Shoko Hashimoto

Interaction between bisphenol derivatives and protein disulphide isomerase (PDI) and inhibition of PDI functions: requirement of chemical structure for binding to PDI.

Bisphenol A (BPA) is an endocrine disrupting chemical and several biological effects have been reported. Previously, protein disulphide isomerase (PDI) was isolated as a target molecule of bisphenol A. In this study, to clarify the effects of BPA on PDI functions, we investigated the relationship between the chemical structure of BPA derivatives and the effects on PDI-mediated isomerase and chaperone activity. We also investigated the effects of changes in the isomerase domain of PDI on the binding of chemicals, using PDI mutants and oxidized or reduced PDI. Among six chemicals, only chemicals, which have a phenol group, can bind to PDI and these chemicals also have an inhibitory effect on PDI-mediated isomerase activity. Changes in the structure of the PDI isomerase domain did not affect chemical-binding activity. On the other hand, the chemicals used in this study have low effects on chaperone activity of PDI. Substitutions in Cys residues (Cys398 and Cys401) of the isomerase active site changed chaperone activity. The present study indicates that phenolic compounds specifically bind to PDI and inhibit isomerase activity. This study provides useful information to predict the biological effects of chemicals and structural studies of PDI containing the function of chemical binding.

Inhibition of the functional interplay between endoplasmic reticulum (ER) oxidoreduclin-1α (Ero1α) and protein-disulfide isomerase (PDI) by the endocrine disruptor bisphenol A.

Background: Protein-disulfide isomerase (PDI) has previously been identified to bind bisphenol A (BPA), an endocrine disrupter. Results: BPA inhibited Ero1α-PDI-mediated disulfide bond formation. Conclusion: BPA significantly inhibited the Ero1α and PDI oxidative cycle, probably through closure of the substrate- and Ero1α-binding pocket in the PDI b′ domain. Significance: BPA may have inhibitory effects on oxidative folding of secretory and membrane proteins. Bisphenol A (BPA) is an endocrine disruptor that may have adverse effects on human health. We recently isolated protein-disulfide isomerase (PDI) as a BPA-binding protein from rat brain homogenates and found that BPA markedly inhibited PDI activity. To elucidate mechanisms of this inhibition, detailed structural, biophysical, and functional analyses of PDI were performed in the presence of BPA. BPA binding to PDI induced significant rearrangement of the N-terminal thioredoxin domain of PDI, resulting in more compact overall structure. This conformational change led to closure of the substrate-binding pocket in b′ domain, preventing PDI from binding to unfolded proteins. The b′ domain also plays an essential role in the interplay between PDI and ER oxidoreduclin 1α (Ero1α), a flavoenzyme responsible for reoxidation of PDI. We show that BPA inhibited Ero1α-catalyzed PDI oxidation presumably by inhibiting the interaction between the b′ domain of PDI and Ero1α; the phenol groups of BPA probably compete with a highly conserved tryptophan residue, located in the protruding β-hairpin of Ero1α, for binding to PDI. Consistently, BPA slowed down the reoxidation of PDI and caused the reduction of PDI in HeLa cells, indicating that BPA has a great impact on the redox homeostasis of PDI within cells. However, BPA had no effect on the interaction between PDI and peroxiredoxin-4 (Prx4), another PDI family oxidase, suggesting that the interaction between Prx4 and PDI is different from that of Ero1α and PDI. These results indicate that BPA, a widely distributed and potentially harmful chemical, inhibits Ero1-PDI-mediated disulfide bond formation.

Endoplasmic reticulum stress responses in mouse models of Alzheimer disease: overexpression paradigm versus knock-in paradigm

Endoplasmic reticulum (ER) stress is believed to play an important role in the etiology of Alzheimers disease (AD). The accumulation of misfolded proteins and perturbation of intracellular calcium homeostasis are thought to underlie the induction of ER stress, resulting in neuronal dysfunction and cell death. Several reports have shown an increased ER stress response in amyloid precursor protein (APP) and presenilin1 (PS1) double-transgenic (Tg) AD mouse models. However, whether the ER stress observed in these mouse models is actually caused by AD pathology remains unclear. APP and PS1 contain one and nine transmembrane domains, respectively, for which it has been postulated that overexpressed membrane proteins can become wedged in a misfolded configuration in ER membranes, thereby inducing nonspecific ER stress. Here, we used an App-knockin (KI) AD mouse model that accumulates amyloid-β (Aβ) peptide without overexpressing APP to investigate whether the ER stress response is heightened because of Aβ pathology. Thorough examinations indicated that no ER stress responses arose in App-KI or single APP-Tg mice. These results suggest that PS1 overexpression or mutation induced a nonspecific ER stress response that was independent of Aβ pathology in the double-Tg mice. Moreover, we observed no ER stress in a mouse model of tauopathy (P301S-Tau-Tg mice) at various ages, suggesting that ER stress is also not essential in tau pathology–induced neurodegeneration. We conclude that the role of ER stress in AD pathogenesis needs to be carefully addressed in future studies.

Protein-disulfide Isomerase Regulates the Thyroid Hormone Receptor-mediated Gene Expression via Redox Factor-1 through Thiol Reduction-Oxidation

Background: Previously we found that overexpression of PDI suppressed the T3 response in GH3 cells. Results: Overexpression of wild-type PDI, but not C/A Mt, suppressed the T3 response. Interactions between Ref-1 and TRβ1 and Ref-1 and PDI were detected. Conclusion: PDI is involved in regulation of T3-mediated gene expression via Ref-1. Significance: Ref-1 contributes to TR function, and Ref-1 activity is regulated by PDI. Protein-disulfide isomerase (PDI) is a dithiol/disulfide oxidoreductase that regulates the redox state of proteins. We previously found that overexpression of PDI in rat pituitary tumor (GH3) cells suppresses 3,3′,5-triiodothyronine (T3)-stimulated growth hormone (GH) expression, suggesting the contribution of PDI to the T3-mediated gene expression via thyroid hormone receptor (TR). In the present study, we have clarified the mechanism of regulation by which TR function is regulated by PDI. Overexpression of wild-type but not redox-inactive mutant PDI suppressed the T3-induced GH expression, suggesting that the redox activity of PDI contributes to the suppression of GH. We considered that PDI regulates the redox state of the TR and focused on redox factor-1 (Ref-1) as a mediator of the redox regulation of TR by PDI. Interaction between Ref-1 and TRβ1 was detected. Overexpression of wild-type but not C64S Ref-1 facilitated the GH expression, suggesting that redox activity of Cys-64 in Ref-1 is involved in the TR-mediated gene expression. Moreover, PDI interacted with Ref-1 and changed the redox state of Ref-1, suggesting that PDI controls the redox state of Ref-1. Our studies suggested that Ref-1 contributes to TR-mediated gene expression and that the redox state of Ref-1 is regulated by PDI. Redox regulation of PDI via Ref-1 is a new aspect of PDI function.