocols necessary to partially purify a serine peptidase from bovine whole brain cytosolic fraction, covering crude tissue extract preparation through to partial purification of the target enzyme using anion-exchange chromatography. Protocols for assaying total protein and enzyme activity in both pre- and post-IEC fractions are also described. The target serine peptidase, prolyl oligopeptidase (POP, EC3.4.21.26), is an 80-kDa enzyme with endopeptidase activity towards peptide substrates of ≤30 amino acids. POP is a ubiquitous post-proline cleaving enzyme with particularly high expression levels in the mammalian brain, where it participates in the metabolism of neuroactive peptides and peptide-like hormones (e.g. thyroliberin, gonadotropin-releasing hormone).

  3. A role for prolyl isomerase PIN1 in the phosphorylation-dependent modulation of PRRXL1 function.

    PubMed

    Soares-Dos-Reis, Ricardo; Pessoa, Ana Sofia; Dias, Ana Filipa; Falcão, Miguel; Matos, Mariana Raimundo; Vitorino, Rui; Monteiro, Filipe Almeida; Lima, Deolinda; Reguenga, Carlos

    2017-02-20

    Prrxl1 encodes for a paired-like homeodomain transcription factor essential for the correct establishment of the dorsal root ganglion - spinal cord nociceptive circuitry during development. Prrxl1-null mice display gross anatomical disruption of this circuitry, which translates to a markedly diminished sensitivity to noxious stimuli. Here, by the use of an immunoprecipitation and mass spectrometry approach, we identify five highly conserved phosphorylation sites (T110, S119, S231, S233 and S251) in PRRXL1 primary structure. Four are phospho-S/T-P sites, which suggest a role for the prolyl isomerase PIN1 in regulating PRRXL1. Accordingly, PRRXL1 physically interacts with PIN1 and displays diminished transcriptional activity in a Pin1-null cell line. Additionally, these S/T-P sites seem to be important for PRRXL1 conformation, and their point mutation to alanine or aspartate down-regulates PRRXL1 transcriptional activity. Altogether, our findings provide evidence for a putative novel role of PIN1 in the development of the nociceptive system and indicate phosphorylation-mediated conformational changes as a mechanism for regulating the PRRXL1 role in the process.

  4. Negative Regulation of the Stability and Tumor Suppressor Function of Fbw7 by the Pin1 Prolyl Isomerase

    PubMed Central

    Min, Sang-Hyun; Lau, Alan W.; Lee, Tae Ho; Inuzuka, Hiroyuki; Wei, Shuo; Huang, Pengyu; Shaik, Shavali; Lee, Daniel Yenhong; Finn, Greg; Balastik, Martin; Chen, Chun-Hau; Luo, Manli; Tron, Adriana E.; DeCaprio, James A.; Zhou, Xiao Zhen; Wei, Wenyi; Lu, Kun Ping

    2012-01-01

    SUMMARY Fbw7 is the substrate recognition component of the SCF (Skp1-Cullin-F-box)-type E3 ligase complex and a well-characterized tumor suppressor that targets numerous oncoproteins for destruction. Genomic deletion or mutation of FBW7 has been frequently found in various types of human cancers, however, little is known about the upstream signaling pathway(s) governing Fbw7 stability and cellular functions. Here we report that Fbw7 protein destruction and tumor suppressor function are negatively regulated by the prolyl isomerase Pin1. Pin1 interacts with Fbw7 in a phoshorylation-dependent manner and promotes Fbw7 self-ubiquitination and protein degradation by disrupting Fbw7 dimerization. Consequently, over-expressing Pin1 reduces Fbw7 abundance and suppresses Fbw7’s ability to inhibit proliferation and transformation. By contrast, depletion of Pin1 in cancer cells leads to elevated Fbw7 expression, which subsequently reduces Mcl-1 abundance, sensitizing cancer cells to Taxol. Thus, Pin1-mediated inhibition of Fbw7 contributes to oncogenesis and Pin1 may be a promising drug target for anti-cancer therapy. PMID:22608923

  5. Caffeic acid phenethyl ester is a potent inhibitor of HIF prolyl hydroxylase: structural analysis and pharmacological implication.

    PubMed

    Choi, Daekyu; Han, Jeongoh; Lee, Youna; Choi, Jungyun; Han, Songyi; Hong, Sungchae; Jeon, Hyunchu; Kim, Young Mi; Jung, Yunjin

    2010-09-01

    Caffeic acid phenethyl ester (CAPE) is an active component of propolis from honeybee. We investigated a potential molecular mechanism underlying a CAPE-mediated protective effect against ischemia/reperfusion (I/R) injury and analyzed the structure contributing to the CAPE effect. CAPE induced hypoxia-inducible factor-1 (HIF-1) alpha protein, concomitantly transactivating the HIF-1 target genes vascular endothelial growth factor and heme oxygenase-1, which play a protective role in I/R injury. CAPE delayed the degradation of HIF-1alpha protein in cells, which occurred by inhibition of HIF prolyl hydroxylase (HPH), the key enzyme for von Hippel-Lindau-dependent HIF-1alpha degradation. CAPE inhibition of HPH and induction of HIF-1alpha protein were neutralized by an elevated dose of iron. The catechol moiety, a chelating group, is essential for HPH inhibition, while hydrogenation of the double bond (-C=C-) in the Michael reaction acceptor markedly reduced potency. Removal of the phenethyl moiety of CAPE (substitution with the methyl moiety) severely deteriorated its inhibitory activity for HPH. Our data suggest that a beneficial effect of CAPE on I/R injury may be ascribed to the activation of HIF-1 pathway via inhibition of HPH and reveal that the chelating moiety of CAPE acted as a pharmacophore while the double bond and phenethyl moiety assisted in inhibiting HPH.

  6. Prolyl hydroxylase 2 (PHD2) inhibition protects human renal epithelial cells and mice kidney from hypoxia injury

    PubMed Central

    Zhong, Yihong; Ding, Xiaoqiang

    2016-01-01

    Prolyl hydroxylase domain protein 2 (PHD2) is a key oxygen sensor, setting low steady-state level of hypoxia-inducible factor-α (HIF-α). Here, we showed that treatment of cobalt chloride (CoCl2), a hypoxia mimic, in HK-2 tubular epithelial cells induced PHD2 and HIF-1/2α expression as well as cell apoptosis and autophagy activation. Three methyladenine (3-MA), the autophagy inhibitor, blocked autophagy and protected HK-2 cells from CoCl2. Significantly, siRNA knockdown of PHD2 also protected HK-2 cells from CoCl2, possibly via increasing HIF-1α expression. Reversely, HIF-1α siRNA knockdown almost abolished cytoprotection by PHD2 siRNA in CoCl2-treated HK-2 cells. In vivo, pretreatment with a PHD inhibitor L-mimosine remarkably attenuated mice renal ischemia-reperfusion injuries. Molecularly, L-mimosine inhibited apoptosis and inflammatory responses in injured mice kidneys. Together, our results suggest that PHD2 silence or inhibition protects human renal epithelial cells and mice kidney from hypoxia injuries. PMID:27527871

  7. Biochemical and Structural Characterization of Selective Allosteric Inhibitors of the Plasmodium falciparum Drug Target, Prolyl-tRNA-synthetase

    PubMed Central

    2016-01-01

    Plasmodium falciparum (Pf) prolyl-tRNA synthetase (ProRS) is one of the few chemical-genetically validated drug targets for malaria, yet highly selective inhibitors have not been described. In this paper, approximately 40,000 compounds were screened to identify compounds that selectively inhibit PfProRS enzyme activity versus Homo sapiens (Hs) ProRS. X-ray crystallography structures were solved for apo, as well as substrate- and inhibitor-bound forms of PfProRS. We identified two new inhibitors of PfProRS that bind outside the active site. These two allosteric inhibitors showed >100 times specificity for PfProRS compared to HsProRS, demonstrating this class of compounds could overcome the toxicity related to HsProRS inhibition by halofuginone and its analogues. Initial medicinal chemistry was performed on one of the two compounds, guided by the cocrystallography of the compound with PfProRS, and the results can instruct future medicinal chemistry work to optimize these promising new leads for drug development against malaria. PMID:27798837

  8. 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.

  9. Chapter 21: Hyperplasia of Pulmonary Neuroepithelial Bodies (NEB) in Lungs of Prolyl Hydroxylase –1(PHD-1) Deficient Mice

    PubMed Central

    Pan, Jie; Yeger, Herman; Ratcliffe, Peter; Bishop, Tammie

    2017-01-01

    Pulmonary NEB, widely distributed within the airway mucosa of mammalian lungs, are presumed hypoxia sensitive airway O2 sensors responding to changes in airway gas concentration. NEB cell hyperplasia has been reported after exposure to chronic hypoxia and in a variety of paediatric and adult lung disorders. Prolyl hydroxylases (PHD 1–3) regulate the stability of hypoxia-inducible factors (HIF’s) in an O2-dependent manner and function as intrinsic oxygen sensors. To determine a possible role of PHD-1in NEB cells we have quantitated NEB’s in lungs of neonatal (P2) and adult (2 months) PHD-1-deficient mice and compared them to wild type (WT) control mice. Lung tissues fixed in formalin and embedded in paraffin were processed for immunoperoxidase method and frozen sections for multilabel immunoflourescence using antibodies for NEB markers synaptophysin, synaptic vesicle protein 2 and the peptide CGRP. The frequency and size of NEB in lungs of PHD-1 deficient neonatal mice (P2) and at 2 months was increased significantly compared to WT controls (p < 0.01). The present data suggests an important role for PHD enzymes in NEB cell biology deserving further studies. Since the PHD-1 deficient mouse appears to be the first animal model showing NEB cell hyperplasia it may be useful for studies of NEB physiology and pathobiology. PMID:23080156

  10. (R)-α-lipoyl-glycyl-L-prolyl-L-glutamyl dimethyl ester codrug as a multifunctional agent with potential neuroprotective activities.

    PubMed

    Cacciatore, Ivana; Baldassarre, Leonardo; Fornasari, Erika; Cornacchia, Catia; Di Stefano, Antonio; Sozio, Piera; Cerasa, Laura Serafina; Fontana, Antonella; Fulle, Stefania; Di Filippo, Ester Sara; La Rovere, Rita Maria Laura; Pinnen, Francesco

    2012-11-01

    The (R)-α-lipoyl-glycyl-L-prolyl-L-glutamyl dimethyl ester codrug (LA-GPE, 1) was synthesized as a new multifunctional drug candidate with antioxidant and neuroprotective properties for the treatment of neurodegenerative diseases. Physicochemical properties, chemical and enzymatic stabilities were evaluated, along with the capacity of LA-GPE to penetrate the blood-brain barrier (BBB) according to an in vitro parallel artificial membrane permeability assay for the BBB. We also investigated the potential effectiveness of LA-GPE against the cytotoxicity induced by 6-hydroxydopamine (6-OHDA) and H2O2 on the human neuroblastoma cell line SH-SY5Y by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Our results show that codrug 1 is stable at both pH 1.3 and 7.4, exhibits good lipophilicity (log P=1.51) and a pH-dependent permeability profile. Furthermore, LA-GPE was demonstrated to be significantly neuroprotective and to act as an antioxidant against H2O2- and 6-OHDA-induced neurotoxicity in SH-SY5Y cells.

  11. Origin and evolution of glutamyl-prolyl tRNA synthetase WHEP domains reveal evolutionary relationships within Holozoa.

    PubMed

    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.

  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. 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

  14. Substrate and Enzyme Functional Groups Contribute to Translational Quality Control by Bacterial Prolyl-tRNA Synthetase

    PubMed Central

    Kumar, Sandeep; Das, Mom; Hadad, Christopher M.; Musier-Forsyth, Karin

    2012-01-01

    Aminoacyl-tRNA synthetases activate specific amino acid substrates and attach them via an ester linkage to cognate tRNA molecules. In addition to cognate proline, prolyl-tRNA synthetase (ProRS) can activate cysteine and alanine and misacylate tRNAPro. Editing of the misacylated aminoacyl-tRNA is required for error-free protein synthesis. An editing domain (INS) appended to bacterial ProRS selectively hydrolyzes Ala-tRNAPro, whereas Cys-tRNAPro is cleared by a freestanding editing domain, YbaK, through a unique mechanism involving substrate sulfhydryl chemistry. The detailed mechanism of catalysis by INS is currently unknown. To understand the alanine specificity and mechanism of catalysis by INS, we have explored several possible mechanisms of Ala-tRNAPro deacylation via hybrid QM/MM calculations. Experimental studies were also performed to test the role of several residues in the INS active site, as well as various substrate functional groups in catalysis. Our results support a critical role for the tRNA 2′-OH group in substrate binding and catalytic water activation. A role is also proposed for the protein’s conserved GXXXP loop in transition state stabilization and for the main chain atoms of Gly261 in a proton relay that contributes substantially to catalysis. PMID:22458656

  15. Nanovector-based prolyl hydroxylase domain 2 silencing system enhances the efficiency of stem cell transplantation for infarcted myocardium repair

    PubMed Central

    Zhu, Kai; Lai, Hao; Guo, Changfa; Li, Jun; Wang, Yulin; Wang, Lingyan; Wang, Chunsheng

    2014-01-01

    Mesenchymal stem cell (MSC) transplantation has attracted much attention in myocardial infarction therapy. One of the limitations is the poor survival of grafted cells in the ischemic microenvironment. Small interfering RNA-mediated prolyl hydroxylase domain protein 2 (PHD2) silencing in MSCs holds tremendous potential to enhance their survival and paracrine effect after transplantation. However, an efficient and biocompatible PHD2 silencing system for clinical application is lacking. Herein, we developed a novel PHD2 silencing system based on arginine-terminated generation 4 poly(amidoamine) (Arg-G4) nanoparticles. The system exhibited effective and biocompatible small interfering RNA delivery and PHD2 silencing in MSCs in vitro. After genetically modified MSC transplantation in myocardial infarction models, MSC survival and paracrine function of IGF-1 were enhanced significantly in vivo. As a result, we observed decreased cardiomyocyte apoptosis, scar size, and interstitial fibrosis, and increased angiogenesis in the diseased myocardium, which ultimately attenuated ventricular remodeling and improved heart function. This work demonstrated that an Arg-G4 nanovector-based PHD2 silencing system could enhance the efficiency of MSC transplantation for infarcted myocardium repair. PMID:25429216

  16. Alterations in plasma prolyl endopeptidase activity in depression, mania, and schizophrenia: effects of antidepressants, mood stabilizers, and antipsychotic drugs.

    PubMed

    Maes, M; Goossens, F; Scharpé, S; Calabrese, J; Desnyder, R; Meltzer, H Y

    1995-10-16

    The activity of prolyl endopeptidase (PEP), a serine proteinase, has been found to be significantly lower in the blood of patients with major depression than in normal volunteers. The present study investigates plasma PEP activity in 25 major depressed, 10 manic, and 14 schizophrenic subjects versus 30 normal volunteers. It also examines the effects of antidepressants, valproate, and neuroleptic drugs on plasma PEP activity. PEP activity was significantly lower in major depressed subjects than in normal volunteers and in patients with mania and schizophrenia. In depressed subjects, plasma PEP activity was significantly increased during treatment with antidepressant drugs, such as fluoxetine. Plasma PEP activity was significantly increased in manic and schizophrenic subjects compared with normal volunteers. In manic subjects, short-term treatment with valproate had a significant suppressive effect on PEP activity. No significant effects of neuroleptics on PEP activity could be found in the schizophrenic patients. The results support the hypothesis that lower PEP activity could play a role in the pathophysiology of major depression, while increased PEP activity may be related to psychotic conditions, such as mania and schizophrenia.

  17. Biochemical and Structural Characterization of Selective Allosteric Inhibitors of the Plasmodium falciparum Drug Target, Prolyl-tRNA-synthetase.

    PubMed

    Hewitt, Stephen Nakazawa; Dranow, David M; Horst, Benjamin G; Abendroth, Jan A; Forte, Barbara; Hallyburton, Irene; Jansen, Chimed; Baragaña, Beatriz; Choi, Ryan; Rivas, Kasey L; Hulverson, Matthew A; Dumais, Mitchell; Edwards, Thomas E; Lorimer, Donald D; Fairlamb, Alan H; Gray, David W; Read, Kevin D; Lehane, Adele M; Kirk, Kiaran; Myler, Peter J; Wernimont, Amy; Walpole, Chris; Stacy, Robin; Barrett, Lynn K; Gilbert, Ian H; Van Voorhis, Wesley C

    2017-01-13

    Plasmodium falciparum (Pf) prolyl-tRNA synthetase (ProRS) is one of the few chemical-genetically validated drug targets for malaria, yet highly selective inhibitors have not been described. In this paper, approximately 40,000 compounds were screened to identify compounds that selectively inhibit PfProRS enzyme activity versus Homo sapiens (Hs) ProRS. X-ray crystallography structures were solved for apo, as well as substrate- and inhibitor-bound forms of PfProRS. We identified two new inhibitors of PfProRS that bind outside the active site. These two allosteric inhibitors showed >100 times specificity for PfProRS compared to HsProRS, demonstrating this class of compounds could overcome the toxicity related to HsProRS inhibition by halofuginone and its analogues. Initial medicinal chemistry was performed on one of the two compounds, guided by the cocrystallography of the compound with PfProRS, and the results can instruct future medicinal chemistry work to optimize these promising new leads for drug development against malaria.

  18. 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

    2015-05-05

    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.

  19. Conditional Knockout of Prolyl Hydroxylase Domain Protein 2 Attenuates High Fat-Diet-Induced Cardiac Dysfunction in Mice

    PubMed Central

    Zeng, Heng; Chen, Jian-Xiong

    2014-01-01

    Oxygen sensor prolyl hydroxylases (PHDs) play important roles in the regulation of HIF-α and cell metabolisms. This study was designed to investigate the direct role of PHD2 in high fat-diet (HFD)-induced cardiac dysfunction. In HFD fed mice, PHD2 expression was increased without significant changes in PHD1 and PHD3 levels in the heart. This was accompanied by a significant upregulation of myeloid differentiation factor 88 (MYD88) and NF-κB. To explore the role of PHD2 in HFD-induced cardiac dysfunction, PHD2 conditional knockout mice were fed a HFD for 16 weeks. Intriguingly, knockout of PHD2 significantly reduced MYD88 and NF-κb expression in HFD mouse hearts. Moreover, knockout of PHD2 inhibited TNFα and ICAM-1 expression, and reduced cell apoptosis and macrophage infiltration in HFD mice. This was accompanied by a significant improvement of cardiac function. Most importantly, conditional knockout of PHD2 at late stage in HFD mice significantly improved glucose tolerance and reversed cardiac dysfunction. Our studies demonstrate that PHD2 activity is a critical contributor to the HFD-induced cardiac dysfunction. Inhibition of PHD2 attenuates HFD-induced cardiac dysfunction by a mechanism involving suppression of MYD88/NF-κb pathway and inflammation. PMID:25546437

  20. The Endothelial Prolyl-4-Hydroxylase Domain 2/Hypoxia-Inducible Factor 2 Axis Regulates Pulmonary Artery Pressure in Mice

    PubMed Central

    Rajendran, Ganeshkumar; Astleford, Lindsay; Michael, Mark; Schonfeld, Michael P.; Fields, Timothy; Shay, Sheila; French, Jaketa L.; West, James; Haase, Volker H.

    2016-01-01

    Hypoxia-inducible factors 1 and 2 (HIF-1 and -2) control oxygen supply to tissues by regulating erythropoiesis, angiogenesis and vascular homeostasis. HIFs are regulated in response to oxygen availability by prolyl-4-hydroxylase domain (PHD) proteins, with PHD2 being the main oxygen sensor that controls HIF activity under normoxia. In this study, we used a genetic approach to investigate the endothelial PHD2/HIF axis in the regulation of vascular function. We found that inactivation of Phd2 in endothelial cells specifically resulted in severe pulmonary hypertension (∼118% increase in right ventricular systolic pressure) but not polycythemia and was associated with abnormal muscularization of peripheral pulmonary arteries and right ventricular hypertrophy. Concurrent inactivation of either Hif1a or Hif2a in endothelial cell-specific Phd2 mutants demonstrated that the development of pulmonary hypertension was dependent on HIF-2α but not HIF-1α. Furthermore, endothelial HIF-2α was required for the development of increased pulmonary artery pressures in a model of pulmonary hypertension induced by chronic hypoxia. We propose that these HIF-2-dependent effects are partially due to increased expression of vasoconstrictor molecule endothelin 1 and a concomitant decrease in vasodilatory apelin receptor signaling. Taken together, our data identify endothelial HIF-2 as a key transcription factor in the pathogenesis of pulmonary hypertension. PMID:26976644

  1. 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.

  2. 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

  3. A Novel β-Peptidyl Aminopeptidase (BapA) from Strain 3-2W4 Cleaves Peptide Bonds of Synthetic β-Tri- and β-Dipeptides

    PubMed Central

    Geueke, Birgit; Namoto, Kenji; Seebach, Dieter; Kohler, Hans-Peter E.

    2005-01-01

    A novel bacterial strain that was capable of growing on the β-tripeptide H-βhVal-βhAla-βhLeu-OH as the sole carbon and nitrogen source was isolated from an enrichment culture. On the basis of physiological characterization, partial 16S rRNA sequencing, and fatty acid analysis, strain 3-2W4 was identified as a member of the family Sphingomonadaceae. Growth on the β-tripeptide and the β-dipeptide H-βhAla-βhLeu-OH was observed, and emerging metabolites were characterized. Small amounts of a persisting metabolite, the N-acetylated β-dipeptide, were identified in both media. According to dissolved organic carbon measurements, 74 to 80% of the available carbon was dissimilated. The β-peptide-degrading enzyme was purified from the crude cell extract of cells from strain 3-2W4 grown on complex medium. The enzyme was composed of two subunits, and the N-terminal sequences of both were determined. With this information, it was possible to identify the complete nucleotide sequence and to deduce the primary structure of the gene bapA. The gene encoded a β-peptidyl aminopeptidase (BapA) of 402 amino acids that was synthesized as preprotein with a signal sequence of 29 amino acids. The enzyme was cleaved into two subunits (residues 30 to 278 and 279 to 402). It belonged to the N-terminal nucleophile (Ntn) hydrolase superfamily. PMID:16109932

  4. Tuning of the selectivity of fluorescent peptidyl bioprobe using aggregation induced emission for heavy metal ions by buffering agents in 100% aqueous solutions.

    PubMed

    Neupane, Lok Nath; Hwang, Gi Won; Lee, Keun-Hyeung

    2017-02-03

    Smart fluorescent probes of which the detection of specific target molecules can be controlled are attracting remarkable interest. A fluorescent peptidyl bioprobe (1) was rationally synthesized by conjugating tetraphenylethylene, an aggregation-induced emission (AIE) fluorophore with a peptide receptor (AspHis) that acted as hard and intermediate bases. The selective detection of 1 for specific metal ion in 100% aqueous solutions was controlled by the buffering agents with the chelate effect without the change of pH. In distilled water and phosphate buffered aqueous solution at neutral pH, 1 exhibited a selective Off-On response to a soft metal ion, Hg(2+) among test metal ions by 100-fold enhancement of the emission at 470nm. 1 showed a selective Off-On response (180-fold enhancement) to a hard metal ion, Al(3+) ions among test metal ions in Tris buffered aqueous solution at neutral pH and Hexamine (hexamethylenetetramine) buffered aqueous solution at acidic pH. The detection limit of 0.46 ppb for Hg(2+) and 2.26 ppb for Al(3+) in each condition was lower than the maximum allowable level of the metal ions in drinking water by EPA. This research helps to understand how buffering agents participate in the complex formation and aggregation of fluorescent probes using an AIE process for the selective detection of specific metal ions in aqueous solutions.

  5. Effect of cortisol on neurophysin I/oxytocin and peptidyl glycine-alpha-amidating mono-oxygenase mRNA expression in bovine luteal and granulosa cells.

    PubMed

    Ziolkowska, A; Mlynarczuk, J; Kotwica, J

    2013-01-01

    Cortisol stimulates the synthesis and secretion of oxytocin (OT) from bovine granulosa and luteal cells, but the molecular mechanisms of cortisol action remain unknown. In this study, granulosa cells or luteal cells from days 1-5 and 11-15 of the oestrous cycle were incubated for 4 or 8 h with cortisol (1 x 10(-5), 1 x 10(-7) M). After testing cell viability and hormone secretion (OT, progesterone, estradiol), we studied the effect of cortisol on mRNA expression for precursor of OT (NP-I/OT) and peptidyl glycine-alpha-amidating mono-oxygenase (PGA). The influence of RU 486 (1 x 10(-5) M), a progesterone receptor blocker and inhibitor of the glucocorticosteroid receptor (GR), on the expression for both genes was tested. Cortisol increased the mRNA expression for NP-I/OT and PGA in granulosa cells and stimulated the expression for NP-I/OT mRNA in luteal cells obtained from days 1-5 and days 11-15 of the oestrous cycle. Expression for PGA mRNA was increased only in luteal cells from days 11-15 of the oestrous cycle. In addition, RU 486 blocked the cortisol-stimulated mRNA expression for NP-I/OT and PGA in both types of cells. These data suggest that cortisol affects OT synthesis and secretion in bovine ovarian cells, by acting on the expression of key genes, that may impair ovary

  6. Dissociation rate of cognate peptidyl-tRNA from the A-site of hyper-accurate and error-prone ribosomes.

    PubMed

    Karimi, R; Ehrenberg, M

    1994-12-01

    The binding stability of the aminoacyl-tRNA site (A-site), estimated from the dissociation rate constant kd, of AcPhe-Phe-tRNA(Phe) has been studied for wild-type (wt), for hyperaccurate ribosomes altered in S12 [streptomycin-dependent (SmD) and streptomycin-pseudodependent (SmP) phenotypes], for error-prone ribosomes altered in S4 (Ram phenotype), and for ribosomes in complex with the error-inducing aminoglycosides streptomycin and neomycin. The AcPhe2-tRNA stability is slightly and identically reduced for SmD and SmP phenotypes in relation to wt ribosomes. The stability is increased (kd is reduced) for Ram ribosomes to about the same extent as the proof-reading accuracy is decreased for this phenotype. kd is also reduced by the action of streptomycin and neomycin, but much less than the reduction in proof-reading accuracy induced by streptomycin. Similar kd values for SmD and SmP ribosomes indicate that the cause of streptomycin dependence is not excessive drop-off of peptidyl-tRNAs from the A-site.

  7. YaeJ is a novel ribosome-associated protein in Escherichia coli that can hydrolyze peptidyl-tRNA on stalled ribosomes.

    PubMed

    Handa, Yoshihiro; Inaho, Noriyuki; Nameki, Nobukazu

    2011-03-01

    In bacteria, ribosomes often become stalled and are released by a trans-translation process mediated by transfer-messenger RNA (tmRNA). In the absence of tmRNA, however, there is evidence that stalled ribosomes are released from non-stop mRNAs. Here, we show a novel ribosome rescue system mediated by a small basic protein, YaeJ, from Escherichia coli, which is similar in sequence and structure to the catalytic domain 3 of polypeptide chain release factor (RF). In vitro translation experiments using the E. coli-based reconstituted cell-free protein synthesis system revealed that YaeJ can hydrolyze peptidyl-tRNA on ribosomes stalled by both non-stop mRNAs and mRNAs containing rare codon clusters that extend downstream from the P-site and prevent Ala-tmRNA•SmpB from entering the empty A-site. In addition, YaeJ had no effect on translation of a normal mRNA with a stop codon. These results suggested a novel tmRNA-independent rescue system for stalled ribosomes in E. coli. YaeJ was almost exclusively found in the 70S ribosome and polysome fractions after sucrose density gradient sedimentation, but was virtually undetectable in soluble fractions. The C-terminal basic residue-rich extension was also found to be required for ribosome binding. These findings suggest that YaeJ functions as a ribosome-attached rescue device for stalled ribosomes.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. Involvement of polyphosphate kinase in virulence and stress tolerance of uropathogenic Proteus mirabilis.

    PubMed

    Peng, Liang; Jiang, Qiao; Pan, Jia-Yun; Deng, Cong; Yu, Jing-Yi; Wu, Xiao-Man; Huang, Sheng-He; Deng, Xiao-Yan

    2016-04-01

    Proteus mirabilis (P. mirabilis), a gram-negative enteric bacterium, frequently causes urinary tract infections. Many virulence factors of uropathogenic P. mirabilis have been identified, including urease, flagella, hemolysin and fimbriae. However, the functions of polyphosphate kinase (PPK), which are related to the pathogenicity of many bacteria, remain entirely unknown in P. mirabilis. In this study, a ppk gene encoding the PPK insertional mutant in P. mirabilis strain HI4320 was constructed, and its biological functions were examined. The results of survival studies demonstrated that the ppk mutant was deficient in resistance to oxidative, hyperosmotic and heat stress. The swarming and biofilm formation abilities of P. mirabilis were also attenuated after the ppk interruption. In vitro and in vivo experiments suggested that ppk was required for P. mirabilis to invade the bladder. The negative phenotypes of the ppk mutant could be restored by ppk gene complementation. Furthermore, two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry were used to analyze the proteomes of the wild-type strain and the ppk mutant. Compared with the wild-type strain, seven proteins including TonB-dependent receptor, universal stress protein G, major mannose-resistant/Proteus-like fimbrial protein (MR/P fimbriae), heat shock protein, flagellar capping protein, putative membrane protein and multidrug efflux protein were down-regulated, and four proteins including exported peptidase, repressor protein for FtsI, FKBP-type peptidyl-prolyl cis-trans isomerase and phosphotransferase were up-regulated in the ppk mutant. As a whole, these results indicate that PPK is an important regulator and plays a crucial role in stress tolerance and virulence in uropathogenic P. mirabilis.

  14. Kinetics of folding and association of differently glycosylated variants of invertase from Saccharomyces cerevisiae.

    PubMed Central

    Kern, G.; Kern, D.; Jaenicke, R.; Seckler, R.

