S-nitrosylation Of Protein Disulfide Isomerase Research Articles

CDDO-Me Abrogates Aberrant Mitochondrial Elongation in Clasmatodendritic Degeneration by Regulating NF-κB-PDI-Mediated S-Nitrosylation of DRP1

Clasmatodendrosis is a kind of astroglial degeneration pattern which facilitates excessive autophagy. Although abnormal mitochondrial elongation is relevant to this astroglial degeneration, the underlying mechanisms of aberrant mitochondrial dynamics are still incompletely understood. Protein disulfide isomerase (PDI) is an oxidoreductase in the endoplasmic reticulum (ER). Since PDI expression is downregulated in clasmatodendritic astrocytes, PDI may be involved in aberrant mitochondrial elongation in clasmatodendritic astrocytes. In the present study, 26% of CA1 astrocytes showed clasmatodendritic degeneration in chronic epilepsy rats. 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me; bardoxolone methyl or RTA 402) and SN50 (a nuclear factor-κB (NF-κB) inhibitor) ameliorated the fraction of clasmatodendritic astrocytes to 6.8 and 8.1% in CA1 astrocytes, accompanied by the decreases in lysosomal-associated membrane protein 1 (LAMP1) expression and microtubule-associated protein 1A/1B light-chain 3 (LC3)-II/LC3-I ratio, indicating the reduced autophagy flux. Furthermore, CDDO-Me and SN50 reduced NF-κB S529 fluorescent intensity to 0.6- and 0.57-fold of vehicle-treated animal level, respectively. CDDO-Me and SN50 facilitated mitochondrial fission in CA1 astrocytes, independent of dynamin-related protein 1 (DRP1) S616 phosphorylation. In chronic epilepsy rats, total PDI protein, S-nitrosylated PDI (SNO-PDI), and SNO-DRP1 levels were 0.35-, 0.34- and 0.45-fold of control level, respectively, in the CA1 region and increased CDDO-Me and SN50. Furthermore, PDI knockdown resulted in mitochondrial elongation in intact CA1 astrocytes under physiological condition, while it did not evoke clasmatodendrosis. Therefore, our findings suggest that NF-κB-mediated PDI inhibition may play an important role in clasmatodendrosis via aberrant mitochondrial elongation.

Glutathione depletion results in S-nitrosylation of protein disulfide isomerase in neuroblastoma cells

AimsProtein disulfide isomerase (PDI) is an essential enzyme involved in oxidative protein folding. PDI is S-nitrosylated in the brains of Alzheimer's disease patients, and S-nitrosylated PDI is considered one of main causes of Alzheimer's disease. However, the mechanisms underlying PDI S-nitrosylation have not yet been elucidated. Because glutathione (GSH) depletion is a pathological feature of Alzheimer's disease, we investigated the effect of GSH depletion on the S-nitrosylation level of PDI. Main methodsSH-SY5Y cells, which is a human derived neuroblastoma cells, were used in this study. Glutamate and buthionine sulfoximine (BSO) were used as GSH depletors. S-nitrosylated PDI was detected by biotin-switch assay. Key findingsGSH depletion by glutamate, a cystine/glutamate antiporter xCT inhibitor, increased S-nitrosylated PDI at C343 in SH-SY5Y cells, and induced IRE1α phosphorylation. BSO, a γ-glutamylcysteine synthetase inhibitor, also increased S-nitrosylated PDI and phosphorylated IRE1α upon GSH depletion. Because S-nitrosylated PDI at C343 is stable in neuro cells, S-nitrosylated PDI by GSH depletion progresses to neurodegeneration by the induction of endoplasmic reticulum stress via phosphorylated IRE1α signaling from the early to late stage. Furthermore, treatment with neohesperidin, but not N-acetylcysteine (NAC), improved PDI S-nitrosylation level in GSH-depleted SH-SY5Y cells because nitrosylated compound of NAC induces PDI S-nitrosylation. SignificanceThe results of our study provide a new insight into the mechanisms of neurodegeneration, and may be useful for the development of drugs for Alzheimer's diseases.

Glutathione Depletion Results in S-Nitrosylation of Protein Disulfide Isomerase in Neuroblastoma Cells

Glutathione Depletion Results in S-Nitrosylation of Protein Disulfide Isomerase in Neuroblastoma Cells

Glucose-induced oxidative stress leads to in S-nitrosylation of protein disulfide isomerase in neuroblastoma cells

BackgroundDementia places a significant burden on both patients and caregivers. Since diabetes is a risk factor for dementia, it is imperative to identify the relationship between diabetes and cognitive disorders. Protein disulfide isomerase (PDI) is an enzyme for oxidative protein folding. PDI S-nitrosylation is observed in the brain tissues of Alzheimer's disease patients. The aim of this study is to clarify the relationship between PDI S-nitrosylation and diabetes. MethodsWe used SH-SY5Y cells cultured in high-glucose media. ResultsS-nitrosylated PDI level increased at 7 days and remained high till 28 days in SH-SY5Y cells cultured in high-glucose media. Using PDI wild-type- or PDI C343S-expressing SH-SY5Y cells, PDI C343 was identified as the site of glucose-induced S-nitrosylation. IRE1α and PERK were phosphorylated at day 14 in the SH-SY5Y cells cultured in high-glucose media, and the phosphorylated status was maintained to day 28. To determine the effect of S-nitrosylated PDI on endoplasmic reticulum stress signaling, SH-SY5Y cells were treated with S-nitrosocystein (SNOC) for 30 min, following which the medium was replaced with SNOC-free media and the cells were cultured for 24 h. Only phosphorylated IRE1α treated with SNOC was associated with PDI S-nitrosylation. Neohesperidin, a flavonoid in citrus fruits, is a natural antioxidant. The treatment with neohesperidin in the final 7 days of glucose loading reversed PDI S-nitrosylation and improved cell proliferation. ConclusionGlucose loading leads to S-nitrosylation of PDI C343 and induces neurodegeneration via IRE1α phosphorylation. General significanceThