Cell-Surface Protein Disulfide Isomerase Is Required for Transnitrosation of Metallothionein by S-Nitroso-Albumin in Intact Rat Pulmonary Vascular Endothelial Cells
Liming ZhangClaudette M. St. CroixRong CaoKarla WasserloosSimon C. WatkinsTroy StevensSong LiVladimir A. TyurinValerian E. KaganBruce R. Pitt
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S-nitrosation of the metal binding protein, metallothionein (MT) appears to be a critical link in affecting endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO)-induced changes in cytoplasmic and nuclear labile zinc, respectively. Although low molecular weight S-nitrosothiols also appear to affect this signaling system, less is known about the ability of extracellular protein nitrosothlols to transnitrosate MT. Accordingly, we synthesized fluorescently labeled S-nitroso-albumin (SNO-albumin, a major protein S-nitrosothiol in plasma) and determined, Waconfocal microscopy in fixed tissue, that it is transported into cultured rat pulmonary vascular endothelial cells in a temperature sensitive fashion. The cells were transfected with an expression vector that encodes human MT-IIa cDNA sandwiched between enhanced cyan (donor) and yellow (acceptor) fluorescent proteins (FRET-MT) that can detect conformational changes in MT through fluorescence resonance energy transfer (FRET). SNO-albumin and the membrane-permeant low molecular weight S-nitroso-L-cysteine ethyl ester (L-SNCEE) caused a conformational change in FRET-MT as ascertained by full spectral laser scanning confocal microscopy in live rat pulmonary vascular endothelial cells, a result which is consistent with transnitrosation of the reporter molecule. Transnitrosation of FRET-MT by SNO-albumin, but not L-SNCEE, was sensitive to antisense oligonucleotide-mediated inhibition of the expression of cell surface protein disulfide Isomerase (csPDI). These results extend the original observations of Ramachandran et al. (Ramachandran N, Root P, Jiang XM, Hogg PJ, Mutus B. Proc Natl Acad Sci U S A 98:9539–9544, 2001) and suggest that csPDI-mediated denitrosation helps to regulate the ability of the major plasma NO carrier (SNO-albumin) to transnitrosate endothelial cell molecular targets (e.g. MT).Keywords:
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Abstract Background Ubiquitinated-protein aggregates are implicated in cerebral ischemia/reperfusion injury. The very presence of these ubiquitinated-protein aggregates is abnormal and seems to be disease-related. However, it is not clear what leads to aggregate formation and whether the aggregations represent a reaction to aggregate-mediated neurodegeneration. Methods To study the nitrosative stress-induced protein aggregation in cerebral ischemia/reperfusion injury, we used primary astrocyte cultures as a cell model, and systematically examined their iNOS expression and consequent NO generation following oxygen glucose deprivation and reperfusion. The expression of protein disulfide isomerase (PDI) and copper-zinc superoxide dismutase (SOD1) were also examined, and the biochemical interaction between PDI and SOD1 was determined by immunoprecipitation. In addition, the levels of S-nitrosylated PDI in cultured astrocytes after oxygen glucose deprivation and reperfusion treatment were measured using the biotin-switch assay. The formation of ubiquitinated-protein aggregates was detected by immunoblot and immunofluorescence staining. Results Our data showed that the up-regulation of iNOS expression after oxygen glucose deprivation and reperfusion treatment led to excessive NO generation. Up-regulation of PDI and SOD1 was also identified in cultured astrocytes following oxygen glucose deprivation and reperfusion, and these two proteins were found to bind to each other. Furthermore, the increased nitrosative stress due to ischemia/reperfusion injury was highly associated with NO-induced S-nitrosylation of PDI, and this S-nitrosylation of PDI was correlated with the formation of ubiquitinated-protein aggregates; the levels of S-nitrosylated PDI increased in parallel with the formation of aggregates. When NO generation was pharmacologically inhibited by iNOS specific inhibitor 1400W, S-nitrosylation of PDI was significantly blocked. In addition, the formation of ubiquitinated-protein aggregates in cultured astrocytes following oxygen glucose deprivation and reperfusion was also suppressed by 1400W. Interestingly, these aggregates were colocalized with SOD1, which was found to co-immunoprecipitate with PDI. Conclusions NO-mediated S-nitrosylation of PDI may be involved in the formation of the SOD1-linked ubiquitinated-protein aggregates in cerebral ischemia/reperfusion injury.
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The concentrations of zinc, copper, metallothionein and metallothionein‐Ia mRNA in sheep livers during development was determined. It was found that early sheep foetuses (30–40 days gestation) had very high concentrations of hepatic zinc (2305±814 μg/g dry mass), and that these levels declined steadily to 644±304 μg/g near to term. The copper concentrations in the foetal livers were not higher than those in the adult. The concentrations of metallothionein and metallothionein‐Ia mRNA were also very high in the foetal livers and declined steadily during gestation from 261±94 molecules/pg RNA to 71±18 molecules/pg near to term. Metallothionein‐Ia mRNA concentrations were closely correlated with hepatic zinc concentrations but not with copper. Metallothionein concentrations also decreased during gestation: e.g. 3044 μg/g (wet mass) in one foetus on day 34 of gestation to 862 μg/g on day 125. After birth, however, the concentrations of metallothionein declined to less than 100 μg/g and this decline occurred despite the presence of significant quantities of mRNA. The ratio of metallothionein/metallothionein‐Ia mRNA decreased from 1.3 to 3.2 × 10 5 molecules metallothionein/molecule of metallothionein‐Ia mRNA during gestation to between 0.28–0.64 × 10 5 molecules/molecule in the postnatal animals. We conclude that the major function of metallothioneins in the foetal liver is protection of the liver against the potentially toxic accumulation of zinc. In the postnatal sheep there appears to be a decreased synthesis or increased degradation of metallothionein.
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Abstract Background P2X7 receptor (P2X7R) is an ATP-gated nonselective cationic channel playing important roles in a variety of physiological functions, including inflammation, and apoptotic or necrotic cell death. An extracellular domain has ten cysteine residues forming five intrasubunit disulfide bonds, which are needed for the P2X7R trafficking to the cell surface and the recognition of surface epitopes of apoptotic cells and bacteria. However, the underlying mechanisms of redox/ S -nitrosylation of cysteine residues on P2X7R and its role in P2X7R-mediated post-status epilepticus (SE, a prolonged seizure activity) events remain to be answered. Methods Rats were given pilocarpine (380 mg/kg i.p.) to induce SE. Animals were intracerebroventricularly infused N ω -nitro- l -arginine methyl ester hydrochloride (L-NAME, a NOS inhibitor) 3 days before SE, or protein disulfide isomerase (PDI) siRNA 1 day after SE using an osmotic pump. Thereafter, we performed Western blot, co-immunoprecipitation, membrane fraction, measurement of S -nitrosylated (SNO)-thiol and total thiol, Fluoro-Jade B staining, immunohistochemistry, and TUNEL staining. Results SE increased S -nitrosylation ratio of P2X7R and the PDI-P2X7R bindings, which were abolished by L-NAME and PDI knockdown. In addition, both L-NAME and PDI siRNA attenuated SE-induced microglial activation and astroglial apoptosis. L-NAME and PDI siRNA also ameliorated the increased P2X7R surface expression induced by SE. Conclusions These findings suggest that PDI-mediated redox/ S -nitrosylation may facilitate the trafficking of P2X7R, which promotes microglial activation and astroglial apoptosis following SE. Therefore, our findings suggest that PDI-mediated regulations of dynamic redox status and S -nitrosylation of P2X7R may be a critical mechanism in the neuroinflammation and astroglial death following SE.