    1993-01-01

    A core-glycosylated form of the dimeric enzyme invertase has been isolated from secretion mutants of Saccharomyces cerevisiae blocked in transport to the Golgi apparatus. This glycosylation variant corresponds to the form that folds and associates during biosynthesis of the protein in vivo. In the present work, its largely homogeneous subunit size and well-defined quaternary structure were utilized to characterize the folding and association pathway of this highly glycosylated protein in comparison with the nonglycosylated cytoplasmic and the high-mannose-glycosylated periplasmic forms of the same enzyme encoded by the suc2 gene. Renaturation of core-glycosylated invertase upon dilution from guanidinium-chloride solutions follows a unibimolecular reaction scheme with consecutive first-order subunit folding and second-order association reactions. The rate constant of the rate-limiting step of subunit folding, as detected by fluorescence increase, is k1 = 1.6 +/- 0.4 x 10(-3) s-1 at 20 degrees C; it is characterized by an activation enthalpy of delta H++ = 65 kJ/mol. The reaction is not catalyzed by peptidyl-prolyl cis-trans isomerase of the cyclophilin type. Reactivation of the enzyme depends on protein concentration and coincides with subunit association, as monitored by size-exclusion high-pressure liquid chromatography. The association rate constant, estimated by numerical simulation of reactivation kinetics, increases from 5 x 10(3) M-1 s-1 to 7 x 10(4) M-1 s-1 between 5 and 30 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8268797

  15. pH-induced conformational change of IscU at low pH correlates with protonation/deprotonation of two conserved histidine residues.

    PubMed

    Dai, Ziqi; Kim, Jin Hae; Tonelli, Marco; Ali, Ibrahim K; Markley, John L

    2014-08-19

    IscU, the scaffold protein for the major iron-sulfur cluster biosynthesis pathway in microorganisms and mitochondria (ISC pathway), plays important roles in the formation of [2Fe-2S] and [4Fe-4S] clusters and their delivery to acceptor apo-proteins. Our laboratory has shown that IscU populates two distinct, functionally relevant conformational states, a more structured state (S) and a more dynamic state (D), that differ by cis/trans isomerizations about two peptidyl-prolyl peptide bonds [Kim, J. H., Tonelli, M., and Markley, J. L. (2012) Proc. Natl. Acad. Sci. U.S.A., 109, 454-459. Dai Z., Tonelli, M., and Markley, J. L. (2012) Biochemistry, 51, 9595-9602. Cai, K., Frederick, R. O., Kim, J. H., Reinen, N. M., Tonelli, M., and Markley, J. L. (2013) J. Biol. Chem., 288, 28755-28770]. Here, we report our findings on the pH dependence of the D ⇄ S equilibrium for Escherichia coli IscU in which the D-state is stabilized at low and high pH values. We show that the lower limb of the pH dependence curve results from differences in the pKa values of two conserved histidine residues (His10 and His105) in the two states. The net proton affinity of His10 is about 50 times higher and that of His105 is 13 times higher in the D-state than in the S-state. The origin of the high limb of the D ⇄ S pH dependence remains to be determined. These results show that changes in proton inventory need to be taken into account in the steps in iron-sulfur cluster assembly and transfer that involve transitions of IscU between its S- and D-states.

  16. Exploring the role of host cell chaperones/PPIases during cellular up-take of bacterial ADP-ribosylating toxins as basis for novel pharmacological strategies to protect mammalian cells against these virulence factors.

    PubMed

    Barth, Holger

    2011-03-01

    Bacterial exotoxins exploit protein transport pathways of their mammalian target cells to deliver their enzymatic active moieties into the cytosol. There, they modify their specific substrate molecules resulting in cell damage and the clinical symptoms characteristic for each individual toxin. We have investigated the cellular uptake of the binary actin ADP-ribosylating C2 toxin from Clostridium botulinum and the binary lethal toxin from Bacillus anthracis, a metalloprotease. Both toxins are composed of a binding/translocation component and a separate enzyme component. During cellular uptake, the binding/translocation components form pores in membranes of acidified endosomes, and the enzyme components translocate as unfolded proteins through the pores into the cytosol. We found by using specific pharmacological inhibitors that the host cell chaperone Hsp90 and the peptidyl-prolyl cis/trans isomerase cyclophilin A are crucial for membrane translocation of the enzyme component of the C2 toxin but not of the lethal toxin, although the structures of the binding/translocation components and the overall uptake mechanisms of both toxins are widely comparable. In conclusion, the new findings imply that Hsp90 and cyclophilin function selectively in promoting translocation of certain bacterial toxins depending on the enzyme domains of the individual toxins. The targeted pharmacological inhibition of individual host cell chaperones/PPIases prevents uptake of certain bacterial exotoxins into the cytosol of mammalian cells and thus protects cells from intoxication. Such substances could represent attractive lead substances for development of novel therapeutics to prevent toxic effects during infection with toxin-producing bacteria.

  17. Cyclophilin B Interacts with Sodium-Potassium ATPase and Is Required for Pump Activity in Proximal Tubule Cells of the Kidney

    PubMed Central

    Suñé, Guillermo; Sarró, Eduard; Puigmulé, Marta; López-Hellín, Joan; Zufferey, Madeleine; Pertel, Thomas; Luban, Jeremy; Meseguer, Anna

    2010-01-01

    Cyclophilins (Cyps), the intracellular receptors for Cyclosporine A (CsA), are responsible for peptidyl-prolyl cis-trans isomerisation and for chaperoning several membrane proteins. Those functions are inhibited upon CsA binding. Albeit its great benefits as immunosuppressant, the use of CsA has been limited by undesirable nephrotoxic effects, including sodium retention, hypertension, hyperkalemia, interstial fibrosis and progressive renal failure in transplant recipients. In this report, we focused on the identification of novel CypB-interacting proteins to understand the role of CypB in kidney function and, in turn, to gain further insight into the molecular mechanisms of CsA-induced toxicity. By means of yeast two-hybrid screens with human kidney cDNA, we discovered a novel interaction between CypB and the membrane Na/K-ATPase β1 subunit protein (Na/K-β1) that was confirmed by pull-down, co-immunoprecipitation and confocal microscopy, in proximal tubule-derived HK-2 cells. The Na/K-ATPase pump, a key plasma membrane transporter, is responsible for maintenance of electrical Na+ and K+ gradients across the membrane. We showed that CypB silencing produced similar effects on Na/K-ATPase activity than CsA treatment in HK-2 cells. It was also observed an enrichment of both alpha and beta subunits in the ER, what suggested a possible failure on the maturation and routing of the pump from this compartment towards the plasma membrane. These data indicate that CypB through its interaction with Na/K-β1 might regulate maturation and trafficking of the pump through the secretory pathway, offering new insights into the relationship between cyclophilins and the nephrotoxic effects of CsA. PMID:21085665

  18. Primary Identification, Biochemical Characterization, and Immunologic Properties of the Allergenic Pollen Cyclophilin Cat r 1*

    PubMed Central

    Ghosh, Debajyoti; Mueller, Geoffrey A.; Schramm, Gabriele; Edwards, Lori L.; Petersen, Arnd; London, Robert E.; Haas, Helmut; Gupta Bhattacharya, Swati

    2014-01-01

    Cyclophilin (Cyp) allergens are considered pan-allergens due to frequently reported cross-reactivity. In addition to well studied fungal Cyps, a number of plant Cyps were identified as allergens (e.g. Bet v 7 from birch pollen, Cat r 1 from periwinkle pollen). However, there are conflicting data regarding their antigenic/allergenic cross-reactivity, with no plant Cyp allergen structures available for comparison. Because amino acid residues are fairly conserved between plant and fungal Cyps, it is particularly interesting to check whether they can cross-react. Cat r 1 was identified by immunoblotting using allergic patients' sera followed by N-terminal sequencing. Cat r 1 (∼91% sequence identity to Bet v 7) was cloned from a cDNA library and expressed in Escherichia coli. Recombinant Cat r 1 was utilized to confirm peptidyl-prolyl cis-trans-isomerase (PPIase) activity by a PPIase assay and the allergenic property by an IgE-specific immunoblotting and rat basophil leukemia cell (RBL-SX38) mediator release assay. Inhibition-ELISA showed cross-reactive binding of serum IgE from Cat r 1-allergic individuals to fungal allergenic Cyps Asp f 11 and Mala s 6. The molecular structure of Cat r 1 was determined by NMR spectroscopy. The antigenic surface was examined in relation to its plant, animal, and fungal homologues. The structure revealed a typical cyclophilin fold consisting of a compact β-barrel made up of seven anti-parallel β-strands along with two surrounding α-helices. This is the first structure of an allergenic plant Cyp revealing high conservation of the antigenic surface particularly near the PPIase active site, which supports the pronounced cross-reactivity among Cyps from various sources. PMID:24939849

  19. 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.

  20. Targeting the Cyclophilin Domain of Ran-binding Protein 2 (Ranbp2) with Novel Small Molecules to Control the Proteostasis of STAT3, hnRNPA2B1 and M-Opsin

    PubMed Central

    Cho, Kyoung-in; Orry, Andrew; Park, Se Eun; Ferreira, Paulo A.

    2015-01-01

    Cyclophilins are peptidyl cis-trans prolyl isomerases (PPIases), whose activity is typically inhibited by cyclosporine A (CsA), a potent immunosuppressor. Cyclophilins are also chaperones. Emerging evidence supports that cyclophilins present non-overlapping PPIase and chaperone activities. The proteostasis of the disease-relevant substrates, signal transducer and activator of transcription 3 and 5 (STAT3/STAT5), heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) and M-opsin, are regulated by non-overlapping chaperone and PPIase activities of the cyclophilin domain (CY) of Ranbp2, a multifunctional and modular scaffold which controls nucleocytoplasmic shuttling and proteostasis of selective substrates. Although highly homologous, CY and the archetypal cyclophilin A (CyPA) present distinct catalytic and CsA-binding activities owing to unique structural features between these cylophilins. We explored structural idiosyncrasies between CY and CyPA to screen in silico nearly 9 million small molecules (SM) against the CY PPIase pocket and identify SMs with selective bioactivity toward STAT3, hnRNPA2B1 and/or M-opsin proteostasis. We found three classes of SMs that enhance the cytokine-stimulated transcriptional activity of STAT3 without changing latent and activated STAT3 levels, down-regulate hnRNPA2B1 or M-opsin proteostasis, or a combination of these. Further, a SM which suppresses hnRNPA2B1 proteostasis also inhibits strongly and selectively the PPIase activity of CY. This study unravels chemical probes for multimodal regulation of CY of Ranbp2 and its substrates and this regulation likely results in the allosterism stemming from the interconversion of conformational substates of cyclophilins. The results also demonstrate the feasibility of CY in drug discovery against disease-relevant substrates controlled by Ranbp2 and they open new opportunities for therapeutic interventions. PMID:26030368

  1. Psychrophily and catalysis.

    PubMed

    Gerday, Charles

    2013-04-16

    Polar and other low temperature environments are characterized by a low content in energy and this factor has a strong incidence on living organisms which populate these rather common habitats. Indeed, low temperatures have a negative effect on ectothermic populations since they can affect their growth, reaction rates of biochemical reactions, membrane permeability, diffusion rates, action potentials, protein folding, nucleic acids dynamics and other temperature-dependent biochemical processes. Since the discovery that these ecosystems, contrary to what was initially expected, sustain a rather high density and broad diversity of living organisms, increasing efforts have been dedicated to the understanding of the molecular mechanisms involved in their successful adaptation to apparently unfavorable physical conditions. The first question that comes to mind is: How do these organisms compensate for the exponential decrease of reaction rate when temperature is lowered? As most of the chemical reactions that occur in living organisms are catalyzed by enzymes, the kinetic and thermodynamic properties of cold-adapted enzymes have been investigated. Presently, many crystallographic structures of these enzymes have been elucidated and allowed for a rather clear view of their adaptation to cold. They are characterized by a high specific activity at low and moderate temperatures and a rather low thermal stability, which induces a high flexibility that prevents the freezing effect of low temperatures on structure dynamics. These enzymes also display a low activation enthalpy that renders them less dependent on temperature fluctuations. This is accompanied by a larger negative value of the activation entropy, thus giving evidence of a more disordered ground state. Appropriate folding kinetics is apparently secured through a large expression of trigger factors and peptidyl-prolyl cis/trans-isomerases.

  2. Cyclophilin A is an important mediator of platelet function by regulating integrin αIIbβ3 bidirectional signalling.

    PubMed

    Wang, Lian; Soe, Nwe Nwe; Sowden, Mark; Xu, Yingqian; Modjeski, Kristina; Baskaran, Padmamalini; Kim, Yeonghwan; Smolock, Elaine M; Morrell, Craig N; Berk, Bradford C

    2014-05-05

    Cyclophilin A (CyPA) is an important mediator in cardiovascular diseases. It possesses peptidyl-prolyl cis-trans isomerase activity (PPIase) and chaperone functions, which regulate protein folding, intracellular trafficking and reactive oxygen species (ROS) production. Platelet glycoprotein receptor αIIbβ3 integrin activation is the common pathway for platelet activation. It was our objective to understand the mechanism by which CyPA-regulates αIIbβ3 activation in platelets. Mice deficient for CyPA (CyPA-/-) had prolonged tail bleeding time compared to wild-type (WT) controls despite equivalent platelet numbers. In vitro studies revealed that CyPA-/- platelets exhibited dramatically decreased thrombin-induced platelet aggregation. In vivo, formation of occlusive thrombi following FeCl3 injury was also significantly impaired in CyPA-/- mice compared with WT-controls. Furthermore, CyPA deficiency inhibited flow-induced thrombus formation in vitro. Flow cytometry demonstrated that thrombin-induced ROS production and αIIbβ3 activation were reduced in CyPA-/- platelets. Coimmunoprecipitation studies showed ROS-dependent increased association of CyPA and αIIbβ3. This association was dependent upon the PPIase activity of CyPA. Significantly, fibrinogen-platelet binding, platelet spreading and cytoskeleton reorganisation were also altered in CyPA-/- platelets. Moreover, CyPA deficiency prevented thrombin-induced αIIbβ3 and cytoskeleton association. In conclusion, CyPA is an important mediator in platelet function by regulation of αIIbβ3 bidirectionalsignalling through increased ROS production and facilitating interaction between αIIbβ3 and the cell cytoskeleton.

  3. Coupled Dynamics and Entropic Contribution to the Allosteric Mechanism of Pin1.

    PubMed

    Barman, Arghya; Hamelberg, Donald

    2016-08-25

    Allosteric communication in proteins regulates a plethora of downstream processes in subcellular signaling pathways. Describing the effects of cooperative ligand binding on the atomic level is a key to understanding many regulatory processes involving biomolecules. Here, we use microsecond-long molecular dynamics simulations to investigate the allosteric mechanism of Pin1, a potential therapeutic target and a phosphorylated-Ser/Thr dependent peptidyl-prolyl cis-trans isomerase that regulates several subcellular processes and has been implicated in many diseases, including cancer and Alzheimer's. Experimental studies suggest that the catalytic domain and the noncatalytic WW domain are allosterically coupled; however, an atomic level description of the dynamics associated with the interdomain communication is lacking. We show that binding of the substrate to the WW domain is directly coupled to the dynamics of the catalytic domain, causing rearrangement of the residue-residue contact dynamics from the WW domain to the catalytic domain. The binding affinity of the substrate in the catalytic domain is also enhanced upon binding of the substrate to the WW domain. Modulation of the dynamics of the catalytic domain upon binding of the substrate to the WW domain leads to prepayment of the entropic cost of binding the substrate to the catalytic domain. This study shows that Ile 28 at the interfacial region between the catalytic and WW domains is certainly one of the residues responsible for bridging the communication between the two domains. The results complement previous experiments and provide valuable atomistic insights into the role of dynamics and possible entropic contribution to the allosteric mechanism of proteins.

  4. 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.

  5. Primary identification, biochemical characterization, and immunologic properties of the allergenic pollen cyclophilin cat R 1.

    PubMed

    Ghosh, Debajyoti; Mueller, Geoffrey A; Schramm, Gabriele; Edwards, Lori L; Petersen, Arnd; London, Robert E; Haas, Helmut; Gupta Bhattacharya, Swati

    2014-08-01

    Cyclophilin (Cyp) allergens are considered pan-allergens due to frequently reported cross-reactivity. In addition to well studied fungal Cyps, a number of plant Cyps were identified as allergens (e.g. Bet v 7 from birch pollen, Cat r 1 from periwinkle pollen). However, there are conflicting data regarding their antigenic/allergenic cross-reactivity, with no plant Cyp allergen structures available for comparison. Because amino acid residues are fairly conserved between plant and fungal Cyps, it is particularly interesting to check whether they can cross-react. Cat r 1 was identified by immunoblotting using allergic patients' sera followed by N-terminal sequencing. Cat r 1 (∼ 91% sequence identity to Bet v 7) was cloned from a cDNA library and expressed in Escherichia coli. Recombinant Cat r 1 was utilized to confirm peptidyl-prolyl cis-trans-isomerase (PPIase) activity by a PPIase assay and the allergenic property by an IgE-specific immunoblotting and rat basophil leukemia cell (RBL-SX38) mediator release assay. Inhibition-ELISA showed cross-reactive binding of serum IgE from Cat r 1-allergic individuals to fungal allergenic Cyps Asp f 11 and Mala s 6. The molecular structure of Cat r 1 was determined by NMR spectroscopy. The antigenic surface was examined in relation to its plant, animal, and fungal homologues. The structure revealed a typical cyclophilin fold consisting of a compact β-barrel made up of seven anti-parallel β-strands along with two surrounding α-helices. This is the first structure of an allergenic plant Cyp revealing high conservation of the antigenic surface particularly near the PPIase active site, which supports the pronounced cross-reactivity among Cyps from various sources.

  6. 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

  7. Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination.

    PubMed

    Takeda, Mitsuhiro; Ono, Akira M; Terauchi, Tsutomu; Kainosho, Masatsune

    2010-01-01

    The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines (epsilon- and zeta-SAIL Phe) and tyrosine (epsilon-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven that these SAIL amino acids provide dramatic spectral simplification and sensitivity enhancement for the aromatic ring NMR signals. In addition to these SAIL aromatic amino acids, we recently synthesized delta-SAIL Phe and delta-SAIL Tyr, which allow us to observe and assign delta-(13)C/(1)H signals very efficiently. Each of the various types of SAIL Phe and SAIL Tyr yields well-resolved resonances for the delta-, epsilon- or zeta-(13)C/(1)H signals, respectively, which can readily be assigned by simple and robust pulse sequences. Since the delta-, epsilon-, and zeta-proton signals of Phe/Tyr residues give rise to complementary NOE constraints, the concomitant use of various types of SAIL-Phe and SAIL-Tyr would generate more accurate protein structures, as compared to those obtained by using conventional uniformly (13)C, (15)N-double labeled proteins. We illustrated this with the case of an 18.2 kDa protein, Escherichia coli peptidyl-prolyl cis-trans isomerase b (EPPIb), and concluded that the combined use of zeta-SAIL Phe and epsilon-SAIL Tyr would be practically the best choice for protein structural determinations.

  8. 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.

  9. 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

  10. Genome Wide Identification of the Immunophilin Gene Family in Leptosphaeria maculans: A Causal Agent of Blackleg Disease in Oilseed Rape (Brassica napus)

    PubMed Central

    Zouhar, Miloslav; Mazakova, Jana; Rysanek, Pavel

    2014-01-01

    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

  11. Modulation of conotoxin structure and function is achieved through a multienzyme complex in the venom glands of cone snails.

    PubMed

    Safavi-Hemami, Helena; Gorasia, Dhana G; Steiner, Andrew M; Williamson, Nicholas A; Karas, John A; Gajewiak, Joanna; Olivera, Baldomero M; Bulaj, Grzegorz; Purcell, Anthony W

    2012-10-05

    The oxidative folding of large polypeptides has been investigated in detail; however, comparatively little is known about the enzyme-assisted folding of small, disulfide-containing peptide substrates. To investigate the concerted effect of multiple enzymes on the folding of small disulfide-rich peptides, we sequenced and expressed protein-disulfide isomerase (PDI), peptidyl-prolyl cis-trans isomerase, and immunoglobulin-binding protein (BiP) from Conus venom glands. Conus PDI was shown to catalyze the oxidation and reduction of disulfide bonds in two conotoxins, α-GI and α-ImI. Oxidative folding rates were further increased in the presence of Conus PPI with the maximum effect observed in the presence of both enzymes. In contrast, Conus BiP was only observed to assist folding in the presence of microsomes, suggesting that additional co-factors were involved. The identification of a complex between BiP, PDI, and nascent conotoxins further suggests that the folding and assembly of conotoxins is a highly regulated multienzyme-assisted process. Unexpectedly, all three enzymes contributed to the folding of the ribbon isomer of α-ImI. Here, we identify this alternative disulfide-linked species in the venom of Conus imperialis, providing the first evidence for the existence of a "non-native" peptide isomer in the venom of cone snails. Thus, ER-resident enzymes act in concert to accelerate the oxidative folding of conotoxins and modulate their conformation and function by reconfiguring disulfide connectivities. This study has evaluated the role of a number of ER-resident enzymes in the folding of conotoxins, providing novel insights into the enzyme-guided assembly of these small, disulfide-rich peptides.

  12. Modulation of Conotoxin Structure and Function Is Achieved through a Multienzyme Complex in the Venom Glands of Cone Snails*

    PubMed Central

    Safavi-Hemami, Helena; Gorasia, Dhana G.; Steiner, Andrew M.; Williamson, Nicholas A.; Karas, John A.; Gajewiak, Joanna; Olivera, Baldomero M.; Bulaj, Grzegorz; Purcell, Anthony W.

    2012-01-01

    The oxidative folding of large polypeptides has been investigated in detail; however, comparatively little is known about the enzyme-assisted folding of small, disulfide-containing peptide substrates. To investigate the concerted effect of multiple enzymes on the folding of small disulfide-rich peptides, we sequenced and expressed protein-disulfide isomerase (PDI), peptidyl-prolyl cis-trans isomerase, and immunoglobulin-binding protein (BiP) from Conus venom glands. Conus PDI was shown to catalyze the oxidation and reduction of disulfide bonds in two conotoxins, α-GI and α-ImI. Oxidative folding rates were further increased in the presence of Conus PPI with the maximum effect observed in the presence of both enzymes. In contrast, Conus BiP was only observed to assist folding in the presence of microsomes, suggesting that additional co-factors were involved. The identification of a complex between BiP, PDI, and nascent conotoxins further suggests that the folding and assembly of conotoxins is a highly regulated multienzyme-assisted process. Unexpectedly, all three enzymes contributed to the folding of the ribbon isomer of α-ImI. Here, we identify this alternative disulfide-linked species in the venom of Conus imperialis, providing the first evidence for the existence of a “non-native” peptide isomer in the venom of cone snails. Thus, ER-resident enzymes act in concert to accelerate the oxidative folding of conotoxins and modulate their conformation and function by reconfiguring disulfide connectivities. This study has evaluated the role of a number of ER-resident enzymes in the folding of conotoxins, providing novel insights into the enzyme-guided assembly of these small, disulfide-rich peptides. PMID:22891240

  13. The Posttranslocational Chaperone Lipoprotein PrsA Is Involved in both Glycopeptide and Oxacillin Resistance in Staphylococcus aureus

    PubMed Central

    Jousselin, Ambre; Renzoni, Adriana; Andrey, Diego O.; Monod, Antoinette; Lew, Daniel P.

    2012-01-01

    Understanding in detail the factors which permit Staphylococcus aureus to counteract cell wall-active antibiotics is a prerequisite to elaborating effective strategies to prolong the usefulness of these drugs and define new targets for pharmacological intervention. Methicillin-resistant S. aureus (MRSA) strains are major pathogens of hospital-acquired and community-acquired infections and are most often treated with glycopeptides (vancomycin and teicoplanin) because of their resistance to most penicillins and a limited arsenal of clinically proven alternatives. In this study, we examined PrsA, a lipid-anchored protein of the parvulin PPIase family (peptidyl-prolyl cis/trans isomerase) found ubiquitously in all Gram-positive species, in which it assists posttranslocational folding at the outer surface of the cytoplasmic membrane. We show by both genetic and biochemical assays that prsA is directly regulated by the VraRS two-component sentinel system of cell wall stress. Disruption of prsA is tolerated by S. aureus, and its loss results in no detectable overt macroscopic changes in cell wall architecture or growth rate under nonstressed growth conditions. Disruption of prsA leads, however, to notable alterations in the sensitivity to glycopeptides and dramatically decreases the resistance of COL (MRSA) to oxacillin. Quantitative transcriptional analysis reveals that prsA and vraR are coordinately upregulated in a panel of stable laboratory and clinical glycopeptide-intermediate S. aureus (GISA) strains compared to their susceptible parents. Collectively, our results point to a role for prsA as a facultative facilitator of protein secretion or extracellular folding and provide a framework for understanding why prsA is a key element of the VraRS-mediated cell wall stress response. PMID:22526301

  14. A knockout strain of CPR1 induced during fermentation of Saccharomyces cerevisiae KNU5377 is susceptible to various types of stress.

    PubMed

    Kim, Il-Sup; Yun, Hae-Sun; Park, In-Su; Sohn, Ho-Yong; Iwahashi, Hitoshi; Jin, Ing-Nyol

    2006-10-01

    To investigate the tolerance factor of Saccharomyces cerevisiae KNU5377 against various types of environmental stress during fermentation, we identified the protein that is upregulated at high temperatures. The highly upregulated protein was high-score-matched as a cytoplasmic peptidyl-prolyl cis-trans isomerase, cyclophilin (Cpr1p), by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). We constructed a CPR1-deleted KNU5377 strain (KNU5377Y cpr1Delta) to determine the roles of the protein under fermentative or stress condition. The growth of the S. cerevisiae KNU5377Y cpr1Delta strain was completely inhibited under the following conditions: heat (40 degrees C), hydrogen peroxide (20-30 mM), menadione (0.3 mM), ethanol (16%), sulfuric acid (5 mm), and lactic acid (0.4-0.8%). However, the wild-type and cpr1Delta mutant of S. cerevisiae BY4741 as a positive control did not show differences in sensitivity to stress. It is interesting to note that the wild-type KNU5377Y and KNU5377Y cpr1Delta mutant showed high sensitivity against various stresses, particularly, acid stress such as in the presence of sulfuric and lactic acid. Although the alcohol fermentation rate of the KNU5377Y cpr1Delta mutant markedly decreased with an increase in temperature up to 40 degrees C, we observed no decrease in that of the wild-type strain under the same conditions. These results suggest that CPR1 contributes to the stress tolerance of KNU5377 against various types of environmental stress caused during fermentation, thus leading to the physiological role of maintaining an alcohol fermentation yield, even at high temperatures such as 40 degrees C.

  15. Pin1 promotes transforming growth factor-beta-induced migration and invasion.

    PubMed

    Matsuura, Isao; Chiang, Keng-Nan; Lai, Chen-Yu; He, Dongming; Wang, Guannan; Ramkumar, Romila; Uchida, Takafumi; Ryo, Akihide; Lu, Kunping; Liu, Fang

    2010-01-15

    Transforming growth factor-beta (TGF-beta) regulates a wide variety of biological activities. It induces potent growth-inhibitory responses in normal cells but promotes migration and invasion of cancer cells. Smads mediate the TGF-beta responses. TGF-beta binding to the cell surface receptors leads to the phosphorylation of Smad2/3 in their C terminus as well as in the proline-rich linker region. The serine/threonine phosphorylation sites in the linker region are followed by the proline residue. Pin1, a peptidyl-prolyl cis/trans isomerase, recognizes phosphorylated serine/threonine-proline motifs. Here we show that Smad2/3 interacts with Pin1 in a TGF-beta-dependent manner. We further show that the phosphorylated threonine 179-proline motif in the Smad3 linker region is the major binding site for Pin1. Although epidermal growth factor also induces phosphorylation of threonine 179 and other residues in the Smad3 linker region the same as TGF-beta, Pin1 is unable to bind to the epidermal growth factor-stimulated Smad3. Further analysis suggests that phosphorylation of Smad3 in the C terminus is necessary for the interaction with Pin1. Depletion of Pin1 by small hairpin RNA does not significantly affect TGF-beta-induced growth-inhibitory responses and a number of TGF-beta/Smad target genes analyzed. In contrast, knockdown of Pin1 in human PC3 prostate cancer cells strongly inhibited TGF-beta-mediated migration and invasion. Accordingly, TGF-beta induction of N-cadherin, which plays an important role in migration and invasion, is markedly reduced when Pin1 is depleted in PC3 cells. Because Pin1 is overexpressed in many cancers, our findings highlight the importance of Pin1 in TGF-beta-induced migration and invasion of cancer cells.

  16. 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

  17. Expression and characterization of human FKBP52, an immunophilin that associates with the 90-kDa heat shock protein and is a component of steroid receptor complexes.

