Unveiling interatomic distances influencing the reaction coordinates in alanine dipeptide isomerization: An explainable deep learning approach.

The present work shows that the free energy landscape associated with alanine dipeptide isomerization can be effectively represented by specific interatomic distances without explicit reference to dihedral angles. Conventionally, two stable states of alanine dipeptide in vacuum, i.e., C7eq (ß-sheet structure) and C7ax (left handed α-helix structure), have been primarily characterized using the main chain dihedral angles, φ (C-N-Cα-C) and ψ (N-Cα-C-N). However, our recent deep learning combined with the "Explainable AI" (XAI) framework has shown that the transition state can be adequately captured by a free energy landscape using φ and θ (O-C-N-Cα) [Kikutsuji et al., J. Chem. Phys. 156, 154108 (2022)]. In the perspective of extending these insights to other collective variables, a more detailed characterization of the transition state is required. In this work, we employ interatomic distances and bond angles as input variables for deep learning rather than the conventional and more elaborate dihedral angles. Our approach utilizes deep learning to investigate whether changes in the main chain dihedral angle can be expressed in terms of interatomic distances and bond angles. Furthermore, by incorporating XAI into our predictive analysis, we quantified the importance of each input variable and succeeded in clarifying the specific interatomic distance that affects the transition state. The results indicate that constructing a free energy landscape based on the identified interatomic distance can clearly distinguish between the two stable states and provide a comprehensive explanation for the energy barrier crossing.

A deletion in the N-terminal polymerizing domain of laminin ß2 is a new mouse model of chronic nephrotic syndrome.

The importance of the glomerular basement membrane (GBM) in glomerular filtration is underscored by the manifestations of Alport and Pierson syndromes, caused by defects in type IV collagen α3α4α5 and the laminin ß2 chain, respectively. Lamb2 null mice, which model the most severe form of Pierson syndrome, exhibit proteinuria prior to podocyte foot process effacement and are therefore useful for studying GBM permselectivity. We hypothesize that some LAMB2 missense mutations that cause mild forms of Pierson syndrome induce GBM destabilization with delayed effects on podocytes. While generating a CRISPR/Cas9-mediated analogue of a human LAMB2 missense mutation in mice, we identified a 44-amino acid deletion (LAMB2-Del44) within the laminin N-terminal domain, a domain mediating laminin polymerization. Laminin heterotrimers containing LAMB2-Del44 exhibited a 90% reduction in polymerization in vitro that was partially rescued by type IV collagen and nidogen. Del44 mice showed albuminuria at 1.8-6.0 g/g creatinine (ACR) at one to two months, plateauing at an average 200 g/g ACR at 3.7 months, when GBM thickening and hallmarks of nephrotic syndrome were first observed. Despite the massive albuminuria, some Del44 mice survived for up to 15 months. Blood urea nitrogen was modestly elevated at seven-nine months. Eight to nine-month-old Del44 mice exhibited glomerulosclerosis and interstitial fibrosis. Similar to Lamb2-/- mice, proteinuria preceded foot process effacement. Foot processes were widened but not effaced at one-two months despite the high ACRs. At three months some individual foot processes were still observed amid widespread effacement. Thus, our chronic model of nephrotic syndrome may prove useful to study filtration mechanisms, long-term proteinuria with preserved kidney function, and to test therapeutics.

Extracellular Processing of Lysyl Oxidase-like 2 and Its Effect on Amine Oxidase Activity.

Overexpression of lysyl oxidase-like 2 (LOXL2) is associated with several hepatic and vascular fibrotic diseases and tumor progression in some aggressive cancers. Secreted LOXL2 promotes extracellular matrix cross-linking by catalyzing the oxidative deamination of peptidyl lysine. A great deal remains to be learned about the post-translational modifications of LOXL2, including whether such modifications modulate enzymatic and disease-promoting activities; such knowledge would inform the development of potential therapies. We discovered that upon secretion in cell culture, LOXL2 undergoes proteolytic processing of the first two of four scavenger receptor cysteine-rich domains at the N-terminus. A similar pattern of processing was also evident in tissue extracts from an invasive ductal carcinoma patient. Processing occurred at 314Arg-315Phe-316Arg-317Lys↓-318Ala-, implicating proprotein convertases. siRNA-mediated knockdown of proprotein convertases (furin, PACE4, and PC5/6), as well as incubation with their recombinant forms, showed that PACE4 is the major protease that acts on extracellular LOXL2. Unlike LOX, which requires cleavage of its propeptide for catalytic activation, cleavage of LOXL2 was not essential for tropoelastin oxidation or for cross-linking of collagen type IV in vitro. However, in the latter case, processing enhanced the extent of collagen cross-linking ∼2-fold at ≤10 nM LOXL2. These results demonstrate an important difference in the regulatory mechanisms for LOX and LOXL2 catalytic activity. Moreover, they pave the way for further studies of potential differential functions of LOXL2 isoforms in fibrosis and tumor progression.

WITHDRAWN: PACE4 Proteolytically Processes LOXL2 with little impact on its catalytic activity.

This article has been withdrawn by the authors. Figs 1C, 2A, and 2E contained some inadvertently mislabeled data. The authors state that the mislabeling does not affect the conclusions of the article.

S-nitrosylation of the IGF-1 receptor disrupts the cell proliferative action of IGF-1.

The insulin-like growth factor 1 receptor (IGF-1R) is a disulfide-linked heterotetramer containing two α-subunits and two ß-subunits. Earlier studies demonstrate that nitric oxide (NO) can adversely affect IGF-1 action in the central nervous system. It is known that NO can induce S-nitrosylation of the cysteine residues in proteins, thereby partly contributing to the regulation of protein function. In the present study, we sought to determine whether S-nitrosylation of the cysteine residues in IGF-1R is an important post-translational modification that regulates its response to IGF-1. Using cultured SH-SY5Y human neuroblastoma cells as an in vitro model, we found that treatment of cells with S-nitroso-cysteine (SNOC), a NO donor that can nitrosylate the cysteine residues in proteins, induces S-nitrosylation of the ß subunit of IGF-1R but not its α-subunit. IGF-1Rß S-nitrosylation by SNOC is coupled with increased dissociation of the IGF-1R protein complex. In addition, disruption of the IGF-1R function resulting from S-nitrosylation of the IGF-1Rß subunit is associated with disruption of the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways. Further, we observed that SNOC-induced IGF-1Rß S-nitrosylation results in a dose-dependent inhibition of cell proliferation and survival. Together, these results suggest that elevated nitrosative stress may result in dysfunction of cellular IGF-1R signaling through S-nitrosylation of the cysteine residues in the IGF-1Rß subunit, thereby disrupting the downstream PI3K and MAPK signaling functions and ultimately resulting in inhibition of cell proliferation and survival.

Protein disulfide isomerase mediates glutathione depletion-induced cytotoxicity.

Glutathione depletion is a distinct cause underlying many forms of pathogenesis associated with oxidative stress and cytotoxicity. Earlier studies showed that glutamate-induced glutathione depletion in immortalized murine HT22 hippocampal neuronal cells leads to accumulation of reactive oxygen species (ROS) and ultimately cell death, but the precise mechanism underlying these processes is not clear. Here we show that during the induction of glutathione depletion, nitric oxide (NO) accumulation precedes ROS accumulation. While neuronal NO synthase (nNOS) in untreated HT22 cells exists mostly as a monomer, glutathione depletion results in increased formation of the dimer nNOS, accompanied by increases in the catalytic activity. We identified that nNOS dimerization is catalyzed by protein disulfide isomerase (PDI). Inhibition of PDI's isomerase activity effectively abrogates glutathione depletion-induced conversion of monomer nNOS into dimer nNOS, accumulation of NO and ROS, and cytotoxicity. Furthermore, we found that PDI is present in untreated cells in an inactive S-nitrosylated form, which becomes activated following glutathione depletion via S-denitrosylation. These results reveal a novel role for PDI in mediating glutathione depletion-induced oxidative cytotoxicity, as well as its role as a valuable therapeutic target for protection against oxidative cytotoxicity.

EPA, an omega-3 fatty acid, induces apoptosis in human pancreatic cancer cells: role of ROS accumulation, caspase-8 activation, and autophagy induction.

In a recent study, we showed that eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), two common omega-3 fatty acids, can cause ROS accumulation and subsequently induce caspase-8-dependent apoptosis in human breast cancer cells (Kang et al. [2010], PLoS ONE 5: e10296). In this study, we showed that the pancreas has a unique ability to accumulate EPA at a level markedly higher than several other tissues analyzed. Based on this finding, we sought to further investigate the anticancer actions of EPA and its analog DHA in human pancreatic cancer cells using both in vitro and in vivo models. EPA and DHA were found to induce ROS accumulation and caspase-8-dependent cell death in human pancreatic cancer cells (MIA-PaCa-2 and Capan-2) in vitro. Feeding animals with a diet supplemented with 5% fish oil, which contains high levels of EPA and DHA, also strongly suppresses the growth of MIA-PaCa-2 human pancreatic cancer xenografts in athymic nude mice, by inducing oxidative stress and cell death. In addition, we showed that EPA can concomitantly induce autophagy in these cancer cells, and the induction of autophagy diminishes its ability to induce apoptotic cell death. It is therefore suggested that combination of EPA with an autophagy inhibitor may be a useful strategy in increasing the therapeutic effectiveness in pancreatic cancer.

The binding site of bisphenol A to protein disulphide isomerase.

Protein disulphide isomerase (PDI) has been isolated as a binding protein of bisphenol A (BPA) in the rat brain. In this study, we determined binding sites of BPA to PDI and characterized the binding site. First, we identified the BPA-binding domain with ab, b'a'c, a, b, b' and a'c fragment peptides of PDI by surface plasmon resonance spectroscopy. BPA interacted with ab, b'a 'c, a and b', suggesting that a and b' domains are important in their interaction. Second, ab, b'a'c, a,b,b',a', abb'a', abb', b'a', Δb' and a'c fragment peptides were used for their isomerase activity with RNase as a substrate. BPA could inhibit the activity of peptide fragments including b', suggesting that b' domain contributes to inhibition of catalytic activity of PDI by BPA. Next, we investigated the BPA-binding capacity of PDI by amino acid substitution. PDI lost the BPA-binding activity by the mutation of H258 and mutation of Q245 and N300 also decreased its activity. Furthermore, acidic condition increased the BPA-binding activity of PDI. These results suggest that the charge of these amino acid especially, H258, is important for the BPA to bind to PDI.

Effects of polybrominated diphenyl ethers (PBDEs) and their derivatives on protein disulfide isomerase activity and growth hormone release of GH3 cells.

Polybrominated diphenyl ethers (PBDEs) have been used in a variety of consumer products such as flame retardants and recently have been known to be widespread environmental pollutants, which probably affect biological functions of mammalian cells. However, the risk posed by PBDE metabolites has not been clarified. Our previous study suggested that bisphenol A (BPA), an endocrine-disrupting chemical, binds to protein disulfide isomerase (PDI) and inhibits its activity. PDI is an isomerase enzyme in the endoplasmic reticulum and facilitates the formation or cleavage of disulfide bonds. PDI consists of a, b, b', and a' domains and the c region, with the a and a' domains having isomerase active sites. In the present study, we tested the effects of 10 kinds of PBDE compounds and their metabolites on PDI. OH-PBDEs specifically inhibited the isomerase activity of PDI, with 4'-OH-PBDE more effective than 2' (or 2)-OH-PBDEs. 4'-OH-PBDE inhibited the isomerase activity of the b'a'c fragment but not that of ab and a'c, suggesting that the b' domain of PDI is essential for the inhibition by 4'-OH-PBDE. We also investigated the effects of these chemicals on the production of growth hormone (GH) in GH3 cells. In GH3 cells, levels of mRNA and protein of GH stimulated by T(3) were reduced by 4'-OH-PBDE and 4'-MeO-PBDE. The reduction in GH expression caused by these compounds was not changed by the overexpression or knockdown of PDI in GH3 cells, while these manipulations of PDI levels significantly suppressed the expression of GH. These results suggest that the biological effects of PBDEs differed depending on their brominated and hydroxylated positions.

LKM-1 sera from autoimmune hepatitis patients that recognize ERp57, carboxylesterase 1 and CYP2D6.

Liver kidney microsomal antibody type 1 (LKM-1) is a diagnostic marker for autoimmune hepatitis type 2 (AIH-2). Characterization of LKM autoantibodies of patients with AIH-2 demonstrated that a proportion of LKM sera contains autoantibodies which recognize one or more small linear epitopes on cytochrome P450, CYP2D6, an enzyme of drug metabolism pathways. The identification and epitope mapping of antigens involved in autoimmune diseases are important in understanding the mechanisms triggering autoimmunity and providing guidance for designing immunomodulatory therapy. In this study, several proteins recognized by LKM-1-positive sera in rat and human hepatic microsomes were analyzed by MALDI-TOF-MS after separation with ion-exchange chromatography or two-dimensional polyacrylamide gel electrophoresis. We identified these proteins as ERp57 and carboxylesterase 1 (CES1) as well as CYP2D6. Epitopes in ERp57 and CES1 recognized by LKM-1-positive serum were investigated by enzyme-linked immunosorbent assay (ELISA) with protease-digested peptides of ERp57 and CES1. The peptides comprising amino acids 105-129 of ERp57 and 558-566 of CES1 were specifically recognized by the serum. The epitopes in EPp57 and CES1 recognized by LKM-1-positive sera were homologous with those in hepatitis C virus (HCV). Viral infection of such as HCV may thus possibly trigger autoimmune hepatitis.

Spare interactions of highly potent [Arg(14),Lys(15)]nociceptin for cooperative induction of ORL1 receptor activation.

[Arg(14),Lys(15)]Nociceptin is a very potent for ORL1 receptor, showing a few times stronger binding activity and much more enhanced biological activity than endogenous nociceptin. This synergistic outcome has been suggested to be due to the interaction with the receptor aromatic and/or acidic amino acid residues crucial to receptor activation. In order to identify such receptor residues in the second ORL1 extracellular loop, we prepared a series of recombinant mutant receptors. The mutant receptor Gln205Ala was found to be as active as wild-type ORL1 for both nociceptin and [Arg(14),Lys(15)]nociceptin. In contrast, Asp206Ala and Tyr207Ala exhibited considerably reduced activity for [Arg(14),Lys(15)]nociceptin, exhibiting no synergistic activity enhancement. These results suggest that Asp206 and Tyr207 are directly involved in the interaction with nociceptin-[Arg(14),Lys(15)]. Trp208Ala was found to bind strongly both nociceptin and [Arg(14),Lys(15)]nociceptin, although it elicited no biological activity. All these results indicate that the consecutive amino acid residues Asp206, Tyr207, and Trp208 are critical to the activation of the ORL1 receptor, but not to nociceptin-binding.

Discriminatory synergistic effect of Trp-substitutions in superagonist [(Arg/Lys)(14), (Arg/Lys)(15)]nociceptin on ORL1 receptor binding and activation.