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Up-regulation of protein disulfide isomerase (PDI) is an adaptive response to accumulation of misfolded proteins in the endoplasmic reticulum (ER) that helps protect neurons from apoptosis resulting from ER stress. After determining that exposure to a nitric oxide (NO) donor or activation of neuronal NO synthase (nNOS) expressed in HEK-293T cells led to PDI S -nitrosylation, Uehara et al. showed that S -nitrosylated PDI was present in the brains of people who had had Parkinson's disease or Alzheimer's disease. S -nitrosylation impaired the ability of PDI to act as a chaperone (assayed by inhibition of guanidinium-dependent rhodanese aggregation) and an isomerase (assayed by renaturation of an inactive form of RNase A with scrambled disulfide bonds). When coexpressed with synphilin-1 in a dopaminergic neuroblastoma cell line, PDI inhibited the development of synphilin-1-dependent Lewy-body-like inclusions, a protective effect that was attenuated by NO. Exposure of cultured cortical neurons to N -methyl-D-aspartate (NMDA) led to NOS-sensitive accumulation of S -nitrosylated PDI, accumulation of polyubiquitinated proteins, and apoptosis; PDI overexpression decreased the number of polyubiquitinated and apoptotic cells and attenuated NMDA-dependent activation of the unfolded protein response. In neuroblastoma cells, PDI overexpression inhibited cell death in response to ER stress, inhibition of the proteasome, or overexpression of a protein that induces the unfolded protein response, protective effects that were reversed by exposure to a NO donor. Thus, the authors conclude that, in neurodegenerative disorders, S -nitrosylation of PDI by NO attenuates its ability to protect neurons from the neurotoxic effects of ER stress. T. Uehara, T. Nakamura, D. Yao, Z.-Q. Shi, Z. Gu, Y. Ma, E. Masliah, Y. Nomura, S. A. Lipton, S -nitrosylated protein-disulphide isomerase links protein misfolding to neurodegeneration. Nature 441 , 513-517 (2006). [PubMed]
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The Induced Synthesis of Metallothionein in Various Tissues of Rat after Injection of Various Metals
Daily subcutaneous injection of twelve kinds of metal into rats were carried out for one, two or four days at a dosage of 10 mg per kg. After measuring the concentrations of metallothionein and metals in four kinds of organ, the following results were obtained. 1) The apparent inducibilities of metallothionein by Co and Ni in the liver were almost the same as that by Zn. Those by Ca and Mn were 0.44 and 0.36 times that of Zn, respectively. By the administration of Al, Cr, Fe, K, Mg, Na or Pb no significant increase in the concentration of metallothionein in the liver was detected. 2) The apparent inducibility of metallothionein by Ni in the kidney was about twice that by Zn. Co also had a weak apparent inducibility. No other metals used in this experiment increased the concentration of metallothionein in the kidney. 3) Metals other than Zn examined did not induce metallothionein in the small intestine and pancreas. 4) Although a positive correlation was detected between Zn and metallothionein concentration in the organs when metallothionein was induced, between metals administered and metallothionein concentration in the organs a positive correlation was not always given.
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Abstract Protein S -nitrosylation modulates important cellular processes, including neurotransmission, vasodilation, proliferation and apoptosis in various cell types. We have previously reported that protein disulfide isomerase (PDI) is S -nitrosylated in brains of patients with sporadic neurodegenerative diseases. This modification inhibits PDI enzymatic activity and consequently leads to the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) lumen. Here, we describe S -nitrosylation of additional ER pathways that affect the unfolded protein response (UPR) in cell-based models of Parkinson’s disease (PD). We demonstrate that nitric oxide (NO) can S -nitrosylate the ER stress sensors IRE1α and PERK. While S -nitrosylation of IRE1α inhibited its ribonuclease activity, S -nitrosylation of PERK activated its kinase activity and downstream phosphorylation/inactivation or eIF2α. Site-directed mutagenesis of IRE1α(Cys931) prevented S -nitrosylation and inhibition of its ribonuclease activity, indicating that Cys931 is the predominant site of S -nitrosylation. Importantly, cells overexpressing mutant IRE1α(C931S) were resistant to NO-induced damage. Our findings show that nitrosative stress leads to dysfunctional ER stress signaling, thus contributing to neuronal cell death.
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Induction of hepatic metallothionein was investigated in zinc-depleted rats after a single feeding (via stomach tube) of a complete diet with or without supplemental zinc. Subsequent to a maximum elevation in serum zinc (3.25 micrograms/ml) at 3 h, the rate of metallothionein synthesis increased 4.5-fold by 10 h after feeding. Changes in the rate of metallothionein synthesis coincided with similar changes in the level of translatable mRNA coding for metallothionein. Accumulation in liver of Zn2+ as metallothionein rose to a maximum by 12 h after diet administration and thereafter remained constant. Radioactive zinc (65Zn) included in the diet rapidly associated with newly formed metallothionein. Unlike Zn2+, 65Zn exhibited marked fluctuations within the 24-h period following feeding, indicating that zinc associated with metallothionein may be capable of exchange and/or dissociation. Changes in total liver 65Zn were accompanied by concomitant changes in metallothionein-bound 65Zn. This study has related (temporally) the metabolism of dietary zinc to the induction, apparently via transcription of mRNA, of hepatic metallothionein.
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