    PubMed Central

    Peattie, D A; Harding, M W; Fleming, M A; DeCenzo, M T; Lippke, J A; Livingston, D J; Benasutti, M

    1992-01-01

    Using an FK506 affinity column to identify mammalian immunosuppressant-binding proteins, we identified an immunophilin with an apparent M(r) approximately 55,000, which we have named FKBP52. We used chemically determined peptide sequence and a computerized algorithm to search GenPept, the translated GenBank data base, and identified two cDNAs likely to encode the murine FKBP52 homolog. We amplified a murine cDNA fragment, used it to select a human FKBP52 (hFKBP52) cDNA clone, and then used the clone to deduce the hFKBP52 sequence (calculated M(r) 51,810) and to express hFKBP52 in Escherichia coli. Recombinant hFKBP52 has peptidyl-prolyl cis-trans isomerase activity that is inhibited by FK506 and rapamycin and an FKBP12-like consensus sequence that probably defines the immunosuppressant-binding site. FKBP52 is apparently common to several vertebrate species and associates with the 90-kDa heat shock protein (hsp90) in untransformed mammalian steroid receptor complexes. The putative immunosuppressant-binding site is probably distinct from the hsp90-binding site, and we predict that FKBP52 has different structural domains to accommodate these functions. hFKBP52 contains 12 protein kinase phosphorylation-site motifs and a potential calmodulin-binding site, implying that posttranslational phosphorylation could generate multiple isoforms of the protein and that calmodulin and intracellular Ca2+ levels could affect FKBP52 function. FKBP52 transcripts are present in a variety of human tissues and could vary in abundance and/or stability. Images PMID:1279700

  18. What is the mitochondrial permeability transition pore?

    PubMed

    Halestrap, Andrew P

    2009-06-01

    Under conditions of mitochondrial calcium overload, especially when accompanied by oxidative stress, elevated phosphate concentrations and adenine nucleotide depletion, a non-specific pore, the mitochondrial permeability transition pore (MPTP), opens in the inner mitochondrial membrane. MPTP opening enables free passage into the mitochondria of molecules of <1.5 kDa including protons. The resulting uncoupling of oxidative phosphorylation leads to ATP depletion and necrotic cell death and it is now widely recognised that MPTP opening is a major cause of reperfusion injury and an effective target for cardioprotection. The properties of the MPTP are well defined, but despite extensive research in many laboratories, its exact molecular identity remains uncertain. Knockout studies have confirmed a role for cyclophilin-D (CyP-D), probably mediated by its peptidyl-prolyl cis-trans isomerase activity facilitating a conformational change of an inner membrane protein. However, the identity of the membrane component(s) remains controversial. Knockout studies have eliminated an essential role for either the voltage dependent anion channel (VDAC) or the adenine nucleotide translocase (ANT), although a regulatory role for the ANT was confirmed. Our own studies implicate the mitochondrial phosphate carrier (PiC) in MPTP formation and are consistent with a calcium-triggered conformational change of the PiC, facilitated by CyP-D, inducing pore opening. We propose that this is enhanced by an association of the PiC with the "c" conformation of the ANT. Agents that modulate pore opening may act on either or both the PiC and the ANT. However, knockdown and reconstitution studies are awaited to confirm or refute this model.

  19. Conformational plasticity of an enzyme during catalysis: intricate coupling between cyclophilin A dynamics and substrate turnover.

    PubMed

    McGowan, Lauren C; Hamelberg, Donald

    2013-01-08

    Enzyme catalysis is central to almost all biochemical processes, speeding up rates of reactions to biological relevant timescales. Enzymes make use of a large ensemble of conformations in recognizing their substrates and stabilizing the transition states, due to the inherent dynamical nature of biomolecules. The exact role of these diverse enzyme conformations and the interplay between enzyme conformational dynamics and catalysis is, according to the literature, not well understood. Here, we use molecular dynamics simulations to study human cyclophilin A (CypA), in order to understand the role of enzyme motions in the catalytic mechanism and recognition. Cyclophilin A is a tractable model system to study using classical simulation methods, because catalysis does not involve bond formation or breakage. We show that the conformational dynamics of active site residues of substrate-bound CypA is inherent in the substrate-free enzyme. CypA interacts with its substrate via conformational selection as the configurations of the substrate changes during catalysis. We also show that, in addition to tight intermolecular hydrophobic interactions between CypA and the substrate, an intricate enzyme-substrate intermolecular hydrogen-bonding network is extremely sensitive to the configuration of the substrate. These enzyme-substrate intermolecular interactions are loosely formed when the substrate is in the reactant and product states and become well formed and reluctant to break when the substrate is in the transition state. Our results clearly suggest coupling among enzyme-substrate intermolecular interactions, the dynamics of the enzyme, and the chemical step. This study provides further insights into the mechanism of peptidyl-prolyl cis/trans isomerases and the general interplay between enzyme conformational dynamics and catalysis.

  20. TNF-α Suppresses Prolyl-4-Hydroxylase α1 Expression via the ASK1–JNK–NonO Pathway

    PubMed Central

    Zhang, Cheng; Zhang, Ming-Xiang; Shen, Ying H.; Burks, Jared K.; Zhang, Yun; Wang, Jian; LeMaire, Scott A.; Yoshimura, Koichi; Aoki, Hiroki; Coselli, Joseph S.; Wang, Xing Li

    2008-01-01

    Background Inflammation is known to contribute to the pathogenesis of vascular diseases in which arterial wall extracellular matrix (ECM) homeostasis is disrupted. Tumor necrosis factor-α (TNF-α), a pivotal cytokine that regulates ECM metabolism by increasing degradation and decreasing production of arterial collagens, is associated with vulnerable plaques and aortic aneurysms. Methods and Results In the current study, we showed that, when administered in doses of 1 to 100 ng/mL, TNF-α dose-dependently downregulated the expression of prolyl-4-hydroxylase αI [P4Hα (I)]—the rate-limiting subunit for the P4H enzyme essential for procollagen hydroxylation, secretion, and deposition in primary human aortic smooth muscle cells (HASMCs). Using a progressive deletion cloning approach, we characterized the TNF-α–responsive element (TaRE) in the human P4Hα (I) promoter and found that a negative regulatory region at the position of −32 to +18bp is responsible for ≈80% of TNF-α–mediated suppression. Using oligonucleotide-based transcription factor pull-down method in which proteins were resolved in 1-D gel electrophoresis and identified using LC-MS/MS, we identified the NonO protein binds this region. When NonO expression silenced with specific siRNA, we found that 70% of the TNF-α–mediated P4Hα suppression was abolished, which appeared to be mediated by the ASK1-JNK pathway. Conclusions Our findings define a novel molecular pathway for inflammation associated extracellular matrix dysregulation, which may account for atherosclerotic plaque rupture and aortic aneurysm formation. Further understanding of this pathway may facilitate development of novel therapeutics for vascular diseases. PMID:17478756

  1. 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.

  2. Structural Analysis of Prolyl Oligopeptidases Using Molecular Docking and Dynamics: Insights into Conformational Changes and Ligand Binding

    PubMed Central

    Kaushik, Swati; Sowdhamini, Ramanathan

    2011-01-01

    Prolyl oligopeptidase (POP) is considered as an important pharmaceutical target for the treatment of numerous diseases. Despite enormous studies on various aspects of POPs structure and function still some of the questions are intriguing like conformational dynamics of the protein and interplay between ligand entry/egress. Here, we have used molecular modeling and docking based approaches to unravel questions like differences in ligand binding affinities in three POP species (porcine, human and A. thaliana). Despite high sequence and structural similarity, they possess different affinities for the ligands. Interestingly, human POP was found to be more specific, selective and incapable of binding to a few planar ligands which showed extrapolation of porcine POP in human context is more complicated. Possible routes for substrate entry and product egress were also investigated by detailed analyses of molecular dynamics (MD) simulations for the three proteins. Trajectory analysis of bound and unbound forms of three species showed differences in conformational dynamics, especially variations in β-propeller pore size, which was found to be hidden by five lysine residues present on blades one and seven. During simulation, β-propeller pore size was increased by ∼2 Å in porcine ligand-bound form which might act as a passage for smaller product movement as free energy barrier was reduced, while there were no significant changes in human and A. thaliana POPs. We also suggest that these differences in pore size could lead to fundamental differences in mode of product egress among three species. This analysis also showed some functionally important residues which can be used further for in vitro mutagenesis and inhibitor design. This study can help us in better understanding of the etiology of POPs in several neurodegenerative diseases. PMID:22132071

  3. Prolyl oligopeptidase inhibition attenuates the toxicity of a proteasomal inhibitor, lactacystin, in the alpha-synuclein overexpressing cell culture.

    PubMed

    Myöhänen, Timo T; Norrbacka, Susanna; Savolainen, Mari H

    2017-01-01

    Lewy bodies, the histopathological hallmarks of Parkinson's disease (PD), contain insoluble and aggregated α-synuclein (aSyn) and many other proteins, proposing a role for failure in protein degradation system in the PD pathogenesis. Proteasomal dysfunction has indeed been linked to PD and aSyn oligomers have been shown to inhibit proteasomes and autophagy. Our recent studies have shown that inhibitors of prolyl oligopeptidase (PREP) can prevent the aggregation and enhance the clearance of accumulated aSyn, and therefore, we wanted to study if PREP inhibition can overcome the aSyn aggregation and toxicity induced by lactacystin, a proteasomal inhibitor. The cells overexpressing human A30P or A53T mutated aSyn were incubated with lactacystin and a PREP inhibitor, KYP-2047, for 48h. Theafter, the cells were fractioned, and the effects of lactacystin with/without 1μM KYP-2047 on aSyn aggregation and ubiquitin accumulation, cell viability and on autophagic markers (p62, Beclin1 and LC3BII) were studied. We found that KYP-2047 attenuated lactacystin-induced cell death in mutant aSyn overexpressing cells but not in non-overexpressing control cells. KYP-2047 reduced significantly SDS-insoluble high-molecular-weight aSyn oligomers that were in line with the cell viability results. In addition, significant reduction in protein accumulation marker, p62, was seen in SDS fraction while LC3BII, a marker for autophagosome formation, was increased, indicating to enhanced autophagy. Our results further streghten the possibilities for PREP inhibitors as a potential drug therapy against synucleinopathies and other protein aggregating diseases.

  4. RUNX3 inhibits hypoxia-inducible factor-1α protein stability by interacting with prolyl hydroxylases in gastric cancer cells.

    PubMed

    Lee, S H; Bae, S C; Kim, K W; Lee, Y M

    2014-03-13

    RUNX3 is silenced by histone modification and hypoxia-inducible factor (HIF)-1α is stabilized under hypoxia, but little is known of cross-talk between RUNX3 and HIF-1α under hypoxia. In the present study, the authors investigated the effect of RUNX3 on HIF-1α stability in gastric cancer cells. RUNX3 overexpression was found to downregulate HIF-1α stability under normoxic and hypoxic conditions. Furthermore, the activity of a luciferase reporter containing five copies of vascular endothelial growth factor (VEGF) promoter hypoxia-responsive element (5 × HRE) and the amount of secreted VEGF, were diminished in RUNX3-expressing but increased in RUNX3-knockdown cells. When expression of RUNX3 was recovered using epigenetic reagents the expressions of HIF-1α and VEGF were clearly suppressed under hypoxic conditions. RUNX3 also significantly attenuated the half-life of HIF-1α protein, and induced the cytosolic localization and ubiquitination of HIF-1α. In addition, RUNX3 directly interacted with the C-terminal activation domain of HIF-1α and prolyl hydroxylase (PHD) 2 and enhanced the interaction between HIF-1α and PHD2, which potentiated proline hydroxylation and promoted the degradation of HIF-1α. Furthermore, RUNX3 overexpression significantly inhibited hypoxia-induced angiogenesis in vitro and in vivo. Taken together, these results suggest that RUNX3 destabilizes HIF-1α protein by promoting the proline hydroxylation of HIF-1α through binding to HIF-1α/PHD2. RUNX3 appears to be a novel suppressor of HIF-1α and of hypoxia-mediated angiogenesis in gastric cancer cells.

  5. Loss of prolyl hydroxylase domain protein 2 in vascular endothelium increases pericyte coverage and promotes pulmonary arterial remodeling

    PubMed Central

    Xie, Xue-Jiao; Tao, Yong-Kang; He, Xiaochen; Roman, Richard J.; Aschner, Judy L.; Chen, Jian-Xiong

    2016-01-01

    Pulmonary arterial hypertension (PAH) is a leading cause of heart failure. Although pulmonary endothelial dysfunction plays a crucial role in the progression of the PAH, the underlying mechanisms are poorly understood. The HIF-α hydroxylase system is a key player in the regulation of vascular remodeling. Knockout of HIF-2α has been reported to cause pulmonary hypertension. The present study examined the role of endothelial cell specific prolyl hydroxylase-2 (PHD2) in the development of PAH and pulmonary vascular remodeling. The PHD2f/f mouse was crossbred with VE-Cadherin-Cre promoter mouse to generate an endothelial specific PHD2 knockout (Cdh5-Cre-PHD2ECKO) mouse. Pulmonary arterial pressure and the size of the right ventricle was significantly elevated in the PHD2ECKO mice relative to the PHD2f/f controls. Knockout of PHD2 in EC was associated with vascular remodeling, as evidenced by an increase in pulmonary arterial media to lumen ratio and number of muscularized arterioles. The pericyte coverage and vascular smooth muscle cells were also significantly increased in the PA. The increase in vascular pericytes was associated with elevated expression of fibroblast specific protein-1 (FSP-1). Moreover, perivascular interstitial fibrosis of pulmonary arteries was significantly increased in the PHD2ECKO mice. Mechanistically, knockout of PHD2 in EC increased the expression of Notch3 and transforming growth factor (TGF-β) in the lung tissue. We conclude that the expression of PHD2 in endothelial cells plays a critical role in preventing pulmonary arterial remodeling in mice. Increased Notch3/TGF-β signaling and excessive pericyte coverage may be contributing to the development of PAH following deletion of endothelial PHD2. PMID:27613846

  6. Cigarette smoke-induced lung emphysema in mice is associated with prolyl endopeptidase, an enzyme involved in collagen breakdown

    PubMed Central

    Koelink, Pim J.; Henricks, Paul A. J.; Jackson, Patricia L.; Nijkamp, Frans P.; Garssen, Johan; Kraneveld, Aletta D.; Blalock, J. Edwin; Folkerts, Gert

    2011-01-01

    There is increasing evidence that the neutrophil chemoattractant proline-glycine-proline (PGP), derived from the breakdown of the extracellular matrix, plays an important role in neutrophil recruitment to the lung. PGP formation is a multistep process involving neutrophils, metalloproteinases (MMPs), and prolyl endopeptidase (PE). This cascade of events is now investigated in the development of lung emphysema. A/J mice were whole body exposed to cigarette smoke for 20 wk. After 20 wk or 8 wk after smoking cessation, animals were killed, and bronchoalveolar lavage fluid and lung tissue were collected to analyze the neutrophilic airway inflammation, the MMP-8 and MMP-9 levels, the PE activity, and the PGP levels. Lung tissue degradation was assessed by measuring the mean linear intercept. Additionally, we investigated the effect of the peptide l-arginine-threonine-arginine (RTR), which binds to PGP sequences, on the smoke-induced neutrophil influx in the lung after 5 days of smoke exposure. Neutrophilic airway inflammation was induced by cigarette smoke exposure. MMP-8 and MMP-9 levels, PE activity, and PGP levels were elevated in the lungs of cigarette smoke-exposed mice. PE was highly expressed in epithelial and inflammatory cells (macrophages and neutrophils) in lung tissue of cigarette smoke-exposed mice. After smoking cessation, the neutrophil influx, the MMP-8 and MMP-9 levels, the PE activity, and the PGP levels were decreased or reduced to normal levels. Moreover, RTR inhibited the smoke-induced neutrophil influx in the lung after 5 days' smoke exposure. In the present murine model of cigarette smoke-induced lung emphysema, it is demonstrated for the first time that all relevant components (neutrophils, MMP-8, MMP-9, PE) involved in PGP formation from collagen are upregulated in the airways. Together with MMPs, PE may play an important role in the formation of PGP and thus in the pathophysiology of lung emphysema. PMID:21112944

  7. 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.

  8. Fermentation, purification, formulation, and pharmacological evaluation of a prolyl endopeptidase from Myxococcus xanthus: implications for Celiac Sprue therapy.

