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1
Skagia, Aggeliki, Eleni Vezyri, Konstantinos Grados, Anastasia Venieraki, Michael Karpusas, Panagiotis Katinakis, and Maria Dimou. "Structure-Function Analysis of the Periplasmic Escherichia coli Cyclophilin PpiA in Relation to Biofilm Formation." Journal of Molecular Microbiology and Biotechnology 27, no. 4 (2017): 228–36. http://dx.doi.org/10.1159/000478858.
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The presence of peptidyl-prolyl <i>cis</i>/<i>trans</i> isomerases (PPIases, EC: 5.2.1.8) in all domains of life indicates their biological importance. Cyclophilin PpiA, present in the periplasm of gram-negative bacteria, possesses PPIase activity but its physiological functions<b> </b>are still not clearly defined. Here, we demonstrate that the Δ<i>ppiA</i> deletion strain from <i>Escherichia coli</i> exhibits an increased ability for biofilm formation and enhanced swimming motility compared to the wild-type strain. To identify structural features of PpiA which are necessary for the negative modulation of biofilm formation, we constructed a series of mutant PpiA proteins using a combination of error-prone and site-directed mutagenesis approaches. We show that the negative effect of PpiA on biofilm formation is not dependent on its PPIase activity, since PpiA mutants with a reduced PPIase activity are able to complement the Δ<i>ppiA</i> strain during biofilm growth.
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Obi, Ikenna R., Roland Nordfelth, and Matthew S. Francis. "Varying dependency of periplasmic peptidylprolyl cis–trans isomerases in promoting Yersinia pseudotuberculosis stress tolerance and pathogenicity." Biochemical Journal 439, no. 2 (September 28, 2011): 321–32. http://dx.doi.org/10.1042/bj20110767.
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Periplasmic PPIases (peptidylprolyl cis–trans isomerases) catalyse the cis–trans isomerization of peptidyl-prolyl bonds, which is a rate-limiting step during protein folding. We demonstrate that the surA, ppiA, ppiD, fkpA and fklB alleles each encode a periplasmic PPIase in the bacterial pathogen Yersinia pseudotuberculosis. Of these, four were purified to homogeneity. Purified SurA, FkpA and FklB, but not PpiD, displayed detectable PPIase activity in vitro. Significantly, only Y. pseudotuberculosis lacking surA caused drastic alterations to the outer membrane protein profile and FA (fatty acid) composition. They also exhibited aberrant cellular morphology, leaking LPS (lipopolysaccharide) into the extracellular environment. The SurA PPIase is therefore most critical for maintaining Y. pseudotuberculosis envelope integrity during routine culturing. On the other hand, bacteria lacking either surA or all of the genes ppiA, ppiD, fkpA and fklB were sensitive to hydrogen peroxide and were attenuated in mice infections. Thus Y. pseudotuberculosis exhibits both SurA-dependent and -independent requirements for periplasmic PPIase activity to ensure in vivo survival and a full virulence effect in a mammalian host.
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Stifani, Stefano. "The Multiple Roles of Peptidyl Prolyl Isomerases in Brain Cancer." Biomolecules 8, no. 4 (October 11, 2018): 112. http://dx.doi.org/10.3390/biom8040112.
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Peptidyl prolyl isomerases (PPIases) are broadly expressed enzymes that accelerate the cis-trans isomerization of proline peptide bonds. The most extensively studied PPIase family member is protein interacting with never in mitosis A1 (PIN1), which isomerizes phosphorylated serine/threonine–proline bonds. By catalyzing this specific cis-trans isomerization, PIN1 can alter the structure of its target proteins and modulate their activities in a number of different ways. Many proteins are targets of proline-directed phosphorylation and thus PIN1-mediated isomerization of proline bonds represents an important step in the regulation of a variety of cellular mechanisms. Numerous other proteins in addition to PIN1 are endowed with PPIase activity. These include other members of the parvulin family to which PIN1 belongs, such as PIN4, as well as several cyclophilins and FK506-binding proteins. Unlike PIN1, however, these other PPIases do not isomerize phosphorylated serine/threonine–proline bonds and have different substrate specificities. PIN1 and other PPIases are overexpressed in many types of cancer and have been implicated in various oncogenic processes. This review will discuss studies providing evidence for multiple roles of PIN1 and other PPIases in glioblastoma and medulloblastoma, the most frequent adult and pediatric primary brain tumors.
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Klein, Gracjana, Pawel Wojtkiewicz, Daria Biernacka, Anna Stupak, Patrycja Gorzelak, and Satish Raina. "Identification of Substrates of Cytoplasmic Peptidyl-Prolyl Cis/Trans Isomerases and Their Collective Essentiality in Escherichia Coli." International Journal of Molecular Sciences 21, no. 12 (June 13, 2020): 4212. http://dx.doi.org/10.3390/ijms21124212.
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Protein folding often requires molecular chaperones and folding catalysts, such as peptidyl-prolyl cis/trans isomerases (PPIs). The Escherichia coli cytoplasm contains six well-known PPIs, although a requirement of their PPIase activity, the identity of their substrates and relative enzymatic contribution is unknown. Thus, strains lacking all periplasmic and one of the cytoplasmic PPIs were constructed. Measurement of their PPIase activity revealed that PpiB is the major source of PPIase activity in the cytoplasm. Furthermore, viable Δ6ppi strains could be constructed only on minimal medium in the temperature range of 30–37 °C, but not on rich medium. To address the molecular basis of essentiality of PPIs, proteins that aggregate in their absence were identified. Next, wild-type and putative active site variants of FkpB, FklB, PpiB and PpiC were purified and in pull-down experiments substrates specific to each of these PPIs identified, revealing an overlap of some substrates. Substrates of PpiC were validated by immunoprecipitations using extracts from wild-type and PpiC-H81A strains carrying a 3xFLAG-tag appended to the C-terminal end of the ppiC gene on the chromosome. Using isothermal titration calorimetry, RpoE, RseA, S2, and AhpC were established as FkpB substrates and PpiC’s PPIase activity was shown to be required for interaction with AhpC.
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Reffuveille, Fany, Nathalie Connil, Maurizio Sanguinetti, Brunella Posteraro, Sylvie Chevalier, Yanick Auffray, and Alain Rince. "Involvement of Peptidylprolylcis/transIsomerases in Enterococcus faecalis Virulence." Infection and Immunity 80, no. 5 (February 13, 2012): 1728–35. http://dx.doi.org/10.1128/iai.06251-11.
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ABSTRACTPeptidylprolylcis/transisomerases (PPIases) are enzymes involved in protein folding. Analysis of the genome sequence ofEnterococcus faecalisV583 allowed for identification of 3 PPIases carrying genes.ef2898encodes an intracellular PPIase which was not shown to be important for theE. faecalisstress response or virulence. The other two PPIases, the parvulin family rotamase EF0685 and the cyclophilin family member EF1534, are expected to be surface-exposed proteins. They were shown to be important for virulence and resistance to NaCl. A Δef0685Δef1534mutant was also more resistant to oxidative stress, was able to grow under a high manganese concentration, and showed altered resistance to ampicillin and quinolone antibiotics.
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Trémillon, Nicolas, Eric Morello, Daniel Llull, Rabia Mazmouz, Jean-Jacques Gratadoux, Alain Guillot, Marie-Pierre Chapot-Chartier, et al. "PpiA, a Surface PPIase of the Cyclophilin Family in Lactococcus lactis." PLoS ONE 7, no. 3 (March 19, 2012): e33516. http://dx.doi.org/10.1371/journal.pone.0033516.
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Liu, Fei, Xiao-Long Wei, Hao Li, Ji-Fu Wei, Yong-Qing Wang, and Xiao-Jian Gong. "Molecular Evolution of the Vertebrate FK506 Binding Protein 25." International Journal of Genomics 2014 (2014): 1–9. http://dx.doi.org/10.1155/2014/402603.
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FK506 binding proteins (FKBPs) belong to immunophilins with peptidyl-prolyl isomerases (PPIases) activity. FKBP25 (also known as FKBP3) is one of the nuclear DNA-binding proteins in the FKBPs family, which plays an important role in regulating transcription and chromatin structure. The calculation of nonsynonymous and synonymous substitution rates suggested that FKBP25 undergoes purifying selection throughout the whole vertebrate evolution. Moreover, the result of site-specific tests showed that no sites were detected under positive selection. Only one PPIase domain was detected by searching FKBP25 sequences at Pfam and SMART domain databases. Mammalian FKBP25 possess exon-intron conservation, although conservation in the whole vertebrate lineage is incomplete. The result of this study suggests that the purifying selection triggers FKBP25 evolutionary history, which allows us to discover the complete role of the PPIase domain in the interaction between FKBP25 and nuclear proteins. Moreover, intron alterations during FKBP25 evolution that regulate gene splicing may be involved in the purifying selection.
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Ahmad, Saleem, Sen Wang, Weizhong Wu, Kunlong Yang, YanFeng Zhang, Elisabeth Tumukunde, Shihua Wang, and Yu Wang. "Functional Analysis of Peptidyl-prolyl cis-trans Isomerase from Aspergillus flavus." International Journal of Molecular Sciences 20, no. 9 (May 5, 2019): 2206. http://dx.doi.org/10.3390/ijms20092206.
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Aspergillus flavus, a ubiquitous filamentous fungus found in soil, plants and other substrates has been reported not only as a pathogen for plants, but also a carcinogen producing fungus for human. Peptidyl-Prolyl Isomerase (PPIases) plays an important role in cell process such as protein secretion cell cycle control and RNA processing. However, the function of PPIase has not yet been identified in A. flavus. In this study, the PPIases gene from A. flavus named ppci1 was cloned into expression vector and the protein was expressed in prokaryotic expression system. Activity of recombinant ppci1 protein was particularly inhibited by FK506, CsA and rapamycin. 3D-Homology model of ppci1 has been constructed with the template, based on 59.7% amino acid similarity. The homologous recombination method was used to construct the single ppci1 gene deletion strain Δppci1. We found that, the ppci1 gene plays important roles in A. flavus growth, conidiation, and sclerotia formation, all of which showed reduction in Δppci1 and increased in conidiation compared with the wild-type and complementary strains in A. flavus. Furthermore, aflatoxin and peanut seeds infection assays indicated that ppci1 contributes to virulence of A. flavus. Furthermore, we evaluated the effect of PPIase inhibitors on A. flavus growth, whereby these were used to treat wild-type strains. We found that the growths were inhibited under every inhibitor. All, these results may provide valuable information for designing inhibitors in the controlling infections of A. flavus.
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Ren, Ping, Anne Rossettini, Vishnu Chaturvedi, and Steven D. Hanes. "The Ess1 prolyl isomerase is dispensable for growth but required for virulence in Cryptococcus neoformans." Microbiology 151, no. 5 (May 1, 2005): 1593–605. http://dx.doi.org/10.1099/mic.0.27786-0.
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Cryptococcus neoformans is an important human fungal pathogen that also serves as a model for studies of fungal pathogenesis. C. neoformans contains several genes encoding peptidyl-prolyl cis/trans isomerases (PPIases), enzymes that catalyse changes in the folding and conformation of target proteins. Three distinct classes of PPIases have been identified: cyclophilins, FK506-binding proteins (FKBPs) and parvulins. This paper reports the cloning and characterization of ESS1, which is believed to be the first (and probably only) parvulin-class PPIase in C. neoformans. It is shown that ESS1 from C. neoformans is structurally and functionally homologous to ESS1 from Saccharomyces cerevisiae, which encodes an essential PPIase that interacts with RNA polymerase II and plays a role in transcription. In C. neoformans, ESS1 was found to be dispensable for growth, haploid fruiting and capsule formation. However, ESS1 was required for virulence in a murine model of cryptococcosis. Loss of virulence might have been due to the defects in melanin and urease production observed in ess1 mutants, or to defects in transcription of as-yet-unidentified virulence genes. The fact that Ess1 is not essential in C. neoformans suggests that, in this organism, some of its functions might be subsumed by other prolyl isomerases, in particular, cyclophilins Cpa1 or Cpa2. This is supported by the finding that ess1 mutants were hypersensitive to cyclosporin A. C. neoformans might therefore be a useful organism in which to investigate crosstalk among different families of prolyl isomerases.
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Guan, Dan, Xiao Hu, Donghui Diao, Fang Wang, and Yueping Liu. "Genome-Wide Analysis and Identification of the Aux/IAA Gene Family in Peach." International Journal of Molecular Sciences 20, no. 19 (September 23, 2019): 4703. http://dx.doi.org/10.3390/ijms20194703.
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The Auxin/indole-3-acetic acid (Aux/IAA) repressor genes down-regulate the auxin response pathway during many stages of plant and fruit development. In order to determine if and how Aux/IAAs participate in governing texture and hardness in stone fruit maturation, we identified 23 Aux/IAA genes in peach, confirmed by the presence of four conserved domains. In this work, we used fluorescence microscopy with PpIAA-GFP fusion reporters to observe their nuclear localization. We then conducted PCR-based differential expression analysis in “melting” and “stony hard” varieties of peach, and found that in the “melting” variety, nine PpIAAs exhibited peak expression in the S4-3 stage of fruit maturation, with PpIAA33 showing the highest (>120-fold) induction. The expression of six PpIAAs peaked in the S4-2 stage, with PpIAA14 expressed the most highly. Only PpIAA15/16 showed higher expression in the “stony hard” variety than in the “melting” variety, both peaking in the S3 stage. In contrast, PpIAA32 had the highest relative expression in buds, flowers, young and mature leaves, and roots. Our study provides insights into the expression patterns of Aux/IAA developmental regulators in response to auxin during fruit maturation, thus providing insight into their potential development as useful markers for quantitative traits associated with fruit phenotype.
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Pemberton, Trevor J., and John E. Kay. "Identification and Comparative Analysis of the Peptidyl-Prolylcis/transIsomerase Repertoires ofH. sapiens, D. melanogaster, C. elegans, S. cerevisiae and Sz. pombe." Comparative and Functional Genomics 6, no. 5-6 (2005): 277–300. http://dx.doi.org/10.1002/cfg.482.
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The peptidyl-prolylcis/transisomerase (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 withinDrosophila melanogasterthat 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 withSchizosaccharomyces pombe, Saccharomyces cerevisiaeis a poor representation of the higher eukaryotes for the study of PPIases.
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Furutani, Masahiro, Toshii Iida, Shigeyuki Yamano, Kei Kamino, and Tadashi Maruyama. "Biochemical and Genetic Characterization of an FK506-Sensitive Peptidyl Prolyl cis-trans Isomerase from a Thermophilic Archaeon, Methanococcus thermolithotrophicus." Journal of Bacteriology 180, no. 2 (January 15, 1998): 388–94. http://dx.doi.org/10.1128/jb.180.2.388-394.1998.
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ABSTRACT 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 (k cat/Km ) measured at 15°C for the peptidyl substrates,N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide andN-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. thermolithotrophicusFK506-binding protein). The MTFK gene (462 bp) was cloned from anM. 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.
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Wojtkiewicz, Pawel, Daria Biernacka, Patrycja Gorzelak, Anna Stupak, Gracjana Klein, and Satish Raina. "Multicopy Suppressor Analysis of Strains Lacking Cytoplasmic Peptidyl-Prolyl cis/trans Isomerases Identifies Three New PPIase Activities in Escherichia coli That Includes the DksA Transcription Factor." International Journal of Molecular Sciences 21, no. 16 (August 14, 2020): 5843. http://dx.doi.org/10.3390/ijms21165843.
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Consistent with a role in catalyzing rate-limiting step of protein folding, removal of genes encoding cytoplasmic protein folding catalysts belonging to the family of peptidyl-prolyl cis/trans isomerases (PPIs) in Escherichia coli confers conditional lethality. To address the molecular basis of the essentiality of PPIs, a multicopy suppressor approach revealed that overexpression of genes encoding chaperones (DnaK/J and GroL/S), transcriptional factors (DksA and SrrA), replication proteins Hda/DiaA, asparatokinase MetL, Cmk and acid resistance regulator (AriR) overcome some defects of Δ6ppi strains. Interestingly, viability of Δ6ppi bacteria requires the presence of transcriptional factors DksA, SrrA, Cmk or Hda. DksA, MetL and Cmk are for the first time shown to exhibit PPIase activity in chymotrypsin-coupled and RNase T1 refolding assays and their overexpression also restores growth of a Δ(dnaK/J/tig) strain, revealing their mechanism of suppression. Mutagenesis of DksA identified that D74, F82 and L84 amino acid residues are critical for its PPIase activity and their replacement abrogated multicopy suppression ability. Mutational studies revealed that DksA-mediated suppression of either Δ6ppi or ΔdnaK/J is abolished if GroL/S and RpoE are limiting, or in the absence of either major porin regulatory sensory kinase EnvZ or RNase H, transporter TatC or LepA GTPase or Pi-signaling regulator PhoU.
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Drouault, Sophie, Jamila Anba, Sophie Bonneau, Alexander Bolotin, S. Dusko Ehrlich, and Pierre Renault. "The Peptidyl-Prolyl Isomerase Motif Is Lacking in PmpA, the PrsA-Like Protein Involved in the Secretion Machinery of Lactococcus lactis." Applied and Environmental Microbiology 68, no. 8 (August 2002): 3932–42. http://dx.doi.org/10.1128/aem.68.8.3932-3942.2002.
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ABSTRACT The prsA-like gene from Lactococcus lactis encoding its single homologue to PrsA, an essential protein triggering the folding of secreted proteins in Bacillus subtilis, was characterized. This gene, annotated pmpA, encodes a lipoprotein of 309 residues whose expression is increased 7- to 10-fold when the source of nitrogen is limited. A slight increase in the expression of the PrsA-like protein (PLP) in L. lactis removed the degradation products previously observed with the Staphylococcus hyicus lipase used as a model secreted protein. This shows that PmpA either triggers the folding of the secreted lipase or activates its degradation by the cell surface protease HtrA. Unlike the case for B. subtilis, the inactivation of the gene encoding PmpA reduced only slightly the growth rate of L. lactis in standard conditions. However, it almost stopped its growth when the lipase was overexpressed in the presence of salt in the medium. Like PrsA of B. subtilis and PrtM of L. lactis, the L. lactis PmpA protein could thus have a foldase activity that facilitates protein secretion. These proteins belong to the third family of peptidyl-prolyl cis/trans-isomerases (PPIases) for which parvulin is the prototype. Almost all PLP from gram-positive bacteria contain a domain with the PPIase signature. An exception to this situation was found only in Streptococcaceae, the family to which L. lactis belongs. PLP from Streptococcus pneumoniae and Enterococcus faecalis possess this signature, but those of L. lactis, Streptococcus pyogenes, and Streptococcus mutans do not. However, secondary structure predictions suggest that the folding of PLP is conserved over the entire length of the proteins, including the unconserved signature region. The activity associated with the expression of PmpA in L. lactis and these genomic data show that either the PPIase motif is not necessary for PPIase activity or, more likely, PmpA foldase activity does not necessarily require PPIase activity.
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Devasahayam, Gina, Vishnu Chaturvedi, and Steven D. Hanes. "The Ess1 Prolyl Isomerase Is Required for Growth and Morphogenetic Switching in Candida albicans." Genetics 160, no. 1 (January 1, 2002): 37–48. http://dx.doi.org/10.1093/genetics/160.1.37.
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Abstract Prolyl-isomerases (PPIases) are found in all organisms and are important for the folding and activity of many proteins. Of the 13 PPIases in Saccharomyces cerevisiae only Ess1, a parvulin-class PPIase, is essential for growth. Ess1 is required to complete mitosis, and Ess1 and its mammalian homolog, Pin1, interact directly with RNA polymerase II. Here, we isolate the ESS1 gene from the pathogenic fungus Candida albicans and show that it is functionally homologous to the S. cerevisiae ESS1. We generate conditional-lethal (ts) alleles of C. albicans ESS1 and use these mutations to demonstrate that ESS1 is essential for growth in C. albicans. We also show that reducing the dosage or activity of ESS1 blocks morphogenetic switching from the yeast to the hyphal and pseudohyphal forms under certain conditions. Analysis of double mutants of ESS1 and TUP1 or CPH1, two genes known to be involved in morphogenetic switching, suggests that ESS1 functions in the same pathway as CPH1 and upstream of or in parallel to TUP1. Given that switching is important for virulence of C. albicans, inhibitors of Ess1 might be useful as antifungal agents.
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Huang, Han-kuei, Susan L. Forsburg, Ulrik P. John, Matthew J. O’Connell, and Tony Hunter. "Isolation and characterization of the Pin1/Ess1p homologue inSchizosaccharomyces pombe." Journal of Cell Science 114, no. 20 (October 15, 2001): 3779–88. http://dx.doi.org/10.1242/jcs.114.20.3779.
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Pin1/Ess1p is a highly conserved WW domain-containing peptidyl-prolyl isomerase (PPIase); its WW domain binds specifically to phospho-Ser/Thr-Pro sequences and its catalytic domain isomerizes phospho-Ser/Thr-Pro bonds. Pin1 PPIase activity can alter protein conformation in a phosphorylation-dependent manner and/or promote protein dephosphorylation. Human Pin1 interacts with mitotic phosphoproteins, such as NIMA, Cdc25 and Wee1, and inhibits G2/M progression in Xenopus extracts. Depletion of Pin1 in HeLa cells and deletion of ESS1 in S. cerevisiae result in mitotic arrest. In addition, Pin1/Ess1p play roles in transcription in S. cerevisiae and in mammalian somatic cells. The S. pombe genome sequence has an open reading frame (ORF) that has 47% identity with Pin1. Expression of this ORF rescued the growth defect caused by ess1 deletion in S. cerevisiae, indicating that S. pombe Pin1p is a functional Pin1 homologue. Overexpression of pin1+ in S. pombe caused slow growth and a G1 delay. Deletion of pin1+ (pin1Δ) did not affect cell cycle progression or cell growth, but increased sensitivity to the cyclophilin inhibitor, cyclosporin A, suggesting that cyclophilin family PPIases have overlapping functions with the Pin1p PPIase. Deletion of pin1+ did not affect the DNA replication checkpoint, but conferred a modest increase in UV sensitivity. Furthermore, the pin1Δ allele caused a synthetic growth defect when combined with either cdc25-22 or wee1-50 but not the cdc24-1 temperature-sensitive mutant. The pin1Δ strain showed increased sensitivity to the PP1/PP2A family phosphatase inhibitor, okadaic acid, suggesting that Pin1p plays a role in protein dephosphorylation as a result of its ability to increase the population of phospho-Ser/Thr-Pro peptide bonds in the trans conformation that is required for PP2A-mediated dephosphorylation. Our genetic data also suggest that Pin1p might function as a positive regulator of Cdc25p and Wee1p.
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Qing, Min, Feng Yang, Bo Zhang, Gang Zou, John M. Robida, Zhiming Yuan, Hengli Tang, and Pei-Yong Shi. "Cyclosporine Inhibits Flavivirus Replication through Blocking the Interaction between Host Cyclophilins and Viral NS5 Protein." Antimicrobial Agents and Chemotherapy 53, no. 8 (May 18, 2009): 3226–35. http://dx.doi.org/10.1128/aac.00189-09.
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ABSTRACT Although flaviviruses cause significant human diseases, no effective therapy is currently available. Host factors essential for viral replication are potential targets for antiviral development. Here we report that cyclophilins (CyPs), a family of cellular peptidyl-prolyl isomerases (PPIases), play a role in flavivirus replication. Huh-7.5 cells with knockdown of different isoforms of CyP were less efficient than parental cells in supporting flavivirus replication, including West Nile virus (WNV), dengue virus, and yellow fever virus. The low viral replication in CyP A (CyPA) knockdown cells could be rescued by trans supplying of a wild-type CyPA but not by trans supplying of a mutant CyPA (defective in the PPIase activity), indicating that the isomerase activity of CyPA is critical for viral replication. Immunoprecipitation and biochemical pulldown analyses showed that CyPA interacts with WNV genomic RNA and viral NS5 protein in the replication complex. Furthermore, antiviral experiments demonstrated that cyclosporine (Cs; an 11-amino-acid cyclic peptide known to block the PPIase activity of CyPA) inhibits flavivirus replication in cell culture at nontoxic concentrations. Time-of-addition and transient replicon results indicated that Cs inhibits flavivirus at the step of viral RNA synthesis. Biochemical analysis showed that Cs directly blocks the interaction between CyPA and WNV NS5 protein. Our results suggest that host CyPA is a component of flavivirus replication complex and could be targeted for potential antiviral development.
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Gavini, Nara, Sudheer Tungtur, and Lakshmi Pulakat. "Peptidyl-Prolyl cis/trans Isomerase-Independent Functional NifH Mutant of Azotobacter vinelandii." Journal of Bacteriology 188, no. 16 (August 15, 2006): 6020–25. http://dx.doi.org/10.1128/jb.00379-06.
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ABSTRACT Peptidyl-prolyl cis/trans isomerases (PPIases) play a pivotal role in catalyzing the correct folding of many prokaryotic and eukaryotic proteins that are implicated in a variety of biological functions, ranging from cell cycle regulation to bacterial infection. The nif accessory protein NifM, which is essential for the biogenesis of a functional NifH component of nitrogenase, is a PPIase. To understand the nature of the molecular signature that defines the NifM dependence of NifH, we screened a library of nifH mutants in the nitrogen-fixing bacterium Azotobacter vinelandii for mutants that acquired NifM independence. Here, we report that NifH can acquire NifM independence when the conserved Pro258 located in the C-terminal region of NifH, which wraps around the other subunit in the NifH dimer, is replaced by serine.
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Kallscheuer, Nicolai, Michael Bott, Jan van Ooyen, and Tino Polen. "Single-Domain Peptidyl-Prolylcis/transIsomerase FkpA from Corynebacterium glutamicum Improves the Biomass Yield at Increased Growth Temperatures." Applied and Environmental Microbiology 81, no. 22 (September 4, 2015): 7839–50. http://dx.doi.org/10.1128/aem.02113-15.
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ABSTRACTPeptidyl-prolylcis/transisomerases (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, includingCorynebacterium glutamicum, a facultative anaerobic Gram-positive and industrial workhorse for the production of amino acids. InC. glutamicum, a predicted single-domain FK-506 (tacrolimus) binding protein (FKBP)-type PPIase (FkpA) is encoded directly downstream ofgltA, which encodes citrate synthase (CS). This gene cluster is also present in otherActinobacteria. Here we carried outin vitroandin vivoexperiments to study the function and influence of predicted FkpA inC. 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 CSin vitro. Deletion offkpAcauses 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 mMl-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 inC. glutamicumΔfkpA, giving insight into the transcriptional response upon mild heat stress when FkpA is absent.
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Benton, B. M., J. H. Zang, and J. Thorner. "A novel FK506- and rapamycin-binding protein (FPR3 gene product) in the yeast Saccharomyces cerevisiae is a proline rotamase localized to the nucleolus." Journal of Cell Biology 127, no. 3 (November 1, 1994): 623–39. http://dx.doi.org/10.1083/jcb.127.3.623.
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The gene (FPR3) encoding a novel type of peptidylpropyl-cis-trans-isomerase (PPIase) was isolated during a search for previously unidentified nuclear proteins in Saccharomyces cerevisiae. PPIases are thought to act in conjunction with protein chaperones because they accelerate the rate of conformational interconversions around proline residues in polypeptides. The FPR3 gene product (Fpr3) is 413 amino acids long. The 111 COOH-terminal residues of Fpr3 share greater than 40% amino acid identity with a particular class of PPIases, termed FK506-binding proteins (FKBPs) because they are the intracellular receptors for two immunosuppressive compounds, rapamycin and FK506. When expressed in and purified from Escherichia coli, both full-length Fpr3 and its isolated COOH-terminal domain exhibit readily detectable PPIase activity. Both fpr3 delta null mutants and cells expressing FPR3 from its own promoter on a multicopy plasmid have no discernible growth phenotype and do not display any alteration in sensitivity to the growth-inhibitory effects of either FK506 or rapamycin. In S. cerevisiae, the gene for a 112-residue cytosolic FKBP (FPR1) and the gene for a 135-residue ER-associated FKBP (FPR2) have been described before. Even fpr1 fpr2 fpr3 triple mutants are viable. However, in cells carrying an fpr1 delta mutation (which confers resistance to rapamycin), overexpression from the GAL1 promoter of the C-terminal domain of Fpr3, but not full-length Fpr3, restored sensitivity to rapamycin. Conversely, overproduction from the GAL1 promoter of full-length Fpr3, but not its COOH-terminal domain, is growth inhibitory in both normal cells and fpr1 delta mutants. In fpr1 delta cells, the toxic effect of Fpr3 overproduction can be reversed by rapamycin. Overproduction of the NH2-terminal domain of Fpr3 is also growth inhibitory in normal cells and fpr1 delta mutants, but this toxicity is not ameliorated in fpr1 delta cells by rapamycin. The NH2-terminal domain of Fpr3 contains long stretches of acidic residues alternating with blocks of basic residues, a structure that resembles sequences found in nucleolar proteins, including S. cerevisiae NSR1 and mammalian nucleolin. Indirect immunofluorescence with polyclonal antibodies raised against either the NH2- or the COOH-terminal segments of Fpr3 expressed in E. coli demonstrated that Fpr3 is located exclusively in the nucleolus.
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Keogh, Zapf, Trzeciak, Null, Wiemels, and Carroll. "Novel Regulation of Alpha-Toxin and the Phenol-Soluble Modulins by Peptidyl-Prolyl cis/trans Isomerase Enzymes in Staphylococcus aureus." Toxins 11, no. 6 (June 16, 2019): 343. http://dx.doi.org/10.3390/toxins11060343.
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Peptidyl-prolyl cis/trans isomerases (PPIases) are enzymes that catalyze the cis-to-trans isomerization around proline bonds, allowing proteins to fold into their correct confirmation. Previously, we identified two PPIase enzymes in Staphylococcus aureus (PpiB and PrsA) that are involved in the regulation of virulence determinants and have shown that PpiB contributes to S. aureus virulence in a murine abscess model of infection. Here, we further examine the role of these PPIases in S. aureus virulence and, in particular, their regulation of hemolytic toxins. Using murine abscess and systemic models of infection, we show that a ppiB mutant in a USA300 background is attenuated for virulence but that a prsA mutant is not. Deletion of the ppiB gene leads to decreased bacterial survival in macrophages and nasal epithelial cells, while there is no significant difference when prsA is deleted. Analysis of culture supernatants reveals that a ppiB mutant strain has reduced levels of the phenol-soluble modulins and that both ppiB and prsA mutants have reduced alpha-toxin activity. Finally, we perform immunoprecipitation to identify cellular targets of PpiB and PrsA. Results suggest a novel role for PpiB in S. aureus protein secretion. Collectively, our results demonstrate that PpiB and PrsA influence S. aureus toxins via distinct mechanisms, and that PpiB but not PrsA contributes to disease.
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Goehring, Anna, Irina Michin, Tina Gerdes, Nina Schulze, Mike Blueggel, Edisa Rehic, Farnusch Kaschani, Markus Kaiser, and Peter Bayer. "Targeting of parvulin interactors by diazirine mediated cross-linking discloses a cellular role of human Par14/17 in actin polymerization." Biological Chemistry 401, no. 8 (July 28, 2020): 955–68. http://dx.doi.org/10.1515/hsz-2019-0423.
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AbstractThe peptidyl-prolyl cis/trans isomerases (PPIases) Parvulin 14 (Par14) and Parvulin 17 (Par17) result from alternative transcription initiation of the PIN4 gene. Whereas Par14 is present in all metazoan, Par17 is only expressed in Hominidae. Par14 resides mainly within the cellular nucleus, while Par17 is translocated into mitochondria. Using photo-affinity labeling, cross-linking and mass spectrometry (MS) we identified binding partners for both enzymes from HeLa lysates and disentangled their cellular roles. Par14 is involved in biogenesis of ribonucleoprotein (RNP)-complexes, RNA processing and DNA repair. Its elongated isoform Par17 participates in protein transport/translocation and in cytoskeleton organization. Nuclear magnetic resonance (NMR) spectroscopy reveals that Par17 binds to β-actin with its N-terminal region, while both parvulins initiate actin polymerization depending on their PPIase activity as monitored by fluorescence spectroscopy. The knockdown (KD) of Par17 in HCT116 cells results in a defect in cell motility and migration.
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Ichiba, Tetsuro, Tatsuya Shibasaki, Eisaku Iizuka, Akira Hachimori, and Tatsuya Samejima. "Cation-induced thermostability of yeast and Escherichia coli pyrophosphatases." Biochemistry and Cell Biology 66, no. 1 (January 1, 1988): 25–31. http://dx.doi.org/10.1139/o88-004.
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Inorganic pyrophosphatases (PPiases) from both yeast and Escherichia coli were found to be stable against heat denaturation in the presence of Mg2+, as previously observed with the enzymes from thermophilic bacteria. No loss of activity was observed after 1 h of incubation at 50 °C and pHs between 6 and 9 in the yeast enzyme, and at 60 °C and pHs between 7.2 and 9.2 in the E. coli enzyme. Such an induced thermostability of the E. coli enzyme was detected when Mn2+, Co2+, Ca2+, Cd2+, and Zn2+ were added in place of Mg2+. On the other hand, the degree of induced thermostability of the yeast enzyme was dependent upon the divalent cations used, and Ni2+ and Cu2+ accelerated the heat inactivation. On adding the divalent cations, the difference spectra of the E. coli enzyme always showed negative peaks in the ultraviolet region, but those of the yeast enzyme changed again depending upon the divalent cations. The circular dichroism spectra in the near ultraviolet region of both enzymes greatly differed from each other, but both were not affected so much by adding the divalent cations unlike the thermophilic enzymes from Bacillus stearothermophilus and thermophilic bacterium PS-3. Yeast and E. coli PPiases did not cross-link with the anti-immunoglobulin G's from the thermophilic enzymes, but the thermophilic enzymes did with each other's antisera. The results in the present study indicated that the conformation of PPiase, in which the aromatic amino acid residues were buried in the interior of the protein molecule, was very important for the thermostability and also that the protein structures of PPiases from B. stearothermophilus and thermophilic bacterium PS-3 were very similar to each other, but were very different from those of the mesophilic enzymes.
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Zhang, Yan, Kyle Fletcher, Rongkui Han, Richard Michelmore, and Ruiwu Yang. "Genome-Wide Analysis of Cyclophilin Proteins in 21 Oomycetes." Pathogens 9, no. 1 (December 26, 2019): 24. http://dx.doi.org/10.3390/pathogens9010024.
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Cyclophilins (CYPs), a highly-conserved family of proteins, belong to a subgroup of immunophilins. Ubiquitous in eukaryotes and prokaryotes, CYPs have peptidyl-prolyl cis–trans isomerase (PPIase) activity and have been implicated as virulence factors in plant pathogenesis by oomycetes. We identified 16 CYP orthogroups from 21 diverse oomycetes. Each species was found to encode 15 to 35 CYP genes. Three of these orthogroups contained proteins with signal peptides at the N-terminal end, suggesting a role in secretion. Multidomain analysis revealed five conserved motifs of the CYP domain of oomycetes shared with other eukaryotic PPIases. Expression analysis of CYP proteins in different asexual life stages of the hemibiotrophic Phytophthora infestans and the biotrophic Plasmopara halstedii demonstrated distinct expression profiles between life stages. In addition to providing detailed comparative information on the CYPs in multiple oomycetes, this study identified candidate CYP effectors that could be the foundation for future studies of virulence.
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MORI, Tadashi, and Takafumi UCHIDA. "Screening for PPIase Pin1 Inhibitors." KAGAKU TO SEIBUTSU 49, no. 6 (2011): 398–405. http://dx.doi.org/10.1271/kagakutoseibutsu.49.398.
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Terkeltaub, Robert A. "Inorganic pyrophosphate generation and disposition in pathophysiology." American Journal of Physiology-Cell Physiology 281, no. 1 (July 1, 2001): C1—C11. http://dx.doi.org/10.1152/ajpcell.2001.281.1.c1.
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Inorganic pyrophosphate (PPi) regulates certain intracellular functions and extracellular crystal deposition. PPiis produced, degraded, and transported by specialized mechanisms. Moreover, dysregulated cellular PPiproduction, degradation, and transport all have been associated with disease, and PPiappears to directly mediate specific disease manifestations. In addition, natural and synthetic analogs of PPiare in use or currently under evaluation as prophylactic agents or therapies for disease. This review summarizes recent developments in the understanding of how PPiis made and disposed of by cells and assesses the body of evidence for potentially significant physiological functions of intracellular PPiin higher organisms. Major topics addressed are recent lines of molecular evidence that directly link decreased and increased extracellular PPilevels with diseases in which connective tissue matrix calcification is disordered. To illustrate in depth the effects of disordered PPimetabolism, this review weighs the roles in matrix calcification of the transmembrane protein ANK, which regulates intracellular to extracellular movement of PPi, and the PPi-generating phosphodiesterase nucleotide pyrophosphatase family isoenzyme plasma cell membrane glycoprotein-1 (PC-1).
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Marsh, Kenneth, Pedro Gonzalez, and Ed Echeverría. "PPi Formation by Reversal of the Tonoplast-bound H+-pyrophosphatase from `Valencia' Orange Juice Cells." Journal of the American Society for Horticultural Science 125, no. 4 (July 2000): 420–24. http://dx.doi.org/10.21273/jashs.125.4.420.
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Tonoplast vesicles isolated from juice cells of mature `Valencia' oranges [Citrus sinensis (L.) Osbeck] showed similar tonoplast-bound vacuolar ATPase (V-ATPase) and inorganic pyrophosphatase (V-PPiase) activity as measured by product formation. Both proton pumps were able to generate a similar pH gradient, although steady-state was reached faster with ATP as substrate. When a ΔpH of 3 units was imposed (vesicle lumen pH of 4.5 and incubation medium of 7.5), tonoplast-bound PPiase was not able to significantly amplify the existing ΔpH. Although not able to function as a H+ pump, V-PPiase effectively synthesized PPi in the presence of inorganic phosphate (Pi). Formation of PPi by V-PPiase was enhanced by ATP but inhibited by NaF, gramicidin, and by antibodies raised against V-PPiase from mung bean [Vigna radiata (L.) R. Wilcz. (Syn. Phaseolus aureus Roxb.)]. Immunological analysis demonstrated an increase in V-PPiase protein with fruit maturity. Data indicate that under in vivo conditions, the V-PPiase of mature orange juice cells acts as a source of inorganic pyrophosphate (PPi) but not as a H+ pump. We propose that synthesis of PPi provides a mechanism for recovery of stored energy in the form of the pH gradient across the vacuole during later stages of development and postharvest storage.
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Küllertz, Gerhard, Sabine Lüthe, and Gunter Fischer. "Semiautomated microtiter plate assay for monitoring peptidylprolyl cis/trans isomerase activity in normal and pathological human sera." Clinical Chemistry 44, no. 3 (March 1, 1998): 502–8. http://dx.doi.org/10.1093/clinchem/44.3.502.
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Abstract An UV/VIS spectrophotometric assay technique was developed that was able to routinely monitor peptidylprolyl cis/trans isomerase (PPIase) activity of biological fluids in 96-well microtiter plates. The assay, based on monitoring the cis-to-trans isomerization of succinyl-Phe-cisPro-Phe-4-nitroanilide as substrate in a chymotrypsin-coupled reaction, yields a throughput of 96 samples per 30 min. The assay’s capacity was exemplified by dealing with the PPIase activity in several normal and pathological human sera. Reference values of 151 healthy subjects (83 females, 69 males, 17 to 60 years old) were found to possess significant sex-specific differences. PPIase activity factor K of the sera was significantly greater in males (5th, 50th, 95th percentiles: 17, 36, 55 K) than females (14, 30, 48 K). PPIase activities of sera from healthy donors (n = 151) were significantly higher (Mann–Whitney rank-sum test P <0.0001) than those of patients (n = 47). PPIase activity in serum samples stored at 4 °C was stable for at least 20 h.
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Ansari, Husam, Giampaolo Greco, and Jeremy Luban. "Cyclophilin A Peptidyl-Prolyl Isomerase Activity Promotes Zpr1 Nuclear Export." Molecular and Cellular Biology 22, no. 20 (October 15, 2002): 6993–7003. http://dx.doi.org/10.1128/mcb.22.20.6993-7003.2002.
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ABSTRACT The peptidyl-prolyl isomerase (PPIase) cyclophilin A (Cpr1p) is conserved from eubacteria to mammals, yet its biological function has resisted elucidation. Unable to identify a phenotype that is suggestive of Cpr1p's function in a cpr1Δ Saccharomyces cerevisiae strain, we screened for CPR1-dependent strains. In all cases, dependence was conferred by mutations in ZPR1, a gene encoding an essential zinc finger protein. CPR1 dependence was suppressed by overexpression of EF1α (a translation factor that binds Zpr1p), Cpr6p (another cyclophilin), or Fpr1p (a structurally unrelated PPIase). Suppression by a panel of cyclophilin A mutants correlated with PPIase activity, confirming the relevance of this activity in CPR1-dependent strains. In CPR1 + cells, wild-type Zpr1p was distributed equally between the nucleus and cytoplasm. In contrast, proteins encoded by CPR1-dependent alleles of ZPR1 accumulated in the nucleus, as did wild-type Zpr1p in cpr1Δ cells. Transport kinetic studies indicated that nuclear export of Zpr1p was defective in cpr1Δ cells, and rescue of this defect correlated with PPIase activity. Our results demonstrate a functional interaction between Cpr1p, Zpr1p, and EF1α, a role for Cpr1p in Zpr1p nuclear export, and a biological function for Cpr1p PPIase activity.
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Zhang, Jie Wei, Michael R. Leach, and Deborah B. Zamble. "The Peptidyl-Prolyl Isomerase Activity of SlyD Is Not Required for Maturation of Escherichia coli Hydrogenase." Journal of Bacteriology 189, no. 21 (August 24, 2007): 7942–44. http://dx.doi.org/10.1128/jb.00922-07.
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ABSTRACT Escherichia coli SlyD, which is involved in the biosynthesis of the metal cluster in the [NiFe]-hydrogenase enzymes, exhibits several activities including that of a peptidyl-prolyl isomerase (PPIase). Mutations that result in deficient PPIase activity do not produce corresponding decreases in the other activities of SlyD in vitro or in hydrogenase production levels in vivo.
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Sigal, N. H., F. Dumont, P. Durette, J. J. Siekierka, L. Peterson, D. H. Rich, B. E. Dunlap, M. J. Staruch, M. R. Melino, and S. L. Koprak. "Is cyclophilin involved in the immunosuppressive and nephrotoxic mechanism of action of cyclosporin A?" Journal of Experimental Medicine 173, no. 3 (March 1, 1991): 619–28. http://dx.doi.org/10.1084/jem.173.3.619.
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In this report we have approached two questions relating to the mechanism of action of cyclosporin A (CsA). First, we address whether the major cytosolic protein for CsA, cyclophilin, is directly involved in mediating the immunosuppressive activity of this drug, and, in particular, whether inhibition of this protein's peptidyl-prolyl cis-trans isomerase (PPIase) activity results in inhibition of murine T cell activation. Second, we ask whether the nephrotoxicity observed with CsA is related to inhibition of PPIase-dependent pathways in cells other than lymphocytes. Using a series of 61 cyclosporin analogues, we generally found a good correlation between cyclophilin binding and immunosuppressive activity for the majority of analogues analyzed. However, a number of compounds of distinct structural classes were found that could interact with cyclophilin but were much less immunosuppressive than expected. The inability of these analogues to inhibit lymphocyte activation could not be explained by their failure to enter the cell and bind to cyclophilin under the conditions used in the cellular assays. Surprisingly, a nonimmunosuppressive analogue, MeAla-6, which bound well to cyclophilin and was active as a PPIase inhibitor, did not induce renal pathology in vivo. Furthermore, another analogue, MeBm2t, which was immunosuppressive in vitro, possessed little or no activity as a PPIase inhibitor. These findings pose serious questions concerning a direct role of cyclosporin in mediating CsA's immunosuppressive and nephrotoxic activities. In addition, they raise doubts about whether PPIase has a direct function in lymphocyte signal transduction.
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Connern, C. P., and A. P. Halestrap. "Purification and N-terminal sequencing of peptidyl-prolyl cis-trans-isomerase from rat liver mitochondrial matrix reveals the existence of a distinct mitochondrial cyclophilin." Biochemical Journal 284, no. 2 (June 1, 1992): 381–85. http://dx.doi.org/10.1042/bj2840381.
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1. Rat liver mitochondrial matrix peptidyl-prolyl cis-trans-isomerase (PPIase) has been purified. The major form of the enzyme has a molecular mass of 18.6 kDa, with a minor active component of 17.6 kDa. 2. The second-order rate constant for cyclosporin A binding to the enzyme was determined from the time-dependence of the inhibition of PPIase by low concentrations of cyclosporin A and found to be 0.9 microM-1.s-1 at 10 degrees C. 3. The Ki for cyclosporin A inhibition of the enzyme was 3.6 nM, and the half-life for dissociation of the enzyme-inhibitor complex was 3.6 min. 4. From the specific activity of the pure enzyme it can be calculated that isolated liver mitochondria contain approx. 45 pmol of enzyme per mg of total mitochondrial protein. Higher values estimated previously [Halestrap & Davidson (1990) Biochem. J. 268, 153-160] are explained by the use of a short (30 s) preincubation period of the enzyme with cyclosporin, which is insufficient to allow full equilibration of the binding of the inhibitor to the PPIase. 5. N-Terminal sequencing of the 18.6 and 17.5 kDa forms of PPIase show the presence of mitochondrial presequences of 13 and three amino acids respectively, with the remaining sequence having a strong sequence similarity to other cyclophilins. 6. Parallel purification and N-terminal sequencing of rat cytosolic PPIase showed the two proteins to have significant differences, implying that they are probably products of separate genes.
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Ulrich, Alexander, and Markus C. Wahl. "Structure and evolution of the spliceosomal peptidyl-prolylcis–transisomerase Cwc27." Acta Crystallographica Section D Biological Crystallography 70, no. 12 (November 22, 2014): 3110–23. http://dx.doi.org/10.1107/s1399004714021695.
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Cwc27 is a spliceosomal cyclophilin-type peptidyl-prolylcis–transisomerase (PPIase). Here, the crystal structure of a relatively protease-resistant N-terminal fragment of human Cwc27 containing the PPIase domain was determined at 2.0 Å resolution. The fragment exhibits a C-terminal appendix and resides in a reduced state compared with the previous oxidized structure of a similar fragment. By combining multiple sequence alignments spanning the eukaryotic tree of life and secondary-structure prediction, Cwc27 proteins across the entire eukaryotic kingdom were identified. This analysis revealed the specific loss of a crucial active-site residue in higher eukaryotic Cwc27 proteins, suggesting that the protein evolved from a prolyl isomerase to a pure proline binder. Noting a fungus-specific insertion in the PPIase domain, the 1.3 Å resolution crystal structure of the PPIase domain of Cwc27 fromChaetomium thermophilumwas also determined. Although structurally highly similar in the core domain, theC. thermophilumprotein displayed a higher thermal stability than its human counterpart, presumably owing to the combined effect of several amino-acid exchanges that reduce the number of long side chains with strained conformations and create new intramolecular interactions, in particular increased hydrogen-bond networks.
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Kumawat, Manoj, Irungbam Karuna, Neeraj Ahlawat, and Sushma Ahlawat. "Identification of Salmonella Typhimurium Peptidyl-prolyl cis-trans Isomerase B (PPIase B) and Assessment of their Role in the Protein Folding." Protein & Peptide Letters 27, no. 8 (September 24, 2020): 744–50. http://dx.doi.org/10.2174/0929866527666200225124104.
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Background: Peptidyl-prolyl cis-trans isomerase (PPIases) enzyme plays a vital role in protein folding. It catalyses the cis-trans isomerisation of peptide bonds, an essential step for newly synthesized protein to acquire its correct functional conformation in both prokaryotes and eukaryotes. Objective: The present study showed the biochemical and molecular characterisation of cyclophilins (PpiB), a type of peptidyl-prolyl isomerases proteins from the pathogenic bacteria Salmonella Typhimurium. Methods: Salmonella Typhimurium is one of the leading serovars responsible for human and animal salmonellosis globally, with the majority of human cases originating through the food chain. Here successful expression and purification of PpiB protein have been demonstrated and LC-MS based analyses showed high protein score and similarity with other PPi protein. Further the enzymatic activity of the purified recombinant PpiB was determined using Succinyl-Ala-Phe-Pro- Phe-p nitroanilide as substrate and enzyme-catalysed reaction. Result: Km and Vmax were calculated and found to be Vm = 1.023 ± .06400 min/μg, Km = 0.6219 ± 0.1701 μM, respectively. We have reported for the first time the presence of Salmonella PPIase-B (PpiB) protein isoforms in salmonella genome having PPi activity. Conclusion: Taken together, our data clearly showed that Salmonella Cyclophilin B (PpiB) protein is active and involved in diverse biological processes and highly similar to the different domain of Cyclophilin proteins.
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Nevers, Quentin, Isaac Ruiz, Nazim Ahnou, Flora Donati, Rozenn Brillet, Laurent Softic, Maxime Chazal, et al. "Characterization of the Anti-Hepatitis C Virus Activity of New Nonpeptidic Small-Molecule Cyclophilin Inhibitors with the Potential for Broad Anti-Flaviviridae Activity." Antimicrobial Agents and Chemotherapy 62, no. 7 (May 14, 2018): e00126-18. http://dx.doi.org/10.1128/aac.00126-18.
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ABSTRACT Although members of the Flaviviridae display high incidence, morbidity, and mortality rates, the development of specific antiviral drugs for each virus is unlikely. Cyclophilins, a family of host peptidyl-prolyl cis-trans isomerases (PPIases), play a pivotal role in the life cycles of many viruses and therefore represent an attractive target for broad-spectrum antiviral development. We report here the pangenotypic anti-hepatitis C virus (HCV) activity of a small-molecule cyclophilin inhibitor (SMCypI). Mechanistic and modeling studies revealed that the SMCypI bound to cyclophilin A in competition with cyclosporine (CsA), inhibited its PPIase activity, and disrupted the CypA-nonstructural protein 5A (NS5A) interaction. Resistance selection showed that the lead SMCypI hardly selected amino acid substitutions conferring low-level or no resistance in vitro. Interestingly, the SMCypI selected D320E and Y321H substitutions, located in domain II of the NS5A protein. These substitutions were previously associated with low-level resistance to cyclophilin inhibitors such as alisporivir. Finally, the SMCypI inhibited the replication of other members of the Flaviviridae family with higher 50% effective concentrations (EC50s) than for HCV. Thus, because of its chemical plasticity and simplicity of synthesis, our new family of SMCypIs represents a promising new class of drugs with the potential for broad-spectrum anti-Flaviviridae activity as well as an invaluable tool to explore the role of cyclophilins in viral life cycles.
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Vicente, R. L., S. Marín, J. R. Valverde, C. Palomino, R. P. Mellado, and S. Gullón. "Functional identification of a Streptomyces lividans FKBP-like protein involved in the folding of overproduced secreted proteins." Open Biology 9, no. 10 (October 2019): 190201. http://dx.doi.org/10.1098/rsob.190201.
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Some bacterial peptidyl-prolyl cis/trans isomerases (PPIases) are involved in secretory protein folding after the translocation step. Streptomyces lividans has been used as a host for engineering extracellular overproduction of homologous and heterologous proteins in industrial applications. Although the mechanisms governing the major secretory pathway (Sec route) and the minor secretory pathway (Tat route) are reasonably well described, the function of proteins responsible for the extracellular secretory protein folding is not characterized as yet. We have characterized a Tat-dependent S . lividans FK506-binding protein-like lipoprotein (FKBP) that has PPIase activity. A mutant in the sli-fkbp gene induces a secretion stress response and affects secretion and activity of the Sec-dependent protein α-amylase. Additionally, propagation in high copy number of the sli-fkbp gene has a positive effect on the activity of both the overproduced α-amylase and the overproduced Tat-dependent agarase, both containing proline cis isomers. Targeted proteomic analyses showed that a relevant group of secreted proteins in S. lividans TK21 are affected by Sli-FKBP, revealing a wide substrate range. The results obtained indicate that, regardless of the secretory route used by proteins in S. lividans , adjusting the expression of sli-fkbp may facilitate folding of dependent proteins when engineering Streptomyces strains for the overproduction of homologous or heterologous secretory proteins.
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Lyon, William R., and Michael G. Caparon. "Trigger Factor-Mediated Prolyl Isomerization Influences Maturation of the Streptococcus pyogenes Cysteine Protease." Journal of Bacteriology 185, no. 12 (June 15, 2003): 3661–67. http://dx.doi.org/10.1128/jb.185.12.3661-3667.2003.
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ABSTRACT Trigger factor, a ribosome-associated chaperone and peptidyl-prolyl cis-trans isomerase (PPIase), is essential for the secretion and maturation of the cysteine protease of the pathogenic gram-positive bacterium Streptococcus pyogenes. In the absence of trigger factor, the nascent protease polypeptide is not targeted to the secretory pathway. Some partial-function mutations restore targeting. However, the secreted protease does not efficiently mature into an enzymatically active form, suggesting that trigger factor has an additional role in protease biogenesis. Here, we show that, while not required for targeting, the PPIase activity of trigger factor is essential for maturation of the protease following its secretion from the bacterial cell. Site-specific mutations introduced into ropA, the gene which encodes trigger factor in S. pyogenes, produced mutant proteins deficient in PPIase activity. When these mutant alleles were used to replace the wild-type gene on the streptococcal chromosome, analysis of protease biogenesis revealed that, although the protease was secreted normally, it did not efficiently mature to an active form. Furthermore, mutation of a single proline residue in the protease prodomain suppressed the requirement for PPIase activity, suggesting that this residue is the target of trigger factor. These data support a model in which trigger factor-mediated prolyl isomerization influences the conformation of the prodomain, which in turn directs the protease into one of several alternative folding pathways.
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Norville, Isobel H., Katrin Breitbach, Kristin Eske-Pogodda, Nicholas J. Harmer, Mitali Sarkar-Tyson, Richard W. Titball, and Ivo Steinmetz. "A novel FK-506-binding-like protein that lacks peptidyl-prolyl isomerase activity is involved in intracellular infection and in vivo virulence of Burkholderia pseudomallei." Microbiology 157, no. 9 (September 1, 2011): 2629–38. http://dx.doi.org/10.1099/mic.0.049163-0.
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Burkholderia pseudomallei is a facultative intracellular bacterial pathogen causing melioidosis, an often fatal infectious disease that is endemic in several tropical and subtropical areas around the world. We previously described a Ptk2 cell-based plaque assay screening system of B. pseudomallei transposon mutants that led to the identification of several novel virulence determinants. Using this approach we identified a mutant with reduced plaque formation in which the BPSL0918 gene was disrupted. BPSL0918 encodes a putative FK-506-binding protein (FKBP) representing a family of proteins that typically possess peptidyl-prolyl isomerase (PPIase) activity. A B. pseudomallei ΔBPSL0918 mutant showed a severely impaired ability to resist intracellular killing and to replicate within primary macrophages. Complementation of the mutant fully restored its ability to grow intracellularly. Moreover, B. pseudomallei ΔBPSL0918 was significantly attenuated in a murine model of infection. Structural modelling confirmed a modified FKBP fold of the BPSL0918-encoded protein but unlike virulence-associated FKBPs from other pathogenic bacteria, recombinant BPSL0918 protein did not possess PPIase activity in vitro. In accordance with this observation BPSL0918 exhibits several mutations in residues that have been proposed to mediate PPIase activity in other FKBPs. To our knowledge this B. pseudomallei FKBP represents the first example of this protein family which lacks PPIase activity but is important in intracellular infection of a bacterial pathogen.
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Lehrer, Jenifer K., Stacey Zavala, Leonard Braitman, Roy M. Gideon, Donald O. Castell, and Philip O. Katz. "INTRAGASTRIC pH CONTROL ON TWICE DAILY PROTON PUMP INHIBITORS (PPIS). IS A PPIA PPI?" American Journal of Gastroenterology 99 (October 2004): S38—S39. http://dx.doi.org/10.14309/00000434-200410001-00115.
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Galat, Andrzej. "Introduction to Peptidyl-Prolyl cis/trans Isomerase (PPIase) Series." Biomolecules 9, no. 2 (February 20, 2019): 74. http://dx.doi.org/10.3390/biom9020074.
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About 30 years after the discovery of peptidyl-prolyl cis/trans isomerases (PPIases), research on this group of proteins has become somewhat calmer than it used to be, but it still generates lots of interest [...]
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Born, Alexandra, Morkos A. Henen, and Beat Vögeli. "Activity and Affinity of Pin1 Variants." Molecules 25, no. 1 (December 20, 2019): 36. http://dx.doi.org/10.3390/molecules25010036.
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Pin1 is a peptidyl-prolyl isomerase responsible for isomerizing phosphorylated S/T-P motifs. Pin1 has two domains that each have a distinct ligand binding site, but only its PPIase domain has catalytic activity. Vast evidence supports interdomain allostery of Pin1, with binding of a ligand to its regulatory WW domain impacting activity in the PPIase domain. Many diverse studies have made mutations in Pin1 in order to elucidate interactions that are responsible for ligand binding, isomerase activity, and interdomain allostery. Here, we summarize these mutations and their impact on Pin1′s structure and function.
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Ikolo, Felicia, Meng Zhang, Dean J. Harrington, Carl Robinson, Andrew S. Waller, Iain C. Sutcliffe, and Gary W. Black. "Characterisation of SEQ0694 (PrsA/PrtM) of Streptococcus equi as a functional peptidyl-prolyl isomerase affecting multiple secreted protein substrates." Molecular BioSystems 11, no. 12 (2015): 3279–86. http://dx.doi.org/10.1039/c5mb00543d.
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Denic, Milica, Evelyne Turlin, Valérie Michel, Frédéric Fischer, Mozhgan Khorasani-Motlagh, Deborah Zamble, Daniel Vinella, and Hilde de Reuse. "A novel mode of control of nickel uptake by a multifunctional metallochaperone." PLOS Pathogens 17, no. 1 (January 14, 2021): e1009193. http://dx.doi.org/10.1371/journal.ppat.1009193.
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Cellular metal homeostasis is a critical process for all organisms, requiring tight regulation. In the major pathogen Helicobacter pylori, the acquisition of nickel is an essential virulence determinant as this metal is a cofactor for the acid-resistance enzyme, urease. Nickel uptake relies on the NixA permease and the NiuBDE ABC transporter. Till now, bacterial metal transporters were reported to be controlled at their transcriptional level. Here we uncovered post-translational regulation of the essential Niu transporter in H. pylori. Indeed, we demonstrate that SlyD, a protein combining peptidyl-prolyl isomerase (PPIase), chaperone, and metal-binding properties, is required for the activity of the Niu transporter. Using two-hybrid assays, we found that SlyD directly interacts with the NiuD permease subunit and identified a motif critical for this contact. Mutants of the different SlyD functional domains were constructed and used to perform in vitro PPIase activity assays and four different in vivo tests measuring nickel intracellular accumulation or transport in H. pylori. In vitro, SlyD PPIase activity is down-regulated by nickel, independently of its C-terminal region reported to bind metals. In vivo, a role of SlyD PPIase function was only revealed upon exposure to high nickel concentrations. Most importantly, the IF chaperone domain of SlyD was shown to be mandatory for Niu activation under all in vivo conditions. These data suggest that SlyD is required for the active functional conformation of the Niu permease and regulates its activity through a novel mechanism implying direct protein interaction, thereby acting as a gatekeeper of nickel uptake. Finally, in agreement with a central role of SlyD, this protein is essential for the colonization of the mouse model by H. pylori.
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Cron, L. E., H. J. Bootsma, N. Noske, P. Burghout, S. Hammerschmidt, and P. W. M. Hermans. "Surface-associated lipoprotein PpmA of Streptococcus pneumoniae is involved in colonization in a strain-specific manner." Microbiology 155, no. 7 (July 1, 2009): 2401–10. http://dx.doi.org/10.1099/mic.0.026765-0.
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Streptococcus pneumoniae produces two surface-associated lipoproteins that share homology with two distinct families of peptidyl-prolyl isomerases (PPIases), the streptococcal lipoprotein rotamase A (SlrA) and the putative proteinase maturation protein A (PpmA). Previously, we have demonstrated that SlrA has PPIase activity, and that the enzyme plays a role in pneumococcal virulence. Here, we investigated the contribution of PpmA to pneumococcal pathogenesis. Pneumococcal mutants of D39 and TIGR4 lacking the gene encoding PpmA were less capable of persisting in the nasopharynx of mice, demonstrating the contribution of PpmA to pneumococcal colonization. This observation was partially confirmed in vitro, as the pneumococcal mutants NCTC10319ΔppmA and TIGR4ΔcpsΔppmA, but not D39ΔcpsΔppmA, were impaired in adherence to Detroit 562 pharyngeal cells. This suggests that the contribution of PpmA to pneumococcal colonization is not solely the result of its role in adherence to epithelial cells. Deficiency in PpmA did not result in reduced binding to various extracellular matrix and serum proteins. Similar to SlrA, we observed that PpmA was involved in immune evasion. Uptake of PpmA-deficient D39Δcps and NCTC10319 by human polymorphonuclear leukocytes was significantly enhanced compared to the isogenic wild-types. In addition, ingestion of D39ΔppmA, but not that of either NCTC10319ΔppmA or TIGR4ΔppmA, by murine macrophage cell line J774 was also enhanced, whereas intracellular killing remained unaffected. We conclude that PpmA contributes to the early stages of infection, i.e. colonization. The contribution of PpmA to virulence can be explained by its strain-specific role in adherence to epithelial cells and contribution to the evasion of phagocytosis.
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Ideno, Akira, Masahiro Furutani, Yoshitaka Iba, Yoshikazu Kurosawa, and Tadashi Maruyama. "FK506 Binding Protein from the Hyperthermophilic Archaeon Pyrococcus horikoshii Suppresses the Aggregation of Proteins in Escherichia coli." Applied and Environmental Microbiology 68, no. 2 (February 2002): 464–69. http://dx.doi.org/10.1128/aem.68.2.464-469.2002.
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ABSTRACT The 29-kDa FK506 binding protein (FKBP) gene is the only peptidyl-prolyl cis-trans isomerase (PPIase) gene in the genome of Pyrococcus horikoshii. We characterized the function of this FKBP (PhFKBP29) and used it to increase the production yield of soluble recombinant protein in Escherichia coli. The PPIase activity (k cat/Km ) of PhFKBP29 was found to be much lower than that of other archaeal 16- to 18-kDa FKBPs by a chymotrypsin-coupled assay of the oligo-peptidyl substrate at 15�C. Besides this low PPIase activity, PhFKBP29 showed chaperone-like protein folding activity which enhanced the refolding yield of chemically unfolded rhodanese in vitro. In addition, it suppressed thermal protein aggregation in a temperature range of 45 to 100�C. When the PhFKBP29 gene was coexpressed with the recombinant Fab fragment gene of the anti-hen egg lysozyme antibody in the cytoplasm of E. coli, whose expressed product tended to form an inactive aggregate in E. coli, it improved the yield of the soluble Fab fragments with antibody specificity. PhFKBP29 exerted protein folding and aggregation suppression in E. coli cells.
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Singh, Ajit Kumar, Aritreyee Datta, Chacko Jobichen, Sheng Luan, and Dileep Vasudevan. "AtFKBP53: a chimeric histone chaperone with functional nucleoplasmin and PPIase domains." Nucleic Acids Research 48, no. 3 (December 6, 2019): 1531–50. http://dx.doi.org/10.1093/nar/gkz1153.
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Abstract FKBP53 is one of the seven multi-domain FK506-binding proteins present in Arabidopsis thaliana, and it is known to get targeted to the nucleus. It has a conserved PPIase domain at the C-terminus and a highly charged N-terminal stretch, which has been reported to bind to histone H3 and perform the function of a histone chaperone. To better understand the molecular details of this PPIase with histone chaperoning activity, we have solved the crystal structures of its terminal domains and functionally characterized them. The C-terminal domain showed strong PPIase activity, no role in histone chaperoning and revealed a monomeric five-beta palm-like fold that wrapped over a helix, typical of an FK506-binding domain. The N-terminal domain had a pentameric nucleoplasmin-fold; making this the first report of a plant nucleoplasmin structure. Further characterization revealed the N-terminal nucleoplasmin domain to interact with H2A/H2B and H3/H4 histone oligomers, individually, as well as simultaneously, suggesting two different binding sites for H2A/H2B and H3/H4. The pentameric domain assists nucleosome assembly and forms a discrete complex with pre-formed nucleosomes; wherein two pentamers bind to a nucleosome.
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Nath, Pulak. "Peptidyl-prolyl isomerase (PPIase): an emerging area in tumor biology." Cancer Research Frontiers 3, no. 1 (April 1, 2017): 126–43. http://dx.doi.org/10.17980/2017.126.
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Köhler, Rolf, Jörg Fanghänel, Bettina König, Edeltraud Lüneberg, Matthias Frosch, Jens-Ulrich Rahfeld, Rolf Hilgenfeld, Gunter Fischer, Jörg Hacker, and Michael Steinert. "Biochemical and Functional Analyses of the Mip Protein: Influence of the N-Terminal Half and of Peptidylprolyl Isomerase Activity on the Virulence of Legionella pneumophila." Infection and Immunity 71, no. 8 (August 2003): 4389–97. http://dx.doi.org/10.1128/iai.71.8.4389-4397.2003.
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ABSTRACT The virulence factor Mip (macrophage infectivity potentiator) contributes to the intracellular survival of Legionella pneumophila, the causative agent of Legionnaires' disease. The protein consists of two domains that are connected via a very long α-helix (A. Riboldi-Tunnicliffe et al., Nat. Struct. Biol. 8:779-783, 2001). The fold of the C-terminal domain (residues 100 to 213) is closely related to human FK506-binding protein (FKBP12), and like FKBP12, Mip exhibits peptidylprolyl cis/trans isomerase (PPIase) activity. The α-helical N-terminal domain is responsible for the formation of very stable Mip homodimers. In order to determine the importance of the homodimeric state of Mip for its biochemical activities and for infectivity of Legionella, a truncated, monomeric Mip variant [Mip(77-213)] was overexpressed in Escherichia coli and characterized biochemically. In vitro isomerase activity assays revealed that the altered protein exhibits full isomerase activity towards peptide substrates. However, the deletion resulted in a dramatic loss in the efficiency of refolding of reduced and carboxy-methylated RNase T1. By cis complementation of the Mip-negative mutant strain L. pneumophila JR32-2, we constructed the strain L. pneumophila JR32-2.4, which expresses an N-terminally truncated variant of Mip. Infection studies with these strains revealed that the N-terminal part and the dimerization of Mip but not its PPIase activity are necessary for full virulence in Acanthamoeba castellanii. Infection of guinea pigs showed that strains with dimerization-deficient Mip (JR32-2.4) or a very low PPIase activity (JR32-2.2) were significantly attenuated in the animal model. These results suggest a different role of the PPIase activity and the N-terminally mediated dimeric state of Mip in monocellular systems and during the infection of guinea pigs.
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Goh, Carlmond Kah Wun, Jovi Silvester, Wan Nur Shuhaida Wan Mahadi, Lee Ping Chin, Lau Tiek Ying, Thean Chor Leow, Ryo Kurahashi, Kazufumi Takano, and Cahyo Budiman. "Expression and characterization of functional domains of FK506-binding protein 35 from Plasmodium knowlesi." Protein Engineering, Design and Selection 31, no. 12 (December 1, 2018): 489–98. http://dx.doi.org/10.1093/protein/gzz008.
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Abstract The FK506-binding protein of Plasmodium knowlesi (Pk-FKBP35) is considerably a viable antimalarial drug target, which belongs to the peptidyl-prolyl cis-trans isomerase (PPIase) protein family member. Structurally, this protein consists of an N-terminal FK506-binding domain (FKBD) and a C-terminal tetratricopeptide repeat domain (TPRD). This study aims to decipher functional properties of these domains as a platform for development of novel antimalarial drugs. Accordingly, full-length Pk-FKBP35 as well as its isolated domains, Pk-FKBD and Pk-TPRD were overexpressed, purified, and characterized. The results showed that catalytic PPIase activity was confined to the full-length Pk-FKBP35 and Pk-FKBD, suggesting that the catalytic activity is structurally regulated by the FKBD. Meanwhile, oligomerization analysis revealed that Pk-TPRD is essential for dimerization. Asp55, Arg60, Trp77 and Phe117 in the Pk-FKBD were considerably important for catalysis as underlined by significant reduction of PPIase activity upon mutations at these residues. Further, inhibition activity of Pk-FKBP35 towards calcineurin phosphatase activity revealed that the presence of FKBD is essential for the inhibitory property, while TPRD may be important for efficient binding to calcineurin. We then discussed possible roles of FKBP35 in Plasmodium cells and proposed mechanisms by which the immunosuppressive drug, FK506, interacts with the protein.
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McMillan, Lana J., Nathaniel L. Hepowit, and Julie A. Maupin-Furlow. "Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents." Applied and Environmental Microbiology 82, no. 2 (November 6, 2015): 538–48. http://dx.doi.org/10.1128/aem.03055-15.
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ABSTRACTSoluble inorganic pyrophosphatases (PPAs) that hydrolyze inorganic pyrophosphate (PPi) to orthophosphate (Pi) are commonly used to accelerate and detect biosynthetic reactions that generate PPias a by-product. Current PPAs are inactivated by high salt concentrations and organic solvents, which limits the extent of their use. Here we report a class A type PPA of the haloarchaeonHaloferax volcanii(HvPPA) that is thermostable and displays robust PPi-hydrolyzing activity under conditions of 25% (vol/vol) organic solvent and salt concentrations from 25 mM to 3 M. HvPPA was purified to homogeneity as a homohexamer by a rapid two-step method and was found to display non-Michaelis-Menten kinetics with aVmaxof 465 U · mg−1for PPihydrolysis (optimal at 42°C and pH 8.5) and Hill coefficients that indicated cooperative binding to PPiand Mg2+. Similarly to other class A type PPAs, HvPPA was inhibited by sodium fluoride; however, hierarchical clustering and three-dimensional (3D) homology modeling revealed HvPPA to be distinct in structure from characterized PPAs. In particular, HvPPA was highly negative in surface charge, which explained its extreme resistance to organic solvents. To demonstrate that HvPPA could drive thermodynamically unfavorable reactions to completion under conditions of reduced water activity, a novel coupled assay was developed; HvPPA hydrolyzed the PPiby-product generated in 2 M NaCl by UbaA (a “salt-loving” noncanonical E1 enzyme that adenylates ubiquitin-like proteins in the presence of ATP). Overall, we demonstrate HvPPA to be useful for hydrolyzing PPiunder conditions of reduced water activity that are a hurdle to current PPA-based technologies.