Isomerase Activity Of Protein Disulfide Isomerase Research Articles

Biological Functions of Protein Disulfide Isomerase as a Target of Phenolic Endocrine-disrupting Chemicals

Endocrine disrupting chemicals (EDCs) are widespread in the environment and suspected of interfering with endocrine homeostasis. Bisphenol A (BPA), 2,2-bis(4-hydroxyphenyl)propane, an EDC, has been widely used throughout the world as an industrial chemical. BPA is a developmental, neural, and reproductive toxicant that mimics estrogen and can interfere with growth and body function. In a recent study, a BPA-binding protein was isolated from P2 fractions of rat brain, and identified as protein disulfide isomerase (PDI). PDI has been studied extensively as a key enzyme involved in the formation of the correct pattern of disulfide bonds in proteins. PDI is also known to be a membrane-associated 3,3',5-triiodo-L-thyronine (T3)-binding protein. BPA inhibits the binding of T3 to PDI and isomerase activity of PDI. PDI has four consecutive domains, a, b, b', and a', each with a thioredoxin fold. The domains a and a' have a catalytically active Cys-Gly-His-Cys motif, while b and b' have substrate-binding sites. To address the chemical structural requirements of BPA for the inhibitory effect of PDI, several BPA analogs were tested, suggesting that the phenolic structure is important for BPA to affect PDI functions. To investigate the biological effects of BPA on PDI functions, GH3 cells were used. GH3 is a rat pituitary tumor cell line and releases growth hormone (GH) via the T3 receptor. Over-expression of PDI suppresses the T3-induced release of GH and when the cells were treated with BPA together with T3, the amount of GH released was much increased. Thus, PDI plays an important role in the hypothalamic-pituitary-thyroid axis. In this review, the possible involvement and biological significance especially in the central nervous system, of PDI as a target of phenolic environmental chemicals, are discussed.

Interaction of human protein disulfide isomerase and human P5 with drug compounds: Analysis using biosensor technology

Binding of aminoglycoside antibiotics to protein disulfide isomerase (PDI) is known to inhibit the chaperone activity but not the isomerase activity of PDI. The amino sugar moiety of the antibiotics is a key factor in these interactions. In the present study, domain deletion analysis of human PDI (hPDI) revealed that the a′c domain is significant for binding to aminoglycoside antibiotics; however, other domains, including the a, b, and b′ domains, are also required, as assessed using the Biacore biosensor analysis system. It was found that the drug compounds interacting with the hPDI family of proteins can be classified into four groups according to the recognition motif for hPDI and hP5. The anti-cancer drug vincristine was found to inhibit chaperone activity but not isomerase activity for both hPDI and hP5 in vitro. Interestingly, a 100:1 molar ratio of vincristine to hPDI or hP5 was sufficient to almost completely inhibit the chaperone activity of hPDI and hP5. These findings may be useful not only for future structure–function studies of the PDI family of proteins but also for new drug design, since PDI plays a critical role in protein-folding in the endoplasmic reticulum and is indispensable for cell viability.

The Contributions of Protein Disulfide Isomerase and Its Homologues to Oxidative Protein Folding in the Yeast Endoplasmic Reticulum

In vitro, protein disulfide isomerase (Pdi1p) introduces disulfides into proteins (oxidase activity) and provides quality control by catalyzing the rearrangement of incorrect disulfides (isomerase activity). Protein disulfide isomerase (PDI) is an essential protein in Saccharomyces cerevisiae, but the contributions of the catalytic activities of PDI to oxidative protein folding in the endoplasmic reticulum (ER) are unclear. Using variants of Pdi1p with impaired oxidase or isomerase activity, we show that isomerase-deficient mutants of PDI support wild-type growth even in a strain in which all of the PDI homologues of the yeast ER have been deleted. Although the oxidase activity of PDI is sufficient for wild-type growth, pulse-chase experiments monitoring the maturation of carboxypeptidase Y reveal that oxidative folding is greatly compromised in mutants that are defective in isomerase activity. Pdi1p and one or more of its ER homologues (Mpd1p, Mpd2p, Eug1p, Eps1p) are required for efficient carboxypeptidase Y maturation. Consistent with its function as a disulfide isomerase in vivo, the active sites of Pdi1p are partially reduced (32 +/- 8%) in vivo. These results suggest that PDI and its ER homologues contribute both oxidase and isomerase activities to the yeast ER. The isomerase activity of PDI can be compromised without affecting growth and viability, implying that yeast proteins that are essential under laboratory conditions may not require efficient disulfide isomerization.

The chaperone activity of protein disulfide isomerase is affected by cyclophilin B and cyclosporin A in vitro.

To elucidate the function of protein disulfide isomerase (PDI), we screened for PDI-binding proteins in a bovine liver extract using affinity column chromatography. One of the binding proteins was identified by SDS-PAGE and N-terminal amino acid sequence analysis to be cyclophilin B (Cyp B). Use of the BIACORE system revealed that purified bovine Cyp B bound specifically to bovine PDI with a K(D) value of 1.19 x 10(-5) M. Interestingly, the binding affinity between PDI and Cyp B was strengthened by preincubation of the Cyp B with cyclosporin A (CsA), yielding a K(D) value of 3.67 x 10(-6) M. Although the interaction between PDI and Cyp B affected neither the isomerase activity of PDI nor the peptidyl-prolyl cis-trans isomerase activity of Cyp B, Cyp B increased the chaperone activity of PDI. However, the complex of Cyp B and CsA completely inhibited the chaperone activity of PDI. Thus, PDI and Cyp B appear to cooperate with each other to regulate the functional expression of proteins in vivo.

Combinations of Protein-disulfide Isomerase Domains Show That There Is Little Correlation between Isomerase Activity and Wild-type Growth

Protein-disulfide isomerase (PDI) has five domains: a, b, b', a' and c, all of which except c have a thioredoxin fold. A single catalytic domain (a or a') is effective in catalyzing oxidation of a reduced protein but not isomerization of disulfides (Darby, N. J., and Creighton, T. E. (1995) Biochemistry 34, 11725-11735). To examine the structural basis for this oxidase and isomerase activity of PDI, shuffled domain mutants were generated using a method that should be generally applicable to multidomain proteins. Domains a and a' along with constructs ab, aa', aba', ab'a' display low disulfide isomerase activity, but all show significant reactivity with mammalian thioredoxin reductase, suggesting that the structure is not seriously compromised. The only domain order that retains significant isomerase activity has the b' domain coupled to the N terminus of the a' domain. This b'a'c has 38% of the isomerase activity of wild-type PDI, equivalent to the activity of full-length PDI with one of the active sites inactivated by mutation (Walker, K. W., Lyles, M. M., and Gilbert, H. F. (1996) Biochemistry 35, 1972-1980). Individual a and a' domains, despite their very low isomerase activities in vitro, support wild-type growth of a pdi1Delta Saccharomyces cerevisiae strain yeast. Thus, most of the PDI structure is dispensable for its essential function in yeast, and high-level isomerase activity appears not required for viability or rapid growth.

Involvement of two sulfur atoms of protein disulfide isomerase and one sulfur atom of the DsbA/PpfA protein in the oxidation of mutant human lysozyme.

Protein disulfide isomerase (PDI) and the DsbA/PpfA protein catalyze the oxidation of mutant human lysozyme, L79CC81A, which has two native disulfide bonds, Cys6-Cys128 and Cys30-Cys116, a non-native Cys79-Cys95, and 2 free cysteine residues at positions 65 and 77. Oxidation of L79CC81A (R-form) yielded two isomers, L79CC81A-a (A-form) with tandem-linked Cys65-Cys77 and Cys79-Cys95, and L79CC81A-b (B-form) with cross-linked Cys65-Cys79 and Cys77-Cys95 (Kanaya, E., Ishihara, K., Tsunasawa, S., Nokihara, K., and Kikuchi, M. (1993) Biochem. J. 292, 469-476). PDI mainly enhanced the formation of the A- form in the absence of oxidized glutathione (GSSG); however, as the concentration of GSSG increased, it markedly accelerated the formation of the B-form. In contrast, the DspA/PpfA protein mainly enhanced the formation of the A-form, regardless of the presence or absence of GSSG. These results and the presumed spatial locations of Cys65, Cys77, and Cys79-Cys95 in the R-form suggest that 1 of the half-cystine residues in the active site of PDI and the DsbA/PpfA protein can react with 1 of the 2 free Cys residues of the R-form. The dependence on GSSG of the B-form formation with PDI can be explained by the formation of two transient intermolecular disulfide bonds between PDI and the R-form and the attack of GSSG by the resultant thiolate anion of Cys79 or Cys95. The independence of the reaction with the DsbA/PpfA protein from GSSG can be explained by the formation of one transient intermolecular disulfide bond. The possible formation of the two transient intermolecular disulfide bonds involving two sulfur atoms of PDI and 2 cysteine or half-cystine residues of the substrate could explain the high isomerase activity of PDI.