    PubMed

    Gass, Jonathan; Ehren, Jennifer; Strohmeier, Gregg; Isaacs, Indu; Khosla, Chaitan

    2005-12-20

    Celiac Sprue is a multi-factorial disease characterized by an inflammatory response to ingested wheat gluten and similar proteins in rye and barley. Proline-rich gluten peptides from wheat, rye, and barley are relatively resistant to gastrointestinal digestion, and therefore persist in the intestinal lumen to elicit immunopathology in genetically susceptible individuals. In this study, we characterize the in vitro gluten detoxifying properties of a therapeutically promising prolyl endopeptidase from Myxococcus xanthus (MX PEP), and describe the development of a prototypical enteric-coated capsule containing a pharmacologically useful dose of this enzyme. A high-cell density fed-batch fermentation process was developed for overproduction of recombinant MX PEP in E. coli, yielding 0.25-0.4 g/L purified protein. A simple, scalable purification and lyophilization procedure was established that yields >95% pure, highly active and stable enzyme as a dry powder. The dry powder was blended with excipients and encapsulated in a hard gelatin capsule. The resulting capsule was enteric coated using Eudragit L30-D55 polymer coat, which provided sufficient resistance to gastric conditions (> 1 h in 0.01 M HCl, pH 2 with pepsin) and rapid release under duodenal conditions (15-30 min release in pH 6.0 in the presence of trypsin and chymotrypsin). In conjunction with pancreatic enzymes, MX PEP breaks down whole gluten into a product mixture that is virtually indistinguishable from that generated by the Flavobacterium meningosepticum (FM) PEP as judged by chromatographic assays. Competitive studies involving selected immunogenic peptides mixed with whole gluten reveal that both PEPs have a wide range of substrate specificity. Our results support further in vitro and in vivo evaluation of the MX PEP capsule as an oral therapeutic agent for Celiac Sprue patients.

  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. 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

  11. HIF prolyl hydroxylase 2 (PHD2) is a critical regulator of hematopoietic stem cell maintenance during steady-state and stress.

    PubMed

    Singh, Rashim Pal; Franke, Kristin; Kalucka, Joanna; Mamlouk, Soulafa; Muschter, Antje; Gembarska, Agnieszka; Grinenko, Tatyana; Willam, Carsten; Naumann, Ronald; Anastassiadis, Konstantinos; Stewart, A Francis; Bornstein, Stefan; Chavakis, Triantafyllos; Breier, Georg; Waskow, Claudia; Wielockx, Ben

    2013-06-27

    Hypoxia is a prominent feature in the maintenance of hematopoietic stem cell (HSC) quiescence and multipotency. Hypoxia-inducible factor (HIF) prolyl hydroxylase domain proteins (PHDs) serve as oxygen sensors and may therefore regulate this system. Here, we describe a mouse line with conditional loss of HIF prolyl hydroxylase 2 (PHD2) in very early hematopoietic precursors that results in self-renewal of multipotent progenitors under steady-state conditions in a HIF1α- and SMAD7-dependent manner. Competitive bone marrow (BM) transplantations show decreased peripheral and central chimerism of PHD2-deficient cells but not of the most primitive progenitors. Conversely, in whole BM transfer, PHD2-deficient HSCs replenish the entire hematopoietic system and display an enhanced self-renewal capacity reliant on HIF1α. Taken together, our results demonstrate that loss of PHD2 controls the maintenance of the HSC compartment under physiological conditions and causes the outcompetition of PHD2-deficient hematopoietic cells by their wild-type counterparts during stress while promoting the self-renewal of very early hematopoietic progenitors.

  12. 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

  13. Aminoacyl and peptidyl analogs of chloramphenicol as slow-binding inhibitors of ribosomal peptidyltransferase: a new approach for evaluating their potency.

    PubMed

    Michelinaki, M; Mamos, P; Coutsogeorgopoulos, C; Kalpaxis, D L

    1997-01-01

    In a model system derived from Escherichia coli, acetylphenylalanyl-puromycin is produced in a pseudo-first-order reaction between the preformed acetylphenylalanyl/tRNA/poly(U)/ribosome complex (complex C) and excess puromycin. Two aminoacyl analogs [3, Gly-chloramphenicol (CAM): 4, L-Phe-CAM] and two peptidyl analogs (2, L-Phe-Gly-CAM: 5, Gly-Phe-CAM) of CAM (1) were tested as inhibitors in this reaction. Detailed kinetic analysis suggests that these analogs (I) react competitively with complex C and form the complex C*l, which is inactive toward puromycin. C*l is formed via a two-step mechanism in which C*l is the product of a slow conformational change of the initial encounter complex Cl according to the equation C + l reversible Cl reversible C*l. Furthermore, we provide evidence that analog 5 may react further with C*l forming the species C*l2. The values of the apparent association rate constant (K(assoc)) are 1.42 x microM-1 min-1 for 2, 0.55 x microM-1 min-1 for 3, and 0.18 x microM-1 min-1 for 4 and 0.038 x microM-1 min-1 for 5 [corrected]. In the case of analog 5, K(assoc) is a linear function of the inhibitor concentration; when [I] approaches zero, the K(assoc) value is equal to 3.8 x 10(2) M-1 sec-1. Such values allow the classification of CAM analogs as slow-binding inhibitors. According to K(assoc) values, we could surmise that analog 2 is 2.5-fold more potent than 3 and 8-fold more potent than 4. The relative potency of analog 5 is the lowest among the analogs and is dependent on its concentration. The results are compared with previous data and discussed on the basis of a possible retro-inverso relationship between CAM analogs and puromycin.

  14. Mechanism of Action of Prolyl Oligopeptidase (PREP) in Degenerative Brain Diseases: Has Peptidase Activity Only a Modulatory Role on the Interactions of PREP with Proteins?

    PubMed Central

    Männistö, Pekka T.; García-Horsman, J. Arturo

    2017-01-01

    In the aging brain, the correct balance of neural transmission and its regulation is of particular significance, and neuropeptides have a significant role. Prolyl oligopeptidase (PREP) is a protein highly expressed in brain, and evidence indicates that it is related to aging and in neurodegenration. Although PREP is regarded as a peptidase, the physiological substrates in the brain have not been defined, and after intense research, the molecular mechanisms where this protein is involved have not been defined. We propose that PREP functions as a regulator of other proteins though peptide gated direct interaction. We speculate that, at least in some processes where PREP has shown to be relevant, the peptidase activity is only a consequence of the interactions, and not the main physiological activity. PMID:28261087

  15. 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

  16. 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

  17. Effect of Prolyl-Glycyl-Proline (PGP) and Its Acetylated Form (N-AcPGP) on Calcium Level in the Cytoplasm of Rat Peritoneal Mast Cells.

    PubMed

    Bondarenko, N S; Kurenkova, A D; Nikishin, D A; Umarova, B A

    2016-08-01

    Tripeptide glycyl-prolyl-proline (PGP), a regulatory peptide of the glyproline family, possesses a pronounced anti-inflammatory effect primarily due to its ability to prevent secretion of the proinflammatory mediator histamine by rat peritoneal mast cells. Activation of mast cell with synacthen (ACTH1-24) and substance 48/80 leads to an increase in intracellular calcium concentration. Pretreatment of mast cells with PGP prevented calcium entry into the cytoplasm from both intercellular space and intracellular stores. Acetylated peptide (N-AcPGP) produced a similar effect on histamine release and intracellular calcium content in mast cells activated with synacthen. These findings indicate that both forms of the peptide can stabilize mast cells and prevent intracellular calcium increase.

  18. Studies of the toxicological potential of tripeptides (L-valyl-L-prolyl-L-proline and L-isoleucyl-L-prolyI-L-proline): II. Introduction.

    PubMed

    Bernard, Bruce K; Nakamura, Yasunori; Bando, Izuki; Mennear, John H

    2005-01-01

    The consumption of fermented milk to maintain good health, including the maintance of normal blood pressure, is an ancient tradition in a number of areas of the world (e.g., East Asia, France). Recent studies have suggested that fermented milk has a normotensive effect in hypertensive rats and humans, but no effect on blood pressure in normotensive rats and humans. Two tripeptides, L-valyl-L-prolyl-L-proline (VPP) and L-isoleucyl-L-prolyl-L-proline (IPP), have been identified as possessing significant angiotensin-converting enzyme inhibitory activity and are therefore believed to be the source of the normotensive effects. This document, the second of nine chapters, provides information on these two tripeptides, including physical/chemical properties, molecular weights, chemical structures, normal consumption in the diet, manufacturing information, regulatory approval in Japan, and Japanese consumption of food containing enhanced levels of VPP plus IPP. In addition, the results of studies in rats and humans conducted to evaluate the effect of these substances on blood pressure are presented. The research suggests that in adult normotensive volunteers, consumption of up to 7.92 mg of VPP and 4.52 mg IPP daily for 2 weeks causes neither clinical signs nor biologically meaningful effects on systolic or diastolic blood pressure, pulse rate, or clinical pathology (serum chemistry or hematology). However, when a similar study was performed using mildly and moderately hypertensive adults as subjects and they consumed 2.52 mg of VPP and 1.64 mg of IPP per day, a significant drop in systolic blood pressure was detected for a prolonged time interval. This chapter also introduces the issue of safety testing for these substances and describes the information to be found in the subsequent seven chapters.

  19. Posttranslational processing of a new class of hydroxyproline-containing proteins. Prolyl hydroxylation and C-terminal cleavage of tobacco (Nicotiana tabacum) vacuolar chitinase.

    PubMed

    Sticher, L; Hofsteenge, J; Neuhaus, J M; Boller, T; Meins, F

    1993-04-01

    The fungicidal class I chitinases (EC 3.2.1.14) are believed to be important in defending plants against microbial pathogens. The vacuolar isoforms of tobacco (Nicotiana tabacum), chitinases A and B, are the first examples of a new type of hydroxyproline-containing protein with intracellular location, enzymic activity, and a small number of hydroxyprolyl residues restricted to a single, short peptide sequence. We have investigated the posttranslational processing and intracellular transport of transgene-encoded chitinase A in callus cultures of Nicotiana tabacum L. cv Havana 425 and leaves of Nicotiana sylvestris Spegazzini and Comes. Pulse-chase experiments and cell fractionation show that chitinase A is processed in two distinct steps. In the first step, the nascent protein undergoes an increase in apparent M(r) of approximately 1500 detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experiments with the inhibitor of prolyl hydroxylation, alpha,alpha'-dipyridyl, and pulse-chase labeling of cells expressing recombinant forms of chitinase A indicate that the anomalous increase in M(r) is due to hydroxylation of prolyl residues. This step occurs in the endomembrane system before sorting for secretion and vacuolar transport and does not appear to be required for correct targeting of chitinase A to the vacuole. The second step is a proteolytic cleavage. Sequencing of tryptic peptides of the mature proteins indicates that during processing essentially all molecules of chitinase A and B lose a C-terminal heptapeptide, which has been shown to be a vacuolar targeting signal. This appears to occur primarily in the endomembrane system late in intracellular transport. A model for the posttranslational modification of chitinase A is proposed.

  20. Membrane-bound hydroxylases in elicitor-treated bean cells. Rapid induction of the synthesis of prolyl hydroxylase and a putative cytochrome P-450.

    PubMed

    Bolwell, G P; Dixon, R A

    1986-08-15

    Treatment of cell-suspension cultures of bean (Phaseolus vulgaris cv. Canadian Wonder) with an elicitor preparation heat-released from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum resulted in rapid changes in the activities of two microsomal oxygenases, cinnamic acid 4-hydroxylase, involved in accumulation of wall-bound phenolics and phytoalexins, and proline 2-oxoglutarate dioxygenase (prolyl hydroxylase) involved in the post-translational modification of hydroxyproline-rich glycoproteins. An anti-(cytochrome P-450) monoclonal antibody, originally raised against rat cytochrome P-450 isoform c, has been shown to bind to bean microsomes and recognise in Western blots an Mr-48,000 polypeptide, which comigrates with a haeme-containing protein on SDS/polyacrylamide gel analysis and which has been tentatively identified as a cytochrome P-450 capable of the hydroxylation of cinnamic acid. A preparation of proline 2-oxoglutarate dioxygenase purified to homogeneity was used to immunise rabbits for the production of antiserum. An elicitor-induced polypeptide of Mr 65,000 was identified as prolyl hydroxylase while an antigenically related polypeptide of Mr 60,000 was also immunoprecipitated but not induced by elicitor treatment. Use of the two antibodies has demonstrated rapid transient increases in the synthesis of the Mr 48,000 and Mr 65,000 oxygenases in vivo and for mRNAs as measured in in vitro translations, particularly for the putative cytochrome P-450. These increases slightly precede corresponding changes in the synthesis of the soluble enzyme phenylalanine ammonia-lyase, in common with which these oxygenases probably share a mechanism of gene activation underlying the increased activities seen in response to elicitor treatment.

  1. Brief anoxia preconditioning and HIF prolyl-hydroxylase inhibition enhances neuronal resistance in organotypic hippocampal slices on model of ischemic damage.

    PubMed

    Lushnikova, Iryna; Orlovsky, Maxim; Dosenko, Victor; Maistrenko, Anastasiia; Skibo, Galina

    2011-04-22

    It is well known that a brief anoxia or hypoxia episodes can render brain resistant to a subsequent ischemia. Recent investigations indicate that mechanisms of such stimulated endogenous neuroprotection are related to the family of hypoxia-inducible factors (HIF), however there are still little data available on the role of HIF family members in hippocampus-a brain structure, highly sensitive to oxygen deficiency. We have used the model of cultured hippocampal slices and single-cell quantitative RT-PCR to study HIF-1α and HIF-3α mRNA expression following triple 5-min mild anoxia, 30-min oxygen-glucose deprivation and their combination. We also tested the effects of HIF prolyl-hydroxylase inhibition with 2,4-pyridinedicarboxylic acid diethyl ester pre-treatment followed by a 30-min oxygen-glucose deprivation. It was found that neuronal damage induced by oxygen-glucose deprivation was accompanied by a significant decrease in both HIF-1α and HIF-3α mRNA levels in CA1 but not CA3 neurons. Anoxia preconditioning did not affect cell viability and HIF mRNA levels but applied before oxygen-glucose deprivation prevented neuronal damage and suppression of HIF-1α and HIF-3α mRNA expression. It was also found that effects of the prolyl-hydroxylase inhibitor were similar to anoxia preconditioning. These results suggest that anoxia preconditioning increases anti-ischemic neuronal resistance which to a certain extent correlates with the changes of HIF-1α and HIF-3α expression.

  2. 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.

  3. Synthesis of a new pi-deficient phenylalanine derivative from a common 1,4-diketone intermediate and study of the influence of aromatic density on prolyl amide isomer population.

    PubMed

    Dörr, Aurélie; Lubell, William D

    2007-01-01

    Enantiopure (2S)-N-(Boc)-3-(6-methylpyridazinyl)alanine (14) has been synthesized to serve as a phenylalanine analog lacking significant pi-donor capability. Two approaches were developed to furnish the target compound from L-aspartic acid as chiral educt in respectively six and nine steps and 13% and 12% yields. In both routes, a key homoallylic ketone intermediate was synthesized by a copper-catalyzed cascade addition of vinylmagnesium bromide to a carboxylic ester. Dipeptide models Ac-Xaa-Pro-NHMe (21a-c) were prepared and the relative populations of prolyl cis- and trans-amide isomers were measured in chloroform, dimethylsulfoxide, and water by proton NMR spectroscopy in order to assess the significance of the electron density of the neighboring aromatic residue on the prolyl amide geometry.

