Xi-Wen Liu

Protective action of CLA against oxidative inactivation of paraoxonase 1, an antioxidant enzyme

The effect of CLA on paraoxonase 1 (PON1), one of the antioxidant proteins associated with HDL, was investigated for its protective action against oxidative inactivation as well as its stabilization activity. When cis-9 (c9),trans-11 (t11)-CLA and t10,c12-CLA were examined for their protective activity against ascorbate/Cu2−-induced inactivation of PON1 in the presence of Ca2+, two CLA isomers exhibited a remarkable protection (Emax, 71–74%) in a concentration-dependent manner (50% effective concentration, 3–4 μM), characterized by a saturation pattern. Such a protective action was also reproduced with oleic acid, but not linoleic acid. Rather, linoleic acid antagonized the protective action of CLA isomers in a noncompetitive fashion. Additionally, the two CLA isomers also protected PON1 from oxidative inactivation by H2O2 or cumene hydroperoxide. The concentration-dependent protective action of CLA against various oxidative inactivation systems suggests that the protective action of CLA isomers may be mediated through their selective binding to a specific binding site in a PON1 molecule. Separately, the inactivation of PON1 by p-hydroxymercuribenzoate (PHMB), a modifier of the cysteine residue, was also prevented by CLA isomers, suggesting the possible existence of the cysteine residue in the binding site of CLA. The c9,t11-CLA isomer seems to be somewhat more effective than t10,c12-CLA in protecting against the inactivation of PON1 by either peroxides or PHMB, in contrast to the similar efficacy of these two CLA isomers in preventing ascorbate/Cu2+-induced inactivation of PON1. Separately, CLA isomers successfully stabilized PON1, but not linoleic acid. These data suggest that the two CLA isomers may play a beneficial role in protecting PON1 from oxidative inactivation as well as in its stabilization.

Inactivation of protein disulfide isomerase by alkylators including α,β-unsaturated aldehydes at low physiological pHs

Abstract Protein disulfide isomerase (PDI) is known to contain the thioredoxin box motif with a low pKa cysteine residue. To investigate the reactivity of PDI with thiol modifiers at low physiological pHs, either the reduced (PDI[red]) or oxidized form (PDI[oxid]) of PDI was exposed to various alkylating ragents. When PDI was incubated with iodoacetamide at pH 6.3 for 30 min at 38C, a remarkable inactivation (>90%) of PDI[red] was caused by iodoacetamide (IC[50]=8 M). However, PDI[oxid] was only slightly inactivated (approximately 18%) by iodoacetamide. Similarly, PDI[red] was significantly inactivated by Nethylmaleimide (NEM), but PDI[oxid] was not. When the inactivation by these alkylators was analyzed by pseudofirst order kinetics, NEM (k[3]=1.7510[-2] s[-1]; K[i]=124 M) was observed to be more potent than iodoacetamide (k[3]=9.110[-3] s[-1]; K=311 M). Interestingly, the inactivation of PDI[red] by iodoacetamide was greater at pH 6.3 than pH 7.0, in contrast to a similar inactivation potency of NEM at both pHs. Moreover, the maximal inactivation of PDI[red] or PDI[oxid] by iodoacetamide was mainly observed around pH 6.0. In addition, PDI[red] was found to be inactivated by acrolein (IC[50]=10 M) at pH 6.3, and this inactivation was also greater at pH 6.3 than at pH 7. Based on these results, we suggest that PDI[red] is susceptible to inactivation by alkylators including endogenous α,β-unsaturated aldehydes at low physiological pHs.

Identification of alkylation-sensitive target chaperone proteins and their reactivity with natural products containing michael acceptor

Molecular chaperones have a crucial role in the folding of nascent polypeptides in endoplasmic reticulum. Some of them are known to be sensitive to the modification by electrophilic metabolites of organic pro-toxicants. In order to identify chaperone proteins sensitive to alkyators, ER extract was subjected to alkylation by 4-acetamido-4′-maleimidyl-stilbene-2,2′-disul-fonate (AMS), and subsequent SDS-PAGE analyses. Protein spots, with molecular mass of 160, 100, 57 and 36 kDa, were found to be sensitive to AMS alkylation, and one abundant chaperon protein was identified to be protein disulfide isomerase (PDI) in comparison with the purified PDI. To see the reactivity of PDI with cysteine alkylators, the reduced form (PDIred) of PDI was incubated with various alkylators containing Michael acceptor structure for 30 min at 38°C at pH 6.3, and the remaining activity was determined by the insulin reduction assay, lodoacetamide orN-ethylmaleimide at 0.1 mM remarkably inactivated PDIred withN-ethylmale-imide being more potent than iodoacetamide. A partial inactivation of PDIoxid was expressed by iodoacetamide, but notN-ethylmaleimide (NEM) at pH 6.3. Of Michael acceptor compounds tested, 1,4-benzoquinone (IC50, 15 μM) was the most potent, followed by 4-hydroxy-2-nonenal and 1,4-naphthoquinone. In contrast, 1,2-naphthoquinone, devoid of a remarkable inactivation action, was effective to cause the oxidative conversion of PDIred to PDI0Xid. Thus, the action of Michael acceptor compounds differed greatly depending on their structure. Based on these, it is proposed that PDI, one of chaperone proteins in ER, could be susceptible to endogenous or xenobiotic Michael acceptor compoundsin vivo system.

Multimerization of bovine thyroglobulin, partially unfolded or partially unfolded/reduced; Involvement of protein disulfide isomerase and glutathionylated disulfide linkage

Fate of the nascent thyrolglobulin (Tg) molecule is characterized by multimerization. To establish the formation of Tg multimers, the partially unfolded/reduced Tg or deoxycholate-treated/ reduced Tg was subjected to protein disulfide isomerase (PDI)-mediated multimerization. Oxidized glutathione/PDI-mediated formation of multimeric Tg forms, requiring at least an equivalent molar ratio of PDI/Tg monomer, decreased with increasing concentration of reduced glutathione (GSH), suggesting the oxidizing role of PDI. Additional support was obtained when PDI alone, at a PDI/Tg molar ratio of 0.3, expressed a rapid multimerization. Independently, the exposure of partially unfolded Tg to GSH resulted in Tg multimerization, enhanced by PDI, according to thiol-disulfide exchange. Though to a lower extent, a similar result was observed with the dimerization of deoxycholate-pretreated Tg monomer. Consequently, it is implied that intermolecular disulfide linkage may be facilitated at a limited region of unfolded Tg. In an attempt to examine the multimerization site, the cysteine residue-rich fragments of the Tg were subjected to GSH-induced multimerization; a 50 kDa fragment, containing three vicinal dithiols, was multimerized, while an N-terminal domain was not. Present results suggest that the oxidase as well as isomerase function of PDI may be involved in the multimerization of partially unfolded Tg or deoxycholate-treated Tg.

Role of protein disulfide isomerase in molecular fate of thyroglobulin and its regulation by endogenous oxidants and reductants.

The molecular fate of thyroglobulin (Tg) is controlled by oligomerization, a means of storing Tg at high concentrations, and deoligomerization. The oligomerization of bovine Tg are intermolecular reactions that occur through oxidative processes, such as disulfide and dityrosine formation, as well as isopeptide formation; disulfide formation is primarily responsible for Tg oligomerization. Here, the protein disulfide isomerase (PDI) and/or peroxidase-induced oligomerization of unfolded thyroglobulins, which were prepared by treating bovine Tg with heat, urea or thiol/urea, was investigated using SDS-PAGE analyses. In addition, the enzymatic oligomerization was compared with non-enzymatic oligomerization. The thermally-induced oligomerization of Tg, dependent on glutathione redox state, was affected by the ionic strength or the presence of a surfactant. Meanwhile, PDI-catalyzed oligomerization, time and pH-dependent, was the most remarkable with unfolded/reduced Tg, which was prepared from a treatment with urea/DTT, while the thermally-unfolded Tg was less sensitive. Similarly, the oligomerization of unfolded/reduced Tg was also mediated by peroxidase. However, PDI showed no remarkable effect on the peroxidase-mediated oligomerization of either the unfolded or unfolded/reduced Tg. Additionally, the reductive deoligomerization of oligomeric Tg was exerted by PDI in an excessively reducing state. Based on these results, it is proposed that PDI catalyzes the oligomerization of Tg through the disulfide linkage and its deoligomerization in the molecular fate, and this process may require a specific molecular form of Tg, optimally unfolded/reduced, in a proper redox state.

Oxidative inactivation of brain ecto-5'-nucleotidase by thiols/Fe2+ system.

Abstract5′-Nucleotidase, responsible for the conversion of adenosine-5′-monophosphate into adenosine, was purified from bovine brain membranes, and subjected to oxidative inactivation. The 5′-nucleotidase activity decreased slightly after the exposure to either glutathione or Fe2+. The glutathione-mediated inactivation of 5′-nucleotidase was potentiated remarkably by Fe2+, but not Cu2+, in a concentration-dependent manner. Similarly, glutathione exhibited a concentration-dependent enhancement of the Fe2+-mediated inactivation. In comparison, the glutathione/Fe2+ system was much more effective than the ascorbate/Fe2+ system in inactivating the enzyme. In support of an intermediary role of superoxide ions or H2O2 in the action of glutathione/Fe2+ system, superoxide dismutase and catalase expressed a substantial protection against the inactivation by the glutathione/Fe2+ system. Meanwhile, hydroxyl radical scavangers such as mannitol, benzoate or ethanol were incapable of preventing the inactivation, excluding the participation of extraneous hydroxyl radicals. Whereas adenosine 5′-monophosphate as substrate exhibited a modest protection against the glutathione/Fe2+ action, a remarkable protection was expressed by divalent metal ions such as Zn2+ or Mn2+. Structure-activity study with a variety of thiols indicates that the inactivating action of thiols in combination with Fe2+ resides in the free sulfhydyl group and amino group of thiols. Overall, thiols, expressing more inhibitory effect on the activity of 5′-nucleotidase, were found to be more effective in potentiating the Fe2+- mediated inactivation. Further, kinetic analyses indicate that Fe2+ and thiols inhibit the 5′-nucleotidase in a competitive or uncompetitive manner, respectively. These results suggest that ecto-5′-nucleotidase from brain membrane is one of proteins susceptible to thiols/ Fe2+-catalyzed oxidation, and the oxidative inactivation may be related to the selective association of Fe2+ and thiols to the enzyme molecule.

Regulation and inactivation of brain phosphocholine-phosphatase activity.

Regulation of phosphocholine-hydrolyzing phosphatase (phosphocholine-phosphatase) activity, purified from bovine brain, was examined under physiological conditions. Various endogenous phosphomonoesters, which were utilized as substrate, inhibited the phosphocholine-phosphatase activity competitively (Ki, 5.5–82.0 μ.M); among phosphomonoesters tested, there was a similar order of capability between the binding affinity of substrate and the inhibitory potency. In addition, phosphate ions also inhibited the phosphatase activity competitively with a Ki value of approximately 167 μM. Although leucine or theophylline inhibited the phosphatase activity at pH 9.0, their inhibitory action decreased greatly at pH 7.4. The pH-Km and pH-Vm profiles indicate that ionizable amino acids are involved in substrate binding as well as catalysis, alluding that the phosphatase activity may be highly dependent on the intracellular pH. Amino acid modification study supports the existence of tyrosine, arginine or lysine residue in the active site, and the participation of tyrosine residue in the catalytic action may be suggested positively from the susceptibiliy to the action of tetranitromethane or HOI-generator. Separately, the oxidative inactivation of phosphocholine-phosphatase activity was investigated. Of oxidants tested, HOONO, HOCl, HOl and ascorbate/Cu2+ system were effective to inactivate the phosphatase activity. Noteworthy, a remarkable inactivation was accomplished by 30 μM HOCl in combination with 1 mM Kl. In addition, Cu2+ (3 μ.M) in combination with ascor-bate at concentrations as low as 0.1–0.3 mM reduced the phosphatase activity to a great extent. From these results, it is proposed that the phosphocholine-phosphatase activity may be regulated endogenously and susceptible to the various oxidant systemsin vivo.

Reductive depolymerization of bovine thyroglobulin multimersvia enzymatic reduction of protein disulfide and glutathionylated mixed disulfide linkages

The nascent thyroglobulin (Tg) multimer molecule, which is generated during the initial fate of Tg in ER, undergoes the rapid reductive depolymerization. In an attempt to determine the depolymerization process, various types of Tg multimers, which were generated from deoxycholate-treated/reduced Tg, partially unfolded Tg or partially unfolded/reduced Tg, were subjected to various GSH (reduced glutathione) reducing systems using protein disulfide isomerase (PDI), glutathione reductase (GR), glutaredoxin or thioredoxin reductase. The Tg multimers generated from deoxycholate-treated/reduced Tg were depolymerized readily by the PDI/GSH system, which is consistent with the reductase activity of PDI. The PDI/GSH-induced depolymerization of the Tg multimers, which were generated from either partially unfolded Tg or partially unfolded/reduced Tg, required the simultaneous inclusion of glutathione reductase, which is capable of reducing glutathionylated mixed disulfide (PSSG). This suggests that PSSG was generated during the Tg multimerization stage or its depolymerization stage. In particular, the thioredoxin/thioredoxin reductase system or glutaredoxin system was also effective in depolymerizing the Tg multimers generated from the unfolded Tg. Overall, under the net GSH condition, the depolymerization of Tg multimers might be mediated by PDI, which is assisted by other reductive enzymes, and the mechanism for depolymerizing the Tg multimers differs according to the type of Tg multimer containing different degrees and types of disulfide linkages.