ORL1 is an endogenous G protein-coupled receptor for neuropeptide nociceptin. [(R/K)(14), (R/K)(15)]nociceptin is a superagonist that strongly activates the ORL1 receptor. We have previously found that substituting with Trp can reproduce the potentiation induced by Arg or Lys at position 14. In the present study, in order to ensure the effect of Trp-substitution on the activities of [(R/K)(14), (R/K)(15)]nociceptin, we synthesized [W(14), (R/K)(15)]nociceptin and [(R/K)(14), W(15)]nociceptin. [W(14), (R/K)(15)]nociceptin was found to exhibit threefold higher binding activity and 10-fold greater potency in a functional [(35)S]GTPgammaS functional assay as compared to wild-type nociceptin. However, when only Trp was placed in position 15, the resulting analogues, [(R/K)(14), W(15)]nociceptin, showed only a moderate enhancement of binding and biological activity (2-3 fold in both). These results indicate that the placement of Trp at position 14, unlike at position 15, enhances in a synergistic fashion the interaction of nociceptin with the ORL1 receptor. The results indicate that specific interactions feasible for Arg/Lys and Trp in common must be there for aromatic residues in ORL1, thus forming a cation/pi interaction or pi/pi hydrophobic interaction. The necessity for a favorable electrostatic interaction appears strict in position 15.

Hydroxylated polychlorinated biphenyls (PCBs) interact with protein disulfide isomerase and inhibit its activity.

Protein disulfide isomerase (PDI) is a catalyst of isomerization of substrate protein intra- and extra-molecular disulfide bridges and also has 3,3',5-triiodo-l-thyronine (T(3))-binding activity and molecular chaperone-like activity. We previously found that halogenated derivatives of bisphenol A as well as bisphenol A itself bind to PDI and thereby suppress the oxidative refolding of reduced RNaseA by PDI. Polychlorinated biphenyls (PCBs) are environmental endocrine-disrupting chemicals that cause various abnormalities in many organs such as the central nervous system. PCBs are metabolized to hydroxylated compounds (HO-PCBs) in humans and other animals, and HO-PCBs gain toxicity by metabolism. In the present study, 2',3,3',4',5'-pentachlorobiphenyl (penCB), 2',3,3',5,5',6'-hexachlorobiphenyl (hexCB), and their 4-hydroxylated metabolites (HO-penCB and HO-hexCB, respectively) were used to examine whether they interact with PDI and inhibit its activity. HO-penCB and HO-hexCB markedly inhibited the binding of T(3) to PDI. However, nonhydroxylated PCBs did not show any interaction with PDI. The effects of PCBs and HO-PCBs on PDI activity were also investigated using an RNaseA refolding assay. Both HO-PCBs inhibited the oxidative refolding of reduced RNaseA by PDI. We also assessed the effects of HO-PCBs and PCBs on the chaperone activity of PDI, which was measured by a thermal aggregation assay, and found that neither HO-PCBs nor PCBs have significant inhibitory or promoting effects. These findings suggest that the metabolites of PCBs have the potential to cause defective protein folding via PDI.

Bisphenol A disrupts Notch signaling by inhibiting gamma-secretase activity and causes eye dysplasia of Xenopus laevis.

Bisphenol A (BPA) is being recognized as an endocrine-disrupting chemical (EDC). Recently, several reports indicated that BPA affects the central nervous system (CNS) during embryonic development. However, the molecular mechanism of BPA in the CNS is not well known. Here, we show that BPA affected Notch signaling by inhibiting the activity of the Notch intracellular domain (NICD) cleavage-related enzyme, gamma-secretase (gamma-secretase), at the neurula stage of the Xenopus laevis. BPA caused various morphologic aberrations including scoliosis, eye dysplasia, and loss of pigments in the X. laevis tadpole. These abnormalities were seen whenever BPA was used at the neurula stage. In addition, the expression levels of several marker mRNAs at the neurula stage were investigated by RT-PCR, and we found that the mRNAs expression of ectodermal marker, Pax6, CNS marker, Sox2, and neural crest marker, FoxD3, were decreased by treatment with BPA. These genes contribute to the neural differentiation at the neurula stage, and also the downstream factors of Notch signaling. Injection of NICD but not a Notch ligand, delta 1, rescued the abnormalities caused by BPA. We subsequently assayed the inhibition of the activities of NICD cleavage-related enzymes, tumor necrosis factor alpha converting enzyme, and gamma-secretase, by BPA and found that BPA inhibited the gamma-secretase activity. Furthermore, we expressed presenilin, a main component of gamma-secretase, in Escherichia coli and found the direct binding of BPA with presenilin. These results suggest that BPA affected the neural differentiation by inhibiting gamma-secretase activity, leading to neurodevelopmental abnormalities.

Synergistic effect of basic residues at positions 14-15 of nociceptin on binding affinity and receptor activation.

Nociceptin is an endogenous ligand that activates a G protein-coupled receptor ORL1 and contains two indispensable Arg-Lys (RK) dipeptide units at positions 8-9 and 12-13. By replacing an additional RK unit at positions 6-7, 10-11, 14-15, or 16-17, of the peptide we have identified the analog, [RK(14-15)]nociceptin as a superagonist. In fact, this peptide exhibits 3-fold higher binding affinity and 17-fold greater potency in a functional GTPgammaS-binding assay compared to wild-type nociceptin. Here, we have further investigated the role of basic residues in position 14-15. The replacement of three other possible basic dipeptides, KR, RR, and KK, into nociceptin at positions 14-15 resulted in similar enhancements of binding affinity (3-5-fold) and biological potency (10-12-fold in the GTPgammaS assay). However, when only a single basic residue (Arg or Lys) was replaced in either position 14 or 15, all the resulting analogs showed moderate enhancements of binding and biological activity (2-4-fold in both). These results indicate that the addition of basic charges in positions 14 and 15 enhance in a synergistic fashion the interaction of nociceptin with the receptor and only the simultaneous presence of two adjacent basic residues yields an optimal effect. This suggests that specific electrostatic interactions between both amino acids present in 14-15 and corresponding residues in the receptor are responsible for the enhancement of nociceptin activity.

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.

Effect of psychotropic drugs on the 21-hydroxylation of neurosteroids, progesterone and allopregnanolone, catalyzed by rat CYP2D4 and human CYP2D6 in the brain.

We determined the effects of psychotropic drugs on the cytochrome P450 2D (CYP2D)-mediated 21-hydroxylation of progesterone (PROG) and allopregnanolone (ALLO) with the goal of clarifying whether neurosteroid levels are affected by psychotropic drugs in the brain. PROG or ALLO was incubated with rat CYP2D4 or human CYP2D6 in the presence of typical psychotropic drugs, fluoxetine, imipramine, desipramine, mazindol, and GBR12909, and the 21-hydroxylated metabolites of PROG and ALLO were determined by high performance liquid chromatography and liquid chromatography-tandem mass spectrometry, respectively. Fluoxetine competitively inhibited CYP2D4-mediated PROG 21-hydroxylation and increased both Km and Vmax values of CYP2D6-mediated PROG 21-hydroxylation. In addition, fluoxetine competitively inhibited ALLO 21-hydroxylation mediated by CYP2D4 and CYP2D6. Imipramine, desipramine, mazindol, and GBR12909 competitively inhibited PROG 21-hydroxylation mediated by CYP2D4 and/or CYP2D6, and all psychotropic drugs inhibited ALLO 21-hydroxylation mediated by CYP2D4 and/or CYP2D6. The inhibition constants (Ki values) of imipramine, desipramine, and mazindol against the 21-hydroxylation of PROG and ALLO by CYP2D6 were lower than those by CYP2D4. These results indicate that psychotropic drugs including fluoxetine affected the metabolism of neurosteroids, such as PROG and ALLO in the brain, suggesting that the regulation of the neurosteroid levels is modified by central nervous system-active drugs that inhibit brain CYP2D isoforms.