  4. Stereodependent and solvent-specific formation of unusual β-structure through side chain-backbone H-bonding in C4(S)-(NH2 /OH/NHCHO)-L-prolyl polypeptides.

    PubMed

    Bansode, Nitin D; Madhanagopal, B; Sonar, Mahesh V; Ganesh, Krishna N

    2017-01-01

    It is shown that C4(S)-NH2 /OH/NHCHO-prolyl polypeptides exhibit PPII conformation in aqueous medium, but in a relatively hydrophobic solvent trifluoroethanol (TFE) transform into an unusual β-structure. The stereospecific directing effect of H-bonding in defining the specific structure is demonstrated by the absence of β-structure in the corresponding C4(S)-guanidinyl/(NH/O)-acetyl derivatives and retention of β-structure in C4(S)-(NHCHO)-prolyl polypeptides in TFE. The distinct conformations are identified by the characteristic CD patterns and supported by Raman spectroscopic data. The solvent dependent conformational effects are interpreted in terms of intraresidue H-bonding that promotes PPII conformation in water, switching over to interchain H-bonding in TFE. The present observations add a new design principle to the growing repertoire of strategies for engineering peptide secondary structural motifs for innovative nanoassemblies and new biomaterials.

  5. A novel peptide-processing activity of insect peptidyl-dipeptidase A (angiotensin I-converting enzyme): the hydrolysis of lysyl-arginine and arginyl-arginine from the C-terminus of an insect prohormone peptide.

    PubMed Central

    Isaac, R; Schoofs, L; Williams, T A; Veelaert, D; Sajid, M; Corvol, P; Coates, D

    1998-01-01

    Insect peptidyl-dipeptidase A [angiotensin I-converting enzyme (ACE)] is a soluble single-domain peptidyl-dipeptidase that has many properties in common with the C-domain of mammalian somatic ACE and with the single-domain mammalian germinal ACE. Mammalian somatic ACE is important in blood homoeostasis, but the role of ACE in insects is not known. Immunocytochemistry has been used to localize ACE in the neuroendocrine system of the locust, Locusta migratoria. Staining was observed in five groups of neurosecretory cells in the brain and suboesophageal ganglion, in the nervi corpori cardiaci, the storage part of the corpora cardiaca and in the nervi corpori allati. In three groups of neurosecretory cells, ACE co-localized with locustamyotropins, suggesting a possible role for the enzyme in the metabolism of these neuropeptides. We demonstrate in vitro a novel activity of ACE that removes pairs of basic amino acid residues from a locustamyotropin peptide extended at the C-terminus with either Gly-Lys-Arg or Gly-Arg-Arg, corresponding to a consensus recognition sequence for endoproteolysis of prohormone proteins by prohormone convertases. The low Km and high kcat values (Km 7.3 and 5.0 microM, kcat 226 and 207 s-1 for the hydrolysis of Phe-Ser-Pro-Arg-Leu-Gly-Lys-Arg and Phe-Ser-Pro-Arg-Leu-Gly-Arg-Arg, respectively) obtained for the hydrolysis of these two peptides by insect ACE means that these peptides, along with mammalian bradykinin, are the most favoured in vitro ACE substrates so far identified. The discovery of this in vitro prohormone-processing activity of insect ACE provides a possible explanation for the intracellular co-localization of the enzyme with locustamyotropin peptides, and provides evidence for a new role for ACE in the biosynthesis of peptide hormones and transmitters. PMID:9461491

  6. Non-conjugated cis/trans 18:2 in Beef Fat are Mainly Δ-9 Desaturation Products of trans-18:1 Isomers.

    PubMed

    Vahmani, P; Rolland, D C; Gzyl, K E; Dugan, M E R

    2016-12-01

    Human liver cells (HepG2) were cultured with individual trans (t) 18:1 including t6-, t12-, t13-, t14-, t15- and t16-18:1, and retention times of their Δ-9 desaturation products were determined using 100-m biscyanopropyl-polysiloxane and SLB-IL111 columns. Corresponding peaks were found in beef adipose tissues known to have different delta-9 desaturase activities. Further lines of evidence indicating the presence of Δ-9 desaturation products of t-18:1 isomers in beef fat were developed by analysis of fatty acid methyl esters (FAME) fractionated using Ag(+)-TLC, and by GC/MS. Some of the Δ-9 desaturation products of t-18:1 have been previously identified in ruminant fat (c9, t12- and c9, t13-18:2). Some of the Δ-9 desaturation products of t-18:1 (c9, t14- and c9, t15-18:2) have been previously tentatively identified as different fatty acids, and for the first time we provide evidence of the presence of c9, t16-18:2, and where t6, c9-18:2 may elute during analysis of FAME from beef fat.

  7. Effect of gamma-irradiation on the levels of total and cis/trans isomers of beta-carotene in dehydrated parsley

    NASA Astrophysics Data System (ADS)

    Sebastião, Kátia I.; Almeida-Muradian, Lígia B.; Romanelli, Maria Fernanda; Koseki, Paula Massae; Villavicencio, Anna Lúcia C. H.

    2002-03-01

    Ionizing radiation is a method for preservation of foods that use the high energy of gamma rays or accelerated electrons, thereby ionizing molecules. The most important precursor of vitamin A is β-carotene, a carotenoid with pro-vitamin A activity. The highly unsaturated chain confers the instability of β-carotene, and some reactions, as isomerisation, can reduce the characteristics of pro-vitamin A. The present study investigated whether increasing doses of 0, 10 and 20 kGy lower the total β-carotene level and if an enhancement of cis-isomers occurred on samples of dehydrated parsley. No differences were observed of either fractions analyzed at doses applied in this study, nor did it contribute to the decrease of vitamin A.

  8. Density functional theory study of self-association of N-methylformamide and its effect on intramolecular and intermolecular geometrical parameters and the cis/trans population.

    PubMed

    García Martínez, A; Teso Vilar, E; García Fraile, A; Martínez-Ruiz, P

    2006-06-21

    The single-point total energy (E) of several acyclic and cyclic oligomers of N-methylformamide (NMF) was computed by the first time without any geometrical restriction, using the B3LYP6-31G* method of the density functional theory in order to determine the effect of self-association on intramolecular geometrical parameters of cis- and trans-NMF, the intermolecular distances of the hydrogen-bonding chains formed by NMF as well as intermolecular association energies including counterpoise corrections. It is concluded that liquid NMF exists mainly as polymers formed by self-association of trans-NMF units, whereas the cis-NMF isomer occurs as isolated units inserted along the chains. These computational results are in accordance with the experimentally determined predominance (ca. 90%) of trans-NMF population by means of (1)H- NMR and other spectroscopic techniques, but in severe contradiction with a recent interpretation of x-ray diffraction data on liquid NMF, postulating a cyclic trimer of cis-NMF (c-C(3)) as the predominating species. The counterpoise-corrected values of the association energy, DeltaE(CP), calculated for cyclic oligomers, increase with the polymerization degree (n) revealing a high grade of cooperative effect for amidic hydrogen-bonded chains. Noteworthy, the difference between the DeltaE(CP) values of the cyclic cis- and trans-homooligomers of NMF is positive for n=2 and 3 but negative for n > or =4.

  9. Effects of thionation and fluorination on cis-trans isomerization in tertiary amides: an investigation of N-alkylglycine (peptoid) rotamers.

    PubMed

    Engel-Andreasen, Jens; Wich, Kathrine; Laursen, Jonas S; Harris, Pernille; Olsen, Christian A

    2015-06-05

    Peptoids constitute a class of peptidomimetics with potential as protease resistant, biologically active ligands. To harness the full potential of such compounds, however, detailed predictive insight into their propensity to adopt well-defined secondary structures is highly desirable. In this work we present an investigation of the effects of thioamides and/or fluorides in peptoid monomer model systems using chemical synthesis, NMR spectroscopy, and X-ray crystallography. We find that the steric environment surrounding the tertiary amide bonds is the key promoter of conformational preference, and X-ray crystallographic interrogation of our model systems did not suggest the presence of stabilizing n → π* interactions unless the carbonyls were altered electronically by α-halogenation or thioamide formation. In addition to the function as an investigative tool, these two types of modification may thus be utilized as stabilizers of secondary structure in future oligomer designs, such as the cis-amide-based polypeptoid helices that resemble the polyproline type-I helix.

  10. Subchronic Inhalation Toxicity of 3-(Phenoxyphenyl) Methyl (+ or -)-CIS, Trans-3-(2,2-Dichloroethenyl)-2,2-Dimethylcyclopropanecarboxylate (Permethrin) May-December 1978

    DTIC Science & Technology

    1980-12-01

    December 1978 Executive Secretary Armed Forces Pest Management Board Forest Glen Section Walter Reed Army Medical Center Washington, DC 20012 A summary of...dogs were anesthetized with Blo-Talf (Thiamylal sodium), 4.0 percent, by intravenous injection to effect through a pediatric catheter secured in the...44 696 0.x 044L961 x j2 ! Z. i. ±’x2 -Z - 1 -X. - - - - - -Z -I - -I- ZI -z Z x x z x x U- 61 1 X1 z zK B 06 X1 X x > 616 z x x NK K816 z z r L96 x x

  11. 40 CFR 180.545 - Prallethrin (RS)-2-methyl-4-oxo-3-(2-propynyl)cyclopent-2-enyl (1RS)-cis, trans-chrysanthemate...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.545 Prallethrin (RS)-2-methyl-4... operation provided exposed food has been covered or removed from the area being treated prior to...

  12. 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

    ... Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.545 Prallethrin (RS)-2-methyl-4... operation provided exposed food has been covered or removed from the area being treated prior to...

  13. 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...

  14. 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...

  15. Determination of cis/trans phase of variations in the MC1R gene with allele-specific PCR and single base extension.

    PubMed

    Mengel-From, Jonas; Børsting, Claus; Sanchez, Juan J; Eiberg, Hans; Morling, Niels

    2008-12-01

    The MC1R gene encodes a protein with key regulatory functions in the melanin synthesis. A multiplex PCR and a multiplex single base extension protocol were established for genotyping six exonic MC1R variations highly penetrant for red hair (R), four exonic MC1R variations weakly penetrant for red hair (r), two frameshift variations highly penetrant for red hair (R) and three variations in the promoter region. We genotyped 600 individuals from Denmark using either CE or MALDI-TOF MS as the detection platform. A total of 62 individuals were genotyped R/R and among the 62 individuals, 57 had red hair and five had blond hair colour. Two different R alleles may be located in cis (RR/-) position or trans (R/R) position, and the phenotype associated with RR/- and R/R may be different. Two allele-specific PCRs were established with primers targeting the -G445A variation in the MC1R promoter and the allele-specific PCR products were used in the multiplex single base extension assay. In all 62 individuals, the MC1R variants were situated in trans position. Another 18 individuals with red hair colour were either genotyped R/- or R/r, suggesting that other genes influence hair colour.

  16. Selective recognition of cis-trans-isomers of platinum drugs and the detection of triplex DNA based on fluorescence reversible model of quantum dots.

    PubMed

    Xu, Xiaoling; Gao, Fang; Xiao, Xincai; Hu, Yan; Zhu, Chaozhen; Zhao, Dan

    2017-02-05

    The identification of spatial structures of drugs and the researches on their interaction mechanism with DNA are always attractive to the researchers. However, their realization is lack of simple and fast method. This paper reports the establishment of multiple-functional detection platform based on the "turn off-on" model of ZnCdSe quantum dots. In this system, ZnCdSe quantum dots work as the fluorescent probe, platinum anti-cancer drugs as the quencher and triplex DNA as the trapping agent. The seemingly similar cisplatin and transplatin exhibited different fluorescent recovery behaviors due to their difference in structure, and thus realized the selective detection of cisplatin and transplatin with the reaction time set at 10min as well as the quantitation of cisplatin over the range of 2.5×10(-8)-100×10(-8)M. Based on this, the interactions between platinum anti-cancer drugs and ctDNA as well as polymorphic DNA were further studied, and realized the recognition of triplex DNA. The multiple-functional detection platform integrates the functions of the filtration of high-efficient platinum anti-cancer drugs, the researches on interaction mechanism of drugs, and the recognition of polymorphic DNA, meaningful to the future treatment of viral and cancers based on antisense gene strategy.

  17. 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. PMID:27818566

  18. Therapeutic targeting of oxygen-sensing prolyl hydroxylases abrogates ATF4-dependent neuronal death and improves outcomes after brain hemorrhage in several rodent models

    PubMed Central

    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.

    2017-01-01

    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. PMID:26936506

  19. Therapeutic targeting of oxygen-sensing prolyl hydroxylases abrogates ATF4-dependent neuronal death and improves outcomes after brain hemorrhage in several rodent models

    DOE PAGES

    Karuppagounder, Saravanan S.; Alim, Ishraq; Khim, Soah J.; ...

    2016-03-02

    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. Here we show that the hypoxia-inducible factor prolyl-hydroxylase (HIF- PHD) family of iron-dependent oxygen sensing enzymes are effectors of iron chelation. Molecular reduction of the three HIF-PHD enzyme isoforms in mouse striatum improved functional recovery following ICH. A low molecular weight hydroxyquinoline inhibitor of the HIF-PHDs, adaptaquin, reduced neuronal death and behavioral deficitsmore » following 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 expression of the prodeath factor ATF4 rather than activation of a HIF-dependent prosurvival pathway. In conclusion, 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 following ICH in multiple rodent species.« less

  20. Therapeutic targeting of oxygen-sensing prolyl hydroxylases abrogates ATF4-dependent neuronal death and improves outcomes after brain hemorrhage in several rodent models

    SciTech Connect

    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, Guohu; Keep, Richard F.; Schallert, Timothy; Tappero, Ryan V.; Zhong, Jian; Cho, Sunghee; Maxfield, Frederick R.; Holman, T. R.; Culmsee, Carsten; Fong, Guo-Hua -H.; Su, Yijing; Ming, Guo-li; Song, Hongjun; Cave, John W.; Schofield, Christopher J.; Colbourne, Frederick; Coppola, Giovanni; Ratan, Rajiv R.

    2016-03-02

    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. Here we show that the hypoxia-inducible factor prolyl-hydroxylase (HIF- PHD) family of iron-dependent oxygen sensing enzymes are effectors of iron chelation. Molecular reduction of the three HIF-PHD enzyme isoforms in mouse striatum improved functional recovery following ICH. A low molecular weight hydroxyquinoline inhibitor of the HIF-PHDs, adaptaquin, reduced neuronal death and behavioral deficits following 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 expression of the prodeath factor ATF4 rather than activation of a HIF-dependent prosurvival pathway. In conclusion, 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 following ICH in multiple rodent species.

  1. 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.

  2. The cytoplasmic prolyl-tRNA synthetase of the malaria parasite is a dual-stage target of febrifugine and its analogs.

    PubMed

    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

    2015-05-20

    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 developing next-generation 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 (transfer RNA) synthetase (PfcPRS) of the malaria parasite Plasmodium falciparum is a biochemical and functional target of febrifugine and its synthetic derivative 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 Plasmodium berghei mouse model of malaria that halofuginol, a new halofuginone analog that we developed, is 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.

