Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.17 (lysozyme)
 21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protein disulfide isomerase (PDI) is important in assisting the folding and maturation of secretory proteins in eukaryotes. A gene, pdiA, encoding PDIA was previously isolated from Aspergillus niger, and we report its functional characterization here. Functional analysis of PDIA showed that it catalyzes the refolding of denatured and reduced RNase A. pdiA also complemented PDI function in a Saccharomyces cerevisiae Deltapdi1 mutant in a yeast-based killer toxin assay. Levels of pdiA mRNA and PDIA protein were raised by the accumulation of unfolded proteins in the endoplasmic reticulum. This response of pdiA mRNA levels was slower and lower in magnitude than that of A. niger bipA, suggesting that the induction of pdiA is not part of the primary stress response. An increased level of pdiA transcripts was also observed in two A. niger strains overproducing a heterologous protein, hen egg white lysozyme (HEWL). Although overexpression of PDI has been successful in increasing yields of some heterologous proteins in S. cerevisiae, overexpression of PDIA did not increase secreted yields of HEWL in A. niger, suggesting that PDIA itself is not limiting for secretion of this protein. Downregulation of pdiA by antisense mRNA reduced the levels of microsomal PDIA activity by up to 50%, lowered the level of PDIA as judged by Western blots, and lowered the secreted levels of glucoamylase by 60 to 70%.
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PMID:Characterization of a foldase, protein disulfide isomerase A, in the protein secretory pathway of Aspergillus niger. 1065 50

Protein disulfide isomerase (PDI), a member of the thioredoxin (Trx) superfamily, consists of five consecutive domains, a-b-b'-a'-c. Domain combinations, AB, A'C, B'A'C and AB-C, and hybrids of PDI domains with Trx, Trx-C and Trx-B'A'C, have been constructed and expressed in Escherichia coli to examine the contributions of PDI domains to its enzyme and chaperone activities. All the combination and hybrid products are considerably less active than intact PDI in their enzyme activities. Recombinant products containing C, at low concentrations, inhibit the reactivation of lysozyme in HEPES buffer, while those without C do not. Only the intact PDI molecule and the hybrid molecule, Trx-B'A'C, but to a much lower level, show general chaperone activity in assisting the reactivation of denatured D-glyceraldehyde-3-phosphate dehydrogenase. It is suggested that all domains of PDI contribute to the binding of target protein for its chaperone activity.
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PMID:Contributions of protein disulfide isomerase domains to its chaperone activity. 1100 77

The functional properties of a novel protein, protein disulfide isomerase-related protein A (PRPA) from Aspergillus niger T21, have been characterized. (1) PRPA possesses disulfide isomerase activity. (2) In Hepes buffer, at substoichiometric concentrations, PRPA facilitates the formation of inactive lysozyme aggregates associated with PRPA (anti-chaperone activity); while at a high molar excess, PRPA inhibits aggregation by maintaining lysozyme in a soluble, yet inactive, state (chaperone-like activity). However, PRPA only exhibits chaperone-like activity during lysozyme refolding in phosphate buffer. (3) Experiments have indicated that disulfide cross-linkage is not required for the interaction between PRPA and lysozyme, and hydrophobic interaction may be responsible for PRPA effect on lysozyme. (4) Co-expression of PRPA and prochymosin in Escherichia coli leads to reduction of inclusion bodies, rendering part of prochymosin molecules soluble yet inactive. The structural and functional characteristics of PRPA suggest that PRPA may play an important role in protein folding, aggregation, and retention in the endoplasmic reticulum.
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PMID:Chaperone characteristics of PDI-related protein A from Aspergillus niger. 1535 11

Protein disulfide isomerase-related protein A (PRPA) was highly expressed (about 34%) in Escherichia coli by inserting the whole PRPA cDNA into the vector pET23b. After expression, the purified protein was acquired through ammonium fractional precipitation and Bio-Rex 70 chromatography. PRPA shows low disulfide isomerase activity (only about 1/250 of that of hPDI), decreases the reactivation yield of denatured and reduced lysozyme either in redox and non-redox Hepes buffer or redox PBS buffer and facilitates the aggregation of denatured and reduced lysozyme. Fluorescence spectra of PRPA indicate that PRPA has more hydrophobic groups at surface than that of hPDI, and which can be used to explain why PRPA has anti-chaperone activity during the refolding of denatured and reduced lysozyme.
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PMID:[Expression and characterization of protein disulfide isomerase-related protein A]. 1596 95

DsbA (disulfide bond formation protein A) located in the periplasm of Escherichia coli is a disulfide isomerase, which is vital to disulfide bonds formation directly affecting the nascent peptides folding to the correct conformation. In this paper, recombinant DsbA was firstly immobilized onto NHS-activated Sepharose Fast Flow gel. Then Sephadex G-100 gel was sequentially packed on the top of recDsbA Sepharose Fast Flow, and a so-called conjoint chromatography column composed of SEC and immobilized recombinant DsbA was constructed. Denatured lysozyme was applied on the conjoint column. The effect of SEC volume, flow rate, loading amount and volume, pre-equilibrium mode and KCl concentration in the buffer on lysozyme refolding were investigated in detail and the stability of DsbA immobilization was evaluated. Finally the reusability of the conjoint refolding column was also tested. When loading 2.4 mg denatured lysozyme in 0.5 ml solution, the activity recovery reached 92.7% at optimized experimental conditions, and the conjoint column renaturation capacity decreased only 7.7% after six run reuse due to the use of SEC section in the chromatographic refolding process. The conjoint chromatography offers an efficient strategy to refold proteins in vitro with high productivity and column reusability.
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PMID:On-column refolding of denatured lysozyme by the conjoint chromatography composed of SEC and immobilized recombinant DsbA. 2192 75

Protein disulfide isomerase (PDI) and glutathione peroxidase 7 (GPx7) cooperatively promote the oxidative folding of disulfide (SS)-containing proteins in endoplasmic reticulum by recognizing the nascent proteins to convert them into the native folds by means of SS formation and SS isomerization and by catalyzing reoxidation of reduced PDI with H2O2, respectively. In this study, new amphiphilic selenides with a long-chain alkyl group were designed as hybrid mimics of PDI and GPx7 and were applied to the refolding of reduced hen egg-white lysozyme (HEL-R). Competitive SS formation at pH 4 using HEL-R and glutathione (GSH) in the presence of the selenide catalyst and H2O2 showed that the amphiphilic selenides can preferentially catalyze SS formation of HEL-R, probably on account of hydrophobic interactions between the protein and the catalyst. In contrast, simple water-soluble selenides did not exhibit such behavior. In addition, when the pH of the solution was adjusted to 8.5 after the SS formation, surviving GSH promoted the SS isomerization of misfolded HEL to recover the native SS linkages. Thus, the amphiphilic selenides designed here could mimic the function of the PDI-GPx7 system. The combination of a water-soluble selenide and a long-chain alkyl group would be a useful motif in designing medicines for both protein misfolding diseases and antioxidant therapy.
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PMID:An amphiphilic selenide catalyst behaves like a hybrid mimic of protein disulfide isomerase and glutathione peroxidase 7. 2520 35

Protein disulfide isomerase-P5 (P5) is thought to have important functions as an oxidoreductase, however, molecular functions of P5 have not been fully elucidated. We have reported that P5 has insulin reductase activity and inhibits lysozyme refolding by formation of lysozyme multimers with hypermolecular mass inactivated by intermolecular disulfides (hyLYS) in oxidative refolding of reduced denatured lysozyme. To explore the role of each domain of P5, we investigated the effects of domain deletion and Cys-Ala mutants of P5 on insulin reduction and the oxidative refolding of the lysozyme. The mutants of catalytic cysteines, C36/39A, C171/174A, and C36/39/171/174A inhibited the lysozyme refolding almost similarly to P5, and even b domain without catalytic cysteines showed moderate inhibitory effect, suggesting that the b domain played a key role in the inhibition. Western blotting analysis of the refolding products indicated that the catalytic cysteines in both the a and a' domains cross-linked lysozyme comparably to form hyLYS resistant to trypsin, in which b domain was suggested to capture lysozyme for the significant sulfhydryl oxidation. The mutant of the conserved cysteines in b domain, C272/278A, did not form hyLYS, however, showed predominant reductase activity, implying that P5 functioned as a potent sulfhydryl oxidase and a predominant reductase depending on the circumstance around C272/278. These results provide new insight into the molecular basis of P5 function.
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PMID:Conserved C272/278 in b domain regulate the function of PDI-P5 to make lysozymes trypsin-resistant forms via significant intermolecular disulfide cross-linking. 2573 Oct 82

Protein disulfide isomerase is a type of enzyme that catalyses the oxidation, isomerization and reduction of disulfide bonds. Conotoxins that containing disulfide bonds are likely substrates of protein disulfide isomerise. Here, we cloned 12 protein disulfide isomerise genes from 12 different cone snail species that inhabited the sea near Sanya in China. The full-length amino acid sequences of these protein disulfide isomerase genes share a high degree of homology, including the same -CGHC- active site sequence and -RDEL- endoplasmic reticulum retention signal. To obtain enough conus protein disulfide isomerase for functional studies, we constructed the expression vector pET28a-sPDI. Conus protein disulfide isomerase was successfully expressed using Escherichia coli expression system and purified using chromatography method of affinity chromatography. The recombinant conus protein disulfide isomerase showed the ability to catalyse disulfide bond formation and rearrangement in the lysozyme enzyme activity assay. The role of conus protein disulfide isomerase in the in vitro oxidative folding of conotoxins was investigated using synthetic linear conotoxin lt14a, a peptide composed of 13 amino acids. It was confirmed by high performance liquid chromatography and mass spectrometry analysis that conus protein disulfide isomerase can catalyse the disulfide bond formation of linear lt14a. Then, conus protein disulfide isomerase was acted as a fusion partner during the production of engineered peptidyl-prolyl cis-trans isomerase and lt14a derived from cone snails. It was shown that peptidyl-prolyl cis-trans isomerase and conotoxin lt14a are successfully expressed in a highly soluble form by fusion with conus protein disulfide isomerase. Thus, conus protein disulfide isomerase functions not only as an enzyme that catalyses oxidative process but also a fusion partner in recombinant conotoxin expression.
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PMID:Oxidative Folding of Conopeptides Modified by Conus Protein Disulfide Isomerase. 2885 45


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