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At least 1,081 records · Page 60Linked to original sources
PubMed1994

Association of protein disulfide isomerase activity and the induction of contact inhibition.

Interferon-beta (IFN-beta) used as a biological response modifier can cause a wide array of effects, including the restoration of contact inhibition in HT1080 fibrosarcoma cells normally lacking this control. Sequence analysis and characterization of a cDNA whose expression has been shown to be transiently induced by IFN-beta now identifies this cDNA as corresponding to the gene for the multifunctional polypeptide protein disulfide isomerase (PDI). Cell proliferation studies using bacitracin as an inhibitor of PDI demonstrated that contact inhibition could be suppressed in human fibroblasts which normally exhibit this control--allowing attainable cell saturation densities to be significantly greater than those of control cells. PDI enzyme activity assays indicated that 1 mM bacitracin inhibited PDI intracellular activity up to 60%. Moreover, evaluations of PDI levels in normal fibroblasts of increasing densities suggested that enzyme levels correlate with density-dependent growth control, showing a transient increase in intracellular activity as cells approach confluency. Together these results suggest that induction of PDI activity is not only involved in the antiproliferative actions of IFN-beta, but it is critical to the regulation of contact inhibition growth control in normal fibroblasts.

Bacitracin↗
PubMed1994

A human promyelocyte mRNA transiently induced by TPA is homologous to yeast IPP isomerase.

A cDNA clone was isolated from HL60 human promyelocyte cells on the basis of the induction of its cognate mRNA by the phorbol ester TPA (12-O-tetradecanoyl phorbol-13-acetate) in the presence of cycloheximide. Northern analysis showed a peak of induction of a 1.9-kb mRNA at 3-6 h after TPA addition. Sequence analysis revealed that this cDNA was likely a human homolog of yeast isopentenyl diphosphate:dimethylallyl diphosphate isomerase, an enzyme important for isoprenoid synthesis and the prenylation of proteins such as ras p21.

Amino Acid Sequence↗
PubMed1997

Structures of the human gene for the protein disulfide isomerase-related polypeptide ERp60 and a processed gene and assignment of these genes to 15q15 and 1q21.

ERp60 (also known as ERp61 or GRP58) is an isoform of protein disulfide isomerase (PDI) that has two thioredoxin-like domains a and a' in positions corresponding to those of domains a and a' in the PDI polypeptide and shows a significant amino acid sequence similarity to PDI in almost all parts. We report here that the human ERp60 gene is about 18 kb in size and consists of 13 exons. No distinct correlation was found between its exon-intron organization and the modular structure of the ERp60 polypeptide, nor were any similarities in exon-intron organization found between the human ERp60, PDI, and thioredoxin genes. The 5' flanking region of the ERp60 gene has no TATAA box or CCAAT motif but contains several potential binding sites for transcription factors. The highest levels of expression of the ERp60 mRNA were found by Northern blotting in the liver, placenta, lung, pancreas, and kidney, and the lowest in the heart, skeletal muscle, and brain. We also isolated an intronless ERp60 gene that probably represents a pseudogene. The ERp60 gene was mapped by fluorescence in situ hybridization to 15q15 and the processed gene to 1q21, so that neither was located on the same chromosome as the human PDI and thioredoxin genes.

Amino Acid Sequence↗
PubMed1997

Crystal structure of cytoplasmic Escherichia coli peptidyl-prolyl isomerase: evidence for decreased mobility of loops upon complexation.

The structure of the unliganded form of the Escherichia coli cytoplasmic peptidyl-prolyl isomerase (ppiB gene product) in a new crystal form was determined by the molecular replacement method and refined to an R-factor of 16.1% at 2.1 A resolution. The enzyme crystallized in the orthorhombic C2221 space group with unit cell dimensions of a=44.7 A, b=68.2 A and c=102.0 A. Comparison with the reported structure of the enzyme complexed with the tripeptide substrate succinyl-Ala-Pro-Ala-p-nitroanilide revealed subtle changes that occur upon complex formation. There is evidence to suggest that two surface loops have significantly reduced mobility in the complexed structure.

Amino Acid Isomerases↗
PubMed2000

NMR solution structure of hPar14 reveals similarity to the peptidyl prolyl cis/trans isomerase domain of the mitotic regulator hPin1 but indicates a different functionality of the protein.

The 131-amino acid residue parvulin-like human peptidyl-prolyl cis/trans isomerase (PPIase) hPar14 was shown to exhibit sequence similarity to the regulator enzyme for cell cycle transitions human hPin1, but specificity for catalyzing pSer(Thr)-Pro cis/trans isomerizations was lacking. To determine the solution structure of hPar14 the (1)H, (13)C, and (15)N chemical shifts of this protein have been assigned using heteronuclear two and three-dimensional NMR experiments on unlabeled and uniformly (15)N/(13)C-labeled recombinant protein isolated from Escherichia coli cells that overexpress the protein. The chemical shift assignments were used to interpret the NOE data, which resulted in a total of 1042 NOE restraints. The NOE restraints were used along with 71 dihedral angle restraints and 38 hydrogen bonding restraints to produce 50 low-energy structures. The hPar14 folds into a betaalpha(3)betaalphabeta(2) structure, and contains an unstructured 35-amino acid basic tail N-terminal to the catalytic core that replaces the WW domain of hPin1 homologs. The three-dimensional structures of hPar14 and the PPIase domain of human hPin1 reveal a high degree of conservation. The root-mean-square deviations of the mean atomic coordinates of the heavy atoms of the backbone between residues 38 to 45, 50 to 58, 64 to 70, 81 to 86, 115 to 119 and 122 to 128 of hPar14 were 0.81(+/-0.07) A. The hPar14 model structure provides insight into how this class of PPIases may select preferential secondary catalytic sites, and also allows identification of a putative DNA-binding motif in parvulin-like PPIases.

Amino Acid Sequence↗
PubMed2001

The peptidyl-prolyl isomerase Pin1 interacts with hSpt5 phosphorylated by Cdk9.

We identify and characterize several phosphorylated forms of the hSpt5 subunit of the DRB sensitivity-inducing factor (DSIF). A 175-kDa phosphorylated form of hSpt5 is bound to nuclei of interphase HeLa cells. This form is rapidly dephosphorylated when cultured cells are exposed to various drugs belonging to distinct chemical families. All these compounds are known to inhibit the protein kinase Cdk9, which phosphorylates in vitro hSpt5 and Rpb1, the largest subunit of RNA polymerase II. The efficiency to promote the dephosphorylation of both proteins matches their capacity to inhibit purified Cdk9 kinase, suggesting that Cdk9 is the major kinase phosphorylating hSpt5 and Rpb1 in vivo. We show that Cdk9 phosphorylates both the CTR1 and the CTR2 domains of recombinant hSpt5. These domains contain numerous serine-proline and threonine-proline residues similar to those found in the carboxyl-terminal domain (CTD) of Rpb1. The structural homology between hSpt5 CTRs and the Rpb1 CTD is further highlighted by the presence on both proteins of a phosphoepitope recognized by the monoclonal antibody CC-3. Of particular interest, the peptidyl-prolyl isomerase Pin1 interacts with Cdk9-phosphorylated hSpt5. Cdk9 dependent phosphorylation of Rpb1 and hSpt5 followed by Pin1 interaction might thus contribute to the regulation of transcription, pre-mRNA maturation, and the dynamics of these proteins in interphase and mitosis.

Antibodies, Monoclonal↗
PubMed1994

Catalysis of protein folding by agarose-immobilized protein disulfide isomerase.

Protein disulfide isomerase (PDI) catalyzes the formation and rearrangement of disulfide bonds during protein folding. PDI coupled to cyanogen bromide-activated agarose retains its catalytic activity, and a column of this material increases both the rate and the yield for folding disulfide-containing proteins. For reduced, denatured ribonuclease, the overall yield of fully active ribonuclease isolated from the PDI column in one pass was 85-98% of the applied protein. Under the same conditions in the absence of PDI, ribonuclease regained only 16% of its native activity. The oxidative folding of reduced denatured lysozyme is complicated by aggregation so that in the absence of PDI optimal yields of only < or = 25% are obtained at lysozyme concentrations of 1.6 mg/ml. When reduced, denatured lysozyme (1.6 mg/ml) is passed over a PDI column in 1-2 M urea in the presence of a glutathione redox buffer, the specific activity of the recovered lysozyme is identical to that of the native enzyme and the total recovery of the applied protein is 50-65%.

Animals↗
PubMed1994

A single purification procedure for the major resident proteins of the ER lumen: endoplasmin, BiP, calreticulin and protein disulfide isomerase.

We have developed a single purification procedure for the four major resident endoplasmic reticulum (ER) proteins: protein disulfide isomerase (PDI), BiP, endoplasmin, and calreticulin. Three of these proteins are thought to play a role in protein folding in vivo, whereas calreticulin is thought to be the major calcium binding protein in the ER. The proteins were purified from fresh bovine liver by taking advantage of individual characteristics of the proteins. Liver microsomes were prepared and then premeabilized to release the lumenal contents. After ammonium sulfate precipitation, the proteins were purified by chromatography; BiP was purified by affinity chromatography on ATP-agarose, and both endoplasmin and calreticulin were purified by affinity chromatography on Con A-Sepharose. PDI was purified by anionic ion exchange chromatography.

Amino Acid Sequence↗
PubMed1995

Production of rat protein disulfide isomerase in Saccharomyces cerevisiae.

Protein disulfide isomerase (PDI) is an abundant protein of the endoplasmic reticulum that catalyzes the oxidation of protein sulfhydryl groups and the isomerization and reduction of protein disulfide bonds. Saccharomyces cerevisiae cells lacking PDI are inviable. PDI is a component of many different protein processing complexes, and the actual activity of PDI that is required for cell viability is unclear. A cDNA that codes for rat PDI fused to the alpha-factor pre-pro segment was expressed in a protease-deficient strain of S. cerevisiae under the control of an ADH2-GAPDH hybrid promoter. The cells processed the resulting protein and secreted it into the medium as a monomer, despite having a KDEL or HDEL sequence at its C-terminus. The typical yield of isolated protein was 2 mg per liter of culture. The catalytic activity of the PDI from S. cerevisiae was indistinguishable from that of PDI isolated from bovine liver. This expression system is unique in allowing the same plasmid to be used both to complement pdi1 delta S. cerevisiae and to produce PDI for detailed in vitro analyses. Correlations of the in vivo behavior and in vitro properties of PDI are likely to reveal structure-function relationships of biological importance.

Alcohol Dehydrogenase↗
PubMed1996

A novel plant peptidyl-prolyl-cis-trans-isomerase (PPIase): cDNA cloning, structural analysis, enzymatic activity and expression.

A novel cDNA encoding for a peptidyl-prolyl-cis-trans-isomerase (PPIase) belonging to the FK506-binding protein (FKBP) family was isolated from wheat. It contains an open reading frame of 559 amino acids and it represents the first plant FKBP-PPIase to be cloned. It possesses a unique sequence which is composed of three FKPB-like domains, in addition to a putative tetratricopeptide repeat (TPR) motif and a calmodulin-binding site. The recombinant FKBP-PPIase expressed in and purified from Escherichia coli exhibits PPIase activity that is efficiently inhibited by the immunosuppressive drugs FK506 and rapamycin. Northern blot analysis showed that wheat FKBP was found mainly in young tissues. Polyclonal antibodies revealed the presence of cross-reacting proteins in embryos, roots and shoots. The unique structural features, the enzymatic activity and the presence of putative isoforms in wheat tissues indicate the possibility of the involvement of wheat PPIase in essential biological functions, similar to other members of the FKBP gene family.

Amino Acid Isomerases↗
PubMed1996

Expression of protein disulfide isomerase is elevated in the endosperm of the maize floury-2 mutant.

A maize protein disulfide isomerase (PDI, EC 5.3.4.1) cDNA clone was isolated and characterized. The deduced amino acid sequence contains two regions characteristic of the active sites for PDI and a carboxyl-terminal endoplasmic reticulum (ER) retention sequence, Lys-Asp-Glu-Leu. Southern blot analysis indicated the maize PDI is encoded by a single gene that maps to the short arm of chromosome 4. When isolated from the cisternal and protein body ER, the PDI protein resolves into a fast and a slow form on SDS-PAGE. During endosperm development, the PDI RNA level increases between 10 and 14 days after pollination. In floury-2 (fl2) endosperm, which contains an abnormally processed alpha-zein protein, PDI expression is significantly increased, and the level of PDI protein and RNA is positively correlated with the dosage of fl2 alleles. The increase of PDI in fl2 occurs mainly in the cisternal ER fraction, whereas the most dramatic increase of binding protein (BiP) is in the protein body ER. We propose that the induction of PDI in the fl2 mutant reflects its role as a molecular chaperone, and that PDI functions in concert with BiP at different stages of zein processing and assembly into protein bodies.

Amino Acid Sequence↗
PubMed1994

Succinate-ethanol fermentation in Clostridium kluyveri: purification and characterisation of 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA delta 3-delta 2-isomerase.

Anaerobically prepared cell extracts of Clostridium kluyveri grown on succinate plus ethanol contained high amounts of 4-hydroxybutyryl-CoA dehydratase, which catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA. The enzyme was purified 12-fold under strictly anaerobic conditions to over 95% homogeneity and had a specific activity of 123 nkat mg-1. The finding of this dehydratase means that all of the enzymes necessary for fermentation of succinate plus ethanol by C. kluyveri have now been demonstrated to exist in this organism and confirms the proposed pathway involving a reduction of succinate via 4-hydroxybutyrate to butyrate. Interestingly, the enzyme is almost identical to the previously isolated 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum. The dehydratase was revealed as being a homotetramer (m = 59 kDa/subunit), containing 2 +/- 0.2 mol FAD, 13.6 +/- 0.8 mol Fe and 10.8 +/- 1.2 mol inorganic sulfur. The enzyme was irreversibly inactivated after exposure to air. Reduction by sodium dithionite also yielded an inactive enzyme which could be reactivated, however, up to 84% by oxidation with potassium hexacyanoferrate(III). The enzyme possesses an intrinsic vinylacetyl-CoA isomerase activity which was also found in 4-hydroxybutyryl-CoA dehydratase from C. aminobutyricum. Moreover, the N-terminal sequences of the dehydratases from both organisms were found to be 63% identical.

Amino Acid Sequence↗
PubMed1993

Predominant expression of the beta subunit of prolyl 4-hydroxylase (disulfide isomerase) in human extravillous trophoblasts.

Prolyl 4-hydroxylase is a heterodimeric enzyme that is crucial in the biosynthesis of collagen. The beta subunit of this enzyme is a multifunctional protein which is also known as protein-disulfide isomerase. Immunofluorescence and monoclonal antibody (Mab) 5B5 were used to localize the beta subunit in human extraembryonic tissues. The strongest sites of 5B5 reactivity were extravillous cytotrophoblasts in the basal plate, uteroplacental arteries and amniochorion, syncytiotrophoblast displayed variable weaker reactivity. Only a small fraction of placental 5B5 antigen was detected as a component of prolyl-4-hydroxylase by affinity chromatography on immobilized polyproline. The results indicate a difference in the expression of an endoplasmic reticulum marker between villous and extravillous trophoblast. The predominance of 5B5 antigen in extravillous trophoblast could be associated with an increased ability to synthesize collagen or other enzymatic reactions associated with prolyl 4-hydroxylase beta subunit.

Antibody Specificity↗
PubMed1984

Catalysis by protein disulphide-isomerase of the assembly of trimeric procollagen from procollagen polypeptide chains.

Type-I procollagen, 14C-biosynthetically labelled, was reduced under denaturing and non-denaturing conditions. Reoxidation to disulphide-linked trimers occurred with non-denatured chains in the presence of an oxidant system containing oxidized and reduced glutathione. Dimeric intermediates were not detected. This reoxidation was accelerated by homogeneous beef liver protein disulphide-isomerase.

Animals↗
PubMed1979

Identification of ergosta-8,24(28)-dien-3 beta,6 alpha-diol in A delta 8 goes to delta 7 sterol isomerase-blocked yeast mutant.

In addition to the monohydroxysterols found in the delta 8 goes to delta 7 isomerase-blocked Saccharomyces cerevisiae mutant erg 2, a noval dihydroxysterol, ergosta-8,24(38)-dien-3 beta,6 alpha-diol, was isolated. This sterol accumulated to the extent of 2.1% of the total sterol fraction when this mutant was treated with 23-azacholesterol, a known inhibitor of the 24-methylene-sterol-24(28)-reductase.

Chromatography, Gas↗
PubMedDate not supplied

A rapid measurement of delta 5-3 beta-hydroxysteroid dehydrogenase activity coupled with delta 4-delta 5 isomerase by a high pressure liquid chromatography.

By a high pressure liquid chromatography, progesterone (lambda max = 240 nm, epsilon 240 = 17,000) was separable from the other delta 4-3-oxosteroids. Relationship between the amount of progesterone and the area of the peak corresponding to progesterone was established as linear at least between 0-2.0 g. Progesterone is formed from pregnenolone by delta 5-3 beta-hydroxysteroid dehydrogenase coupled with delta r-delta 5 isomerase. Because pregnenolone is devoid of absorption oat 240 nm, the enzyme activity was measurable by the high pressure liquid chromatography from the amount of progesterone produced from pregnenolone in vitro in the presence of nicotinamide adenine dinucleotide. As an example of its application, the dehydrogenase activities in the ovaries of the immature rats treated with gonadotropins were measured by the above method. The estimates obtained by this method were agreeable with those by thin layer chromatography, employing radioactive pregnenolone as the substrate.

3-Hydroxysteroid Dehydrogenases↗
PubMed2001

Genetic interactions between the ESS1 prolyl-isomerase and the RSP5 ubiquitin ligase reveal opposing effects on RNA polymerase II function.

Transcription of protein-coding genes by RNA polymerase II (pol II) is a highly coordinated process that requires the stepwise association of distinct protein complexes with the C-terminal domain (CTD) of Rpbl, the largest subunit of RNA pol II. Interaction of these complexes with the CTD might be subject to regulation by proteins such as Ess1 and Rsp5. Ess1, a prolyl-isomerase, binds the CTD and is thought to play a positive role in pol II transcription by generating conformational isomers of the CTD. Rsp5, a ubiquitin ligase, binds the CTD and is thought to play a negative role in transcription by mediating Rpbl ubiquitination and degradation. In this paper, we demonstrate that ESS1 and RSP5 interact genetically and that these interactions occur via RPBI. We show that over-expression of RSP5 enhances the growth defect of ess1ts cells and this effect is reversed by introducing extra copies of RPB1. Over-expression of RSP5 also mimics the sensitivity of ess1ts mutant cells to the toxicity of plasmids carrying dominant-negative CTD mutations, whereas mutations in RSP5 suppress this effect. Using a modified two-hybrid assay, we also demonstrate that Essl and Rsp5 compete directly for binding to the CTD. The results suggest a model in which Essl and Rsp5 act opposingly on pol II function to control the level of pol II available for transcription.

Binding, Competitive↗
PubMed1997

Isolation and characterisation of a gene encoding protein disulphide isomerase, pdiA, from Aspergillus niger.

Current strategies to improve the secretion of heterologous proteins from Aspergillus niger include the manipulation of chaperones and foldases specific to the endoplasmic reticulum (ER). Here we report the isolation of a gene, pdiA, encoding a putative protein disulphide isomerase (PDI) from A. niger using the Saccharomyces cerevisiae PDI gene as a probe. Sequencing of a genomic clone and RT-PCR products predict a 515-aa protein comprising a 20-aa ER-translocation signal sequence and a 495-aa mature protein (Mr = 54.3 kDa). The predicted protein also contains two thiol oxidoreductase active sites with a -CGHC- motif and a carboxy terminal -HDEL ER-retention signal. Three introns were identified within the pdiA gene and Southern- and dot-blot analysis indicates that the gene is present in a single copy. Northern-blot analysis shows a transcript of the predicted size. Sequence homology to a motif associated with protein trafficking and the induction of chaperones has been identified in the pdiA promoter. Transcription of pdiA is induced 3-4-fold after treatment with tunicamycin, an inhibitor of N-linked glycosylation. The kinetics of induction suggest that pdiA expression is not part of the primary stress response.

Amino Acid Sequence↗