  3. The vacuolar-ATPase complex and assembly factors, TMEM199 and CCDC115, control HIF1α prolyl hydroxylation by regulating cellular iron levels.

    PubMed

    Miles, Anna L; Burr, Stephen P; Grice, Guinevere L; Nathan, James A

    2017-03-15

    Hypoxia Inducible transcription Factors (HIFs) are principally regulated by the 2-oxoglutarate and Iron(II) prolyl hydroxylase (PHD) enzymes, which hydroxylate the HIFα subunit, facilitating its proteasome-mediated degradation. Observations that HIFα hydroxylation can be impaired even when oxygen is sufficient emphasise the importance of understanding the complex nature of PHD regulation. Here, we use an unbiased genome-wide genetic screen in near-haploid human cells to uncover cellular processes that regulate HIF1α. We identify that genetic disruption of the Vacuolar H+ ATPase (V-ATPase), the key proton pump for endo-lysosomal acidification, and two previously uncharacterised V-ATPase assembly factors, TMEM199 and CCDC115, stabilise HIF1α in aerobic conditions. Rather than preventing the lysosomal degradation of HIF1α, disrupting the V-ATPase results in intracellular iron depletion, thereby impairing PHD activity and leading to HIF activation. Iron supplementation directly restores PHD catalytic activity following V-ATPase inhibition, revealing important links between the V-ATPase, iron metabolism and HIFs.

  4. 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.

  5. 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.

  6. Hypoxia-inducible factor prolyl hydroxylase 1 (PHD1) deficiency promotes hepatic steatosis and liver-specific insulin resistance in mice

    PubMed Central

    Thomas, Amandine; Belaidi, Elise; Aron-Wisnewsky, Judith; van der Zon, Gerard C.; Levy, Patrick; Clement, Karine; Pepin, Jean-Louis; Godin-Ribuot, Diane; Guigas, Bruno

    2016-01-01

    Obesity is associated with local tissue hypoxia and elevated hypoxia-inducible factor 1 alpha (HIF-1α) in metabolic tissues. Prolyl hydroxylases (PHDs) play an important role in regulating HIF-α isoform stability. In the present study, we investigated the consequence of whole-body PHD1 gene (Egln2) inactivation on metabolic homeostasis in mice. At baseline, PHD1−/− mice exhibited higher white adipose tissue (WAT) mass, despite lower body weight, and impaired insulin sensitivity and glucose tolerance when compared to age-matched wild-type (WT) mice. When fed a synthetic low-fat diet, PHD1−/− mice also exhibit a higher body weight gain and WAT mass along with glucose intolerance and systemic insulin resistance compared to WT mice. PHD1 deficiency led to increase in glycolytic gene expression, lipogenic proteins ACC and FAS, hepatic steatosis and liver-specific insulin resistance. Furthermore, gene markers of inflammation were also increased in the liver, but not in WAT or skeletal muscle, of PHD1−/− mice. As expected, high-fat diet (HFD) promoted obesity, hepatic steatosis, tissue-specific inflammation and systemic insulin resistance in WT mice but these diet-induced metabolic alterations were not exacerbated in PHD1−/− mice. In conclusion, PHD1 deficiency promotes hepatic steatosis and liver-specific insulin resistance but does not worsen the deleterious effects of HFD on metabolic homeostasis. PMID:27094951

  7. A new mechanism of post-transfer editing by aminoacyl-tRNA synthetases: catalysis of hydrolytic reaction by bacterial-type prolyl-tRNA synthetase.

    PubMed

    Boyarshin, Konstantin S; Priss, Anastasia E; Rayevskiy, Alexsey V; Ilchenko, Mykola M; Dubey, Igor Ya; Kriklivyi, Ivan A; Yaremchuk, Anna D; Tukalo, Michael A

    2017-02-01

    Aminoacyl tRNA synthetases are enzymes that specifically attach amino acids to cognate tRNAs for use in the ribosomal stage of translation. For many aminoacyl tRNA synthetases, the required level of amino acid specificity is achieved either by specific hydrolysis of misactivated aminoacyl-adenylate intermediate (pre-transfer editing) or by hydrolysis of the mischarged aminoacyl-tRNA (post-transfer editing). To investigate the mechanism of post-transfer editing of alanine by prolyl-tRNA synthetase from the pathogenic bacteria Enterococcus faecalis, we used molecular modeling, molecular dynamic simulations, quantum mechanical (QM) calculations, site-directed mutagenesis of the enzyme, and tRNA modification. The results support a new tRNA-assisted mechanism of hydrolysis of misacylated Ala-tRNA(Pro). The most important functional element of this catalytic mechanism is the 2'-OH group of the terminal adenosine 76 of Ala-tRNA(Pro), which forms an intramolecular hydrogen bond with the carbonyl group of the alanine residue, strongly facilitating hydrolysis. Hydrolysis was shown by QM methods to proceed via a general acid-base catalysis mechanism involving two functionally distinct water molecules. The transition state of the reaction was identified. Amino acid residues of the editing active site participate in the coordination of substrate and both attacking and assisting water molecules, performing the proton transfer to the 3'-O atom of A76.

  8. Prolyl oligopeptidase colocalizes with α-synuclein, β-amyloid, tau protein and astroglia in the post-mortem brain samples with Parkinson's and Alzheimer's diseases.

    PubMed

    Hannula, M J; Myöhänen, T T; Tenorio-Laranga, J; Männistö, P T; Garcia-Horsman, J A

    2013-07-09

    Prolyl oligopeptidase (EC 3.4.21.26, PREP) is a serine protease that hydrolyzes proline-containing peptides shorter than 30-mer but it has also nonhydrolytic functions. PREP has been shown to accelerate aggregation of wild-type α-synuclein (α-syn) under cell-free conditions, and PREP inhibitors can block this aggregation both in vitro and in vivo. α-syn is the main component of Lewy bodies in Parkinson's disease (PD) and Lewy body dementia. To clarify the possible interaction of PREP with other markers of neurodegenerative diseases, we studied colocalizations of PREP and (1) α-syn, (2) β-amyloid, (3) tau protein and (4) astroglial and microglial cells in human post-mortem brain samples from PD, Alzheimer's disease (AD) patients and in healthy control brain samples. In the substantia nigra of PD brains, an intense colocalization with PREP and α-syn was evident. PREP colocalized also with β-amyloid plaques in AD brains and with tau protein in AD and in healthy brains. PREP was also found in astroglial cells in PD, AD and control brains, but not in the microglia. Our findings are the first ones to demonstrate colocalization of PREP and pathological proteins in the human brain and support the view that, at least in spatial terms, PREP could be associated with pathogenesis of neurodegenerative diseases.

  9. Inhibition of prolyl oligopeptidase increases the survival of alpha-synuclein overexpressing cells after rotenone exposure by reducing alpha-synuclein oligomers.

    PubMed

    Dokleja, Lana; Hannula, Mirva J; Myöhänen, Timo T

    2014-11-07

    α-Synuclein (aSyn) aggregation, mitochondrial dysfunction and oxidative damage has been shown to be related to the death of dopaminergic neurons in Parkinson's disease (PD). Prolyl oligopeptidase (PREP) is proposed to increase aSyn aggregation, and PREP inhibition has been shown to inhibit the aggregation process in vitro and in vivo. In this study, we investigated the effects of a specific PREP inhibitor, KYP-2047, on rotenone induced aSyn aggregation and increased the production of reactive oxygen species (ROS) in cells overexpressing A53T mutation of aSyn. Rotenone, a mitochondrial toxin that induces oxidative damage and aSyn aggregation, associated with PD pathology, was selected as a model for this study. The results showed that rotenone induced the formation of high-molecular-weight aSyn oligomers, and this was countered by simultaneous incubation with KYP-2047. Inhibition of PREP also decreased the production of ROS in [A53T]aSyn overexpressing cells, leading to improved cell viability.

  10. 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

    2016-03-02

    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.

  11. Issues about the physiological functions of prolyl oligopeptidase based on its discordant spatial association with substrates and inconsistencies among mRNA, protein levels, and enzymatic activity.

    PubMed

    Myöhänen, Timo T; García-Horsman, J Arturo; Tenorio-Laranga, Jofre; Männistö, Pekka T

    2009-09-01

    Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyses proline-containing peptides shorter than 30 amino acids. POP may be associated with cognitive functions, possibly via the cleavage of neuropeptides. Recent studies have also suggested novel non-hydrolytic and non-catalytic functions for POP. Moreover, POP has also been proposed as a regulator of inositol 1,4,5-triphosphate signaling and several other functions such as cell proliferation and differentiation, as well as signal transduction in the central nervous system, and it is suspected to be involved in pathological conditions such as Parkinson's and Alzheimer's diseases and cancer. POP inhibitors have been developed to restore the depleted neuropeptide levels encountered in aging or in neurodegenerative disorders. These compounds have shown some antiamnesic effects in animal models. However, the mechanisms of these hypothesized actions are still far from clear. Moreover, the physiological role of POP has remained unknown, and a lack of basic studies, including its distribution, is obvious. The aim of this review is to gather information about POP and to propose some novel roles for this enzyme based on its distribution and its discordant spatial association with its best known substrates.

  12. Oral collagen-derived dipeptides, prolyl-hydroxyproline and hydroxyprolyl-glycine, ameliorate skin barrier dysfunction and alter gene expression profiles in the skin.

    PubMed

    Shimizu, Jun; Asami, Naoto; Kataoka, Aya; Sugihara, Fumihito; Inoue, Naoki; Kimira, Yoshifumi; Wada, Masahiro; Mano, Hiroshi

    2015-01-09

    Oral supplementation with collagen hydrolysate (CH) has been shown to improve the condition of the skin in humans and experimental animals. Several hydroxyproline-containing oligo-peptides were previously detected in human peripheral blood after the ingestion of CH, and the two dipeptides, prolyl-hydroxyproline (PO) and hydroxyprolyl-glycine (OG), have been proposed to have beneficial effects on human health. When HR-1 hairless mice were fed a HR-AD diet, which lacked magnesium and zinc, transepidermal water loss (TEWL) increased and water content of stratum corneum decreased. In the present study, we investigated the effects of dietary PO and OG on skin barrier dysfunction in HR-1 hairless mice. Mice were fed a HR-AD diet with or without PO (0.15%) and OG (0.15%) for 35 consecutive days. The administration of PO and OG significantly decreased TEWL, and significantly increased water content of stratum corneum. A DNA microarray analysis of the dorsal skin revealed differences in gene expression between the group administered PO and OG and the control group. We also identified muscle-related Gene Ontology as a result of analyzing the up-regulated genes. These results suggested that the administration of PO and OG improved skin barrier dysfunction and altered muscle-related gene expression.

  13. Conditional Deletion of Prolyl Hydroxylase Domain-Containing Protein 2 (Phd2) Gene Reveals Its Essential Role in Chondrocyte Function and Endochondral Bone Formation

    PubMed Central

    Cheng, Shaohong; Xing, Weirong; Pourteymoor, Sheila; Schulte, Jan

    2016-01-01

    The hypoxic growth plate cartilage requires hypoxia-inducible factor (HIF)-mediated pathways to maintain chondrocyte survival and differentiation. HIF proteins are tightly regulated by prolyl hydroxylase domain-containing protein 2 (Phd2)-mediated proteosomal degradation. We conditionally disrupted the Phd2 gene in chondrocytes by crossing Phd2 floxed mice with type 2 collagen-α1-Cre transgenic mice and found massive increases (>50%) in the trabecular bone mass of long bones and lumbar vertebra of the Phd2 conditional knockout (cKO) mice caused by significant increases in trabecular number and thickness and reductions in trabecular separation. Cortical thickness and tissue mineral density at the femoral middiaphysis of the cKO mice were also significantly increased. Dynamic histomorphometric analyses revealed increased longitudinal length and osteoid surface per bone surface in the primary spongiosa of the cKO mice, suggesting elevated conversion rate from hypertrophic chondrocytes to mineralized bone matrix as well as increased bone formation in the primary spongiosa. In the secondary spongiosa, bone formation measured by mineralizing surface per bone surface and mineral apposition rate were not changed, but resorption was slightly reduced. Increases in the mRNA levels of SRY (sex determining region Y)-box 9, osterix (Osx), type 2 collagen, aggrecan, alkaline phosphatase, bone sialoprotein, vascular endothelial growth factor, erythropoietin, and glycolytic enzymes in the growth plate of cKO mice were detected by quantitative RT-PCR. Immunohistochemistry revealed an increased HIF-1α protein level in the hypertrophic chondrocytes of cKO mice. Infection of chondrocytes isolated from Phd2 floxed mice with adenoviral Cre resulted in similar gene expression patterns as observed in the cKO growth plate chondrocytes. Our findings indicate that Phd2 suppresses endochondral bone formation, in part, via HIF-dependent mechanisms in mice. PMID:26562260

  14. Pentylenetetrazol-induced seizures affect the levels of prolyl oligopeptidase, thimet oligopeptidase and glial proteins in rat brain regions, and attenuation by MK-801 pretreatment.

    PubMed

    Ahmed, M Mahiuddin; Arif, Mohammad; Chikuma, Toshiyuki; Kato, Takeshi

    2005-09-01

    The regulatory mechanisms of neuropeptide-metabolizing enzymes often play a critical role in the pathogenesis of neuronal damage. A systemic administration of pentylenetetrazol (PTZ), an antagonist of GABA(A) receptor ion channel binding site, causes generalized epilepsy in an animal model. In the present study, we examined the involvement of prolyl oligopeptidase (POP), thimet oligopeptidase/neurolysin (EP 24.15/16) and glial proteins in PTZ-treated rat brain regions, and the suppressive effect of MK-801, a non-competitive NMDA receptor antagonist, pretreatment for their proteins. The activity of POP significantly decreased in the hippocampus at 30min and 3h, and in the frontal cortex at 3h after PTZ treatment, and pretreatment with MK-801 recovered the activity in the cortex at 3h. The activity of EP 24.15/16 significantly decreased in the hippocampus at 3h and 1 day, and in the cortex at 3h after the PTZ administration, whereas pretreatment with MK-801 recovered the change of the activity. The Western blot analysis of EP 24.15 showed significant decrease of the protein level in the hippocampus 3h after the PTZ treatment, whereas pretreatment with MK-801 recovered. The expression of GFAP and CD11b immunohistochemically increased in the hippocampus of the PTZ-treated rat as compared with controls. Pretreatment with MK-801 also recovered the GFAP and CD11b expression. These data suggest that PTZ-induced seizures of the rats cause indirect activation of glutamate NMDA receptors, then decrease POP and EP 24.15/16 enzyme activities and EP 24.15 immunoreactivity in the neuronal cells of the hippocampal formation. We speculate that changes of those peptidases in the brain may be related to the levels of the neuropeptides regulating PTZ-induced seizures.

  15. 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

  16. Expression of Prolyl Hydroxylases (PHDs) Is Selectively Controlled by HIF-1 and HIF-2 Proteins in Nucleus Pulposus Cells of the Interverte