Designed modification of partial agonist of ORL1 nociceptin receptor for conversion into highly potent antagonist.

Nociceptin is an endogenous agonist ligand of the ORL1 (opioid receptor-like 1) receptor, and its antagonist is a potential target of therapeutics for analgesic and antineuropathy drugs. Ac-RYYRIK-NH(2) is a hexapeptide isolated from the peptide library as an antagonist that inhibits the nociceptin activities mediated through ORL1. However, the structural elements required for this antagonist activity are still indeterminate. In the present study, we evaluated the importance of the acetyl-methyl group in receptor binding and activation, examining the peptides acyl-RYYRIK-NH(2), where acyl (R-CO) possesses a series of alkyl groups, R=C(n)H(2n+1) (n=0-5). The isovaleryl derivative with the C(4)H(9) (=(CH(3))(2)CHCH(2)-) group was found to reveal a high receptor-binding affinity and a strong antagonist nature. This peptide achieved a primary goal of eliminating the agonist activity of Ac-RYYRIK-NH(2) and producing pure antagonist activity.

Over-expression of protein disulfide isomerase reduces the release of growth hormone induced by bisphenol A and/or T3.

We previously isolated a bisphenol A (BPA)-binding protein from rat brain and showed that it was identical to the protein disulfide isomerase (PDI), which also serves as a 3,3',5-triiodo-l-thyronine (T3)-binding protein. In this study, we investigated the effects of BPA on the production of growth hormone (GH) in GH3 cells and examined the possible involvement of PDI in this process. When administered singly, BPA and T3 each induced GH release in GH3 cells. When cells were treated with the combination of BPA and T3, the release of GH was much greater than that by T3 alone, but GH mRNA and promoter activity were not increased. These results suggested that the synergistic effect of T3 plus BPA on GH release was due to posttranslational regulation. Over-expression of PDI suppressed GH mRNA expression and GH release, suggesting that PDI modulates the T3-induced gene expression. We conclude that BPA can disrupt the thyroid hormone function in GH3 cells, and GH release induced by T3 is influenced by expression of PDI.

Different mechanisms for inhibition of human cytochromes P450 1A1, 1A2, and 1B1 by polycyclic aromatic inhibitors.

We have previously shown that several polycyclic aromatic hydrocarbons (PAHs) strongly inhibit their own and other PAH metabolism catalyzed by cytochrome P450 (P450) 1A1, 1A2, and 1B1 [Shimada, T., and Guengerich, F. P. (2006) Chem. Res. Toxicol. 19, 288-294]. In the present study, we examined mechanisms of how PAHs inhibit these P450 enzymes by using 7-ethoxyresorufin O-deethylation (EROD) as a model reaction. First, we examined mechanisms of inhibition of P450 1A1, 1A2, and 1B1 by the synthetic model inhibitors 1-(1-propynyl)pyrene (1PP), 1-ethynylpyrene (1EP), 2-ethynylpyrene (2EP), and 4-(1-propynyl)biphenyl (4Pbi). Both 1PP and 1EP inhibited P450 1A1 in a mechanism-based manner, but P450 1B1 and 1A2 were directly inhibited by 1PP and 1EP. Interestingly, P450 1B1 inactivated 1PP and 1EP to products that were not inhibitory to P450 1B1. 4Pbi was a mechanism-based inhibitor of P450 1A1 and 1B1, but 2EP directly inhibited these P450s. All four of the inhibitors directly inhibited P450 1A2. We also found that benzo[a]pyrene and seven other PAH compounds tested inhibited P450 1A2 in a mechanism-based manner, but fluoranthene directly inhibited P450 1A2. All of the nine PAHs examined were direct inhibitors of P450 1A1 and P450 1B1. These results suggest different mechanisms of inhibition of P450 1A1, 1A2, and 1B1 by PAHs and related chemicals and that interactions between P450 enzymes and PAH inhibitors are involved in differences in inhibition of the enzymes.