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PubMed1997

Scanning and escape during protein-disulfide isomerase-assisted protein folding.

During oxidative protein folding, efficient catalysis of disulfide rearrangements by protein-disulfide isomerase is found to involve an escape mechanism that prevents the enzyme from becoming trapped in covalent complexes with substrates that fail to rearrange in a timely fashion. Protein-disulfide isomerase mutants with only a single active-site cysteine catalyze slow disulfide rearrangements and become trapped in a covalent complex with substrate. Escape is mediated by the second, more carboxyl-terminal cysteine at the active site. A glutathione redox buffer increases the kcat for single-cysteine mutants by 20-40-fold, but the presence of the second cysteine at the active site in the wild-type enzyme increases the kcat by over 200-fold. A model is developed in which kinetic scanning for disulfides of increasing reactivity is timed against an intramolecular clock provided by the second cysteine at the active site. This provides an alternative, more efficient mechanism for rearrangement involving the reduction and reoxidation of substrate disulfides.

Animals↗

PubMed1997

Protein disulfide isomerase and newly synthesized procollagen chains form higher-order structures in the lumen of the endoplasmic reticulum.

A number of proteins that act as necessary catalysts for correct protein folding and oligomerization in the endoplasmic reticulum (ER) are known to be retained in the organelle via the KDEL-receptor mediated retrieval mechanism. However, a complementary system that may help to retain these proteins in the organelle lumen has been suggested to exist and likely involves physical protein-protein interactions at the level of endoplasmic reticulum (ER) itself. In this report, we provide both morphological and biochemical evidence in support of this proposal. We show that in collagen-secreting human skin fibroblasts, protein disulfide isomerase and newly synthesized procollagen chains exist predominantly in an "aggregated" state, and form a reticular-like matrix in the ER lumen in vivo. The size of the aggregates was found to be variable, and may exceed 1.5 million Da. Aggregate formation appeared to be transient and to involve multiple types of protein-protein interactions, including formation of aberrant disulfide bonds. Association of protein disulfide isomerase, on the other hand, was found to require at least partly function-related disulfide bonds. These results support the existence of a reticular-like matrix in the ER lumen, and suggest that aggregation may be part of the normal maturation pathway during collagen biosynthesis.

Antibodies, Monoclonal↗

PubMed2000

The Arabidopsis thaliana PIN1At gene encodes a single-domain phosphorylation-dependent peptidyl prolyl cis/trans isomerase.

A homologue of the human site-specific prolyl cis/trans isomerase PIN1 was identified in Arabidopsis thaliana. The PIN1At gene encodes a protein of 119 amino acids that is 53% identical with the catalytic domain of the human PIN1 parvulin. Steady-state PIN1At mRNA is found in all plant tissues tested. We show by two-dimensional NMR spectroscopy that the PIN1At is a prolyl cis/trans isomerase with specificity for phosphoserine-proline bonds. PIN1At is the first example of an eukaryotic parvulin without N- or C-terminal extensions. The N-terminal WW domain of 40 amino acids, typical of all the phosphorylation-dependent eukaryotic parvulins, is absent. However, triple-resonance NMR experiments showed that PIN1At contained a hydrophobic helix similar to the alpha1 helix observed in PIN1 that could mediate the protein-protein interactions.

Amino Acid Sequence↗

PubMed2004

PDILT, a divergent testis-specific protein disulfide isomerase with a non-classical SXXC motif that engages in disulfide-dependent interactions in the endoplasmic reticulum.

Protein disulfide isomerase (PDI) is the archetypal enzyme involved in the formation and reshuffling of disulfide bonds in the endoplasmic reticulum (ER). PDI achieves its redox function through two highly conserved thioredoxin domains, and PDI can also operate as an ER chaperone. The substrate specificities and the exact functions of most other PDI family proteins remain important unsolved questions in biology. Here, we characterize a new and striking member of the PDI family, which we have named protein disulfide isomerase-like protein of the testis (PDILT). PDILT is the first eukaryotic SXXC protein to be characterized in the ER. Our experiments have unveiled a novel, glycosylated PDI-like protein whose tissue-specific expression and unusual motifs have implications for the evolution, catalytic function, and substrate selection of thioredoxin family proteins. We show that PDILT is an ER resident glycoprotein that liaises with partner proteins in disulfide-dependent complexes within the testis. PDILT interacts with the oxidoreductase Ero1alpha, demonstrating that the N-terminal cysteine of the CXXC sequence is not required for binding of PDI family proteins to ER oxidoreductases. The expression of PDILT, in addition to PDI in the testis, suggests that PDILT performs a specialized chaperone function in testicular cells. PDILT is an unusual PDI relative that highlights the adaptability of chaperone and redox function in enzymes of the endoplasmic reticulum.

Amino Acid Motifs↗

PubMed2004

Excess mannose limits the growth of phosphomannose isomerase PMI40 deletion strain of Saccharomyces cerevisiae.

Phosphomannose isomerase (PMI40) catalyzes the conversion between fructose 6-phosphate and mannose 6-phosphate and thus connects glycolysis, i.e. energy production and GDP-mannose biosynthesis or cell wall synthesis in Saccharomyces cerevisiae. After PMI40 deletion (pmi(-)) the cells were viable only if fed with extracellular mannose and glucose. In an attempt to force the GDP-mannose synthesis in the pmi(-) strain by increasing the extracellular mannose concentrations, the cells showed significantly reduced growth rates without any alterations in the intracellular GDP-mannose levels. To reveal the mechanisms resulting in reduced growth rates, we measured genome-wide gene expression levels, several metabolite concentrations, and selected in vitro enzyme activities in central metabolic pathways. The increasing of the initial mannose concentration led to an increase in the mannose 6-phosphate concentration, which inhibited the activity of the second enzyme in glycolysis, i.e. phosphoglucose isomerase converting glucose 6-phosphate to fructose 6-phosphate. As a result of this limitation, the flux through glycolysis was decreased as was the median expression of the genes involved in glycolysis. The expression levels of RAP1, a transcription factor involved in the regulation of the mRNA levels of several enzymes in glycolysis, as well as those of cell cycle regulators CDC28 and CLN3, decreased concomitantly with the growth rates and expression of many genes encoding for enzymes in glycolysis.

Allosteric Site↗

PubMed2005

Domain swapping between Enterococcus faecalis FabN and FabZ proteins localizes the structural determinants for isomerase activity.

Anaerobic unsaturated fatty acid synthesis in bacteria occurs through the introduction of a double bond into the growing acyl chain. In the Escherichia coli model system, FabA catalyzes both the dehydration of beta-hydroxydecanoyl-ACP and the isomerization of trans-2-decenoyl-ACP to cis-3-decenoyl-ACP as the essential step. A second dehydratase, FabZ, functions in acyl chain elongation but cannot carry out the isomerization reaction. Enterococcus faecalis has two highly related FabZ homologs. One of these, termed EfFabN, carries out the isomerization reaction in vivo, whereas the other, EfFabZ, does not (Wang, H., and Cronan, J. E. (2004) J. Biol. Chem. 279, 34489-34495). We carried out a series of domain swapping and mutagenesis experiments coupled with in vitro biochemical analyses to define the structural feature(s) that specify the catalytic properties of these two enzymes. Substitution of the beta3 and beta4 strands of EfFabZ with the corresponding strands from EfFabN was necessary and sufficient to convert EfFabZ into an isomerase. These data are consistent with the hypothesis that the isomerase potential of beta-hydroxyacyl-ACP dehydratases is determined by the properties of the beta-sheets that dictate the orientation of the central alpha-helix and thus the shape of the substrate binding tunnel rather than the catalytic machinery at the active site.

Amino Acid Sequence↗

PubMed2006

Regulation of Bruton tyrosine kinase by the peptidylprolyl isomerase Pin1.

Bruton tyrosine kinase (Btk) is expressed in B-lymphocytes. Mutations in Btk cause X-linked agammaglobulinemia in humans. However, the mechanism of activation and signaling of this enzyme has not been fully investigated. We have here shown that the peptidylprolyl cis/trans isomerase (PPIase) Pin1 is a negative regulator of Btk, controlling its expression level by reducing its half-life, whereas the catalytic activity of Btk was unaffected. The negative regulatory effect of Pin1 was observed both in cell lines and in Pin(-/-) mice and was found to be dependent on a functionally intact Btk. This may constitute a feedback loop for the regulation of Btk. The target region in Btk was localized to the pleckstrin homology domain suggesting that interphase phosphorylation of serine 115 (Ser-115) in Btk is required, whereas mitosis phosphorylation of serine 21 (Ser-21) is critical. Accordingly, Pin 1 was shown to associate with Btk through binding to Ser-21 and -115, respectively, both of which lie in a classical Pin1-binding pocket. Using a phosphomitotic antibody, it was found that Btk harbors a bona fide MPM2 epitope corresponding to a phosphorylated serine or threonine residue followed by a proline. Our results indicate that the peptidylprolyl isomerase Pin1 interacts with Btk in a cell cycle-dependent manner, regulating the Btk expression level.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1994

Yeast NPI46 encodes a novel prolyl cis-trans isomerase that is located in the nucleolus.

We have identified a gene (NPI46) encoding a new prolyl cis-trans isomerase within the nucleolus of the yeast Saccharomyces cerevisiae. The protein encoded by NPI46 was originally found by us in a search for proteins that recognize nuclear localization sequences (NLSs) in vitro. Thus, NPI46 binds to affinity columns that contain a wild-type histone H2B NLS but not a mutant H2B NLS that is incompetent for nuclear localization in vivo. NPI46 has two domains, a highly charged NH2 terminus similar to two other mammalian nucleolar proteins, nucleolin and Nopp140, and a COOH terminus with 45% homology to a family of mammalian and yeast proline isomerases. NPI46 is capable of catalyzing the prolyl cis-trans isomerization of two small synthetic peptides, succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, as measured by a chymotrypsin-coupled spectrophotometric assay. By indirect immunofluorescence we have shown that NPI46 is a nucleolar protein. NPI46 is not essential for cell viability.

Amino Acid Isomerases↗

PubMed1997

Cyclophilin and protein disulfide isomerase genes are co-transcribed in a functionally related manner in Caenorhabditis elegans.

The ubiquitous enzymes peptidyl prolyl cis-trans isomerase (PPI, EC 5.2.1.8) and protein disulfide isomerase (PDI, EC 5.3.4.1) are important rate-limiting catalysts of protein-folding events in the cell. In the free-living nematode Caenorhabditis elegans, two genes encoding these enzymes (cyp-9 and pdi-1, respectively) are clustered together on chromosome III. In work described elsewhere, the encoded enzymes have been expressed as recombinant proteins and have been determined to possess in vitro PPI and PDI activity. Taken together, this organization of the two genes and the related functions of their transcripts indicate that they may be cotranscribed as a polycistronic unit, similar to bacterial operons. This study details the very close linkage of pdi-1 and cyp-9, which are in the same orientation. pdi-1 is the upstream gene, and the putative polyadenylation cleavage signal of this gene is separated from the trans-splice acceptor site of cyp-9 by only 103 bp. pdi-1 is trans-spliced by the ubiquitous nematode trans-spliced leader SL1, whereas cyp-9 was found to be predominantly trans-spliced by the "operon-specific" trans-spliced leader SL2. Similar trends in relative transcript abundance were demonstrated with synchronously produced mRNA for both genes during larval development, supporting the contention that the genes are co-expressed. Finally, reporter gene analysis provides strong evidence that both genes are controlled by a single upstream regulatory element, which directs expression of both enzymes in the hypodermal cells that synthesize the cuticle.

Alternative Splicing↗

PubMed1993

Protein disulfide isomerase is both an enzyme and a chaperone.

Protein disulfide isomerase (PDI) catalyzes the formation of native disulfides of peptide chains from either the reduced form or randomly joined disulfides. So that thiols situated at distant parts of the polypeptide chain can be joined together to form the native disulfides, the polypeptide chain has to be folded, at least to some extent, into the native conformation. It is suggested that PDI promotes folding of the chains as well as formation of the disulfides and plays a role similar to the chaperones in the folding process. PDI is known to be a multifunctional protein and capable of nonspecific peptide binding. These properties are closely connected to its possible function as a chaperone. Thioredoxin, which has an active site sequence similar to that of PDI but lacks the property of peptide binding, is much less efficient as a disulfide isomerase.

Biological Evolution↗

PubMed2000

Crystallization and X-ray diffraction analysis of peroxisomal Delta3-Delta2-enoyl-CoA isomerase from Saccharomyces cerevisiae.

The purification, crystallization and X-ray diffraction analysis of Saccharomyces cerevisiae Delta(3)-Delta(2)-enoyl-CoA isomerase is described. Delta(3)-Delta(2)-Enoyl-CoA isomerase is a member of the hydratase/isomerase protein family and is an auxiliary enzyme required for the beta-oxidation of unsaturated fatty acids. It is a hexameric enzyme consisting of six identical 32 kDa subunits of 280 residues each. In crystallization trials three crystal forms were obtained, with tetragonal and hexagonal lattices. A 2.5 A data set was collected from the unliganded hexagonal crystals with an R(merge) of 6.6%. The crystal, with unit-cell parameters a = 116.0, b = 116.0, c = 122.9 A, is likely to have P6(3)22 symmetry.

Carbon-Carbon Double Bond Isomerases↗

PubMed1989

Purification and characterization of 55-kDa protein with 3,5,3'-triiodo-L-thyronine-binding activity and protein disulfide-isomerase activity from beef liver membrane.

Beef liver membranes were shown to have different kinds of 3,5,3'-triiodo-L-thyronine binding proteins including the 55-kDa protein which had been reported to have this activity in many cells by affinity labelling with N-bromoacetyl-3,5,3'-[125I]triiodo-L-thyronine. In order to characterize the molecular features of these binding proteins, the 55-kDa protein was purified from a beef liver membrane fraction abundant in the plasma membrane. The protein was solubilized with 0.5% Chaps and purified by chromatography on gel filtration, hydroxyapatite, and Mono Q anion-exchange columns. The purity was confirmed with reversed-phase HPLC and SDS/PAGE. Consequently, 0.4% of the total proteins in the membrane fraction was recovered as the 55-kDa protein. One fourth of the amino acid composition of this protein was Glx (14.6%) plus Asx (11.7%) and the pI of this protein was 4.5. The purified protein has triiodothyronine-binding activity with a Kd of 57 nM which is similar to the high-affinity binding site of the membranes. The anti-(55-kDa protein) sera specifically recognized the 55-kDa protein of beef, rat and human cells. The immunoglobulin G fraction of the anti-(55-kDa protein) sera inhibited triiodothyronine binding to the beef liver membrane fraction. The purified protein also showed the activity of protein disulfide-isomerase (EC 5.3.4.1) as determined by reactivating scrambled ribonuclease. These data strongly suggested that the multi-functional 55-kDa protein which has triiodothyronine-binding activity and the activity of protein disulfide-isomerase, which is also reported to be the beta subunit of prolyl-4-hydroxylase, glycosylation-site-binding protein of oligosaccharyl transferase and iodothyronine 5'-monodeionidase, could be significant in the action of triiodothyronine towards the target cells.

Amino Acids↗

PubMed1993

Purification and properties of an iron-sulfur and FAD-containing 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA delta 3-delta 2-isomerase from Clostridium aminobutyricum.

4-Hydroxybutyryl-CoA dehydratase, the key enzyme in the metabolism of gamma-aminobutyrate in Clostridium aminobutyricum, represents approximately 15-25% of the soluble protein. The enzyme was purified to homogeneity under anaerobic conditions to a specific activity of 209 nkat mg-1. The dehydratase catalyses the reversible conversion of 4-hydroxybutyryl-CoA (Km = 50 microM) to crotonyl-CoA and possesses a probably intrinsic vinylacetyl-CoA delta 3-delta 2-isomerase with a specific activity of 223 nkat mg-1. The equilibrium of the reversible dehydration was determined from both sides as K = [crotonyl-CoA]/[4-hydroxybutyryl-CoA] = 4.2 +/- 0.3. Cyclopropylcarboxyl-CoA was not converted to crotonyl-CoA. The native enzyme has an apparent molecular mass of 232 kDa and is composed of four apparently identical subunits (molecular mass = 56 kDa), indicating a homotetrameric structure. Under anaerobic conditions the active enzyme revealed a brown colour and contained 2 +/- 0.2 mol FAD (64 +/- 5% oxidized), 16 +/- 0.8 mol Fe and 14.4 +/- 1.2 mol inorganic sulfur, which probably form iron-sulfur clusters. Exposure to air resulted initially in a slight activation followed by irreversible inactivation. Concomitantly the vinylacetyl-CoA delta-isomerase activity was lost and the colour of the enzyme changed to yellow. Reduction by sodium dithionite yielded inactive enzyme which could be completely reactivated by oxidation with potassium hexacyanoferrate(III). The data indicate that the active enzyme contains oxidized FAD despite its sensitivity towards oxygen. During the dehydration a non activated C-H bond at C-3 of 4-hydroxybutyryl-CoA has to be cleaved. A putative mechanism for 4-hydroxybutyryl-CoA dehydratase is proposed in which this cleavage is achieved by a FAD-dependent oxidation of 4-hydroxybutyryl-CoA to 4-hydroxycrotonyl-CoA. In a second step the hydroxyl group is substituted by a hydride derived from the now reduced FAD in an SN2' reaction leading to vinylacetyl-CoA. Finally isomerisation yields crotonyl-CoA. 4-Hydroxybutyryl-CoA dehydratase is quite distinct from 3-hydroxyacyl-CoA dehydratase (crotonase) and 2-hydroxyacyl-CoA dehydratases. Contrary to the latter enzyme [e.g. (R)-lactyl-CoA dehydratase and (R)-2-hydroxyglutaryl-CoA dehydratase] which are composed of three different subunits and similarly catalyse the cleavage of a non activated C-H bond at C-3, 4-hydroxybutyryl-CoA dehydratase does not require ATP, MgCl2 and Ti(III)citrate for activity. Furthermore 4-hydroxybutyryl-CoA dehydratase is not inactivated by oxidants such as 5 mM 4-nitrophenol, 5 mM chloramphenicol and 5 mM hydroxylamine.

Acyl Coenzyme A↗

PubMed1995

Chaperone-like activity of protein disulfide-isomerase in the refolding of rhodanese.

Protein disulfide-isomerase (PDI) in near stoichiometric concentrations promotes reactivation and prevents aggregation of guanidine-hydrochloride-denatured rhodanese during refolding upon dilution. PDI also suppresses aggregation of rhodanese during thermal inactivation. The above-mentioned properties displayed by PDI completely satisfy the definition of chaperone and provide additional evidence to confirm the hypothesis proposed previously [Wang, C. C. & Tsou, C. L. (1993) FASEB J. 7, 1515-1517] that PDI is both an enzyme and a chaperone. Since rhodanese contains no disulfide bonds, the chaperone-like activity of PDI acting on rhodanese is independent of its disulfide-isomerase activity.

Animals↗

PubMed1996

Molecular characterisation of plant endoplasmic reticulum. Identification of protein disulfide-isomerase as the major reticuloplasmin.

Purified endoplasmic reticulum devoid of contaminating endomembranes has been isolated from both germinating and developing castor bean endosperm by a modified two-step centrifugation procedure. These membranes have been characterised for protein and lipid composition, subfractionated into lumenal and integral membrane protein fractions, and antisera raised to these two components. A cDNA clone encoding a major lumenal protein of 55 kDa was cloned using affinity-purified antisera and shown to encode a protein with strong sequence similarity to the endoplasmic reticulum lumenal chaperone protein disulfide-isomerase. Northern and Southern blot analysis showed that the mRNA from a single-copy gene was constitutively expressed in all tissues investigated, but was preferentially expressed in developing seed where it was the most abundant lumenal protein. Expression of the recombinant protein in Escherichia coli yielded a homodimer with a molecular mass of 110 kDa with protein disulfide-isomerase catalytic activity, thus confirming identity of this protein.

Amino Acid Sequence↗

PubMed1997

Interactions between protein disulphide isomerase and peptides.

There is growing evidence that protein disulphide isomerase (PDI) has a common chaperone function in the endoplasmic reticulum. To characterise this function, we investigated the interaction of purified PDI with radiolabelled model peptides, somatostatin and mastoparan, by cross-linking. The interaction between the peptides and PDI was specific, for it showed saturation and was abolished by denaturation of PDI. The interaction between a hydrophobic peptide without cysteine residues was much more sensitive to Triton X-100 than the interaction between PDI and a more hydrophilic peptide with or without cysteine residues. We therefore propose that hydrophobic interactions between protein disulphide isomerase and peptides play an important role in the binding process. The interaction between PDI and the bound peptide therefore is enhanced by the formation of mixed disulphide bonds.

Animals↗

PubMed2000

Requirement of the prolyl isomerase Pin1 for the replication checkpoint.

The peptidyl-prolyl isomerase Pin1 has been implicated in regulating cell cycle progression. Pin1 was found to be required for the DNA replication checkpoint in Xenopus laevis. Egg extracts depleted of Pin1 inappropriately transited from the G2 to the M phase of the cell cycle in the presence of the DNA replication inhibitor aphidicolin. This defect in replication checkpoint function was reversed after the addition of recombinant wild-type Pin1, but not an isomerase-inactive mutant, to the depleted extract. Premature mitotic entry in the absence of Pin1 was accompanied by hyperphosphorylation of Cdc25, activation of Cdc2/cyclin B, and generation of epitopes recognized by the mitotic phosphoprotein antibody, MPM-2. Therefore, Pin1 appears to be required for the checkpoint delaying the onset of mitosis in response to incomplete replication.

Animals↗

PubMed1987

The visual cycle operates via an isomerase acting on all-trans retinol in the pigment epithelium.

Thirty years have elapsed since Wald and his colleagues showed that 11-cis retinal was isomerized to all-trans when rhodopsin was bleached, yet little has been understood about the reverse process that generates 11-cis retinal for rhodopsin regeneration. It is not known whether the isomerization is enzyme-mediated, whether it occurs in the pigment epithelium or in the retina, or whether retinal, retinol, or a retinyl ester is the vitamin A compound that is isomerized. Radiolabeled all-trans retinol and high-performance liquid chromatography have now been used to demonstrate the existence of an eye-specific, membrane-bound enzyme (retinol isomerase) that converts all-trans to 11-cis retinol in the dark. Retinol isomerase is concentrated in the pigment epithelium; this localization clarifies the role of this tissue in rhodopsin regeneration and explains the need to transfer all-trans retinol from the rod outer segments to the pigment epithelium during the visual cycle.

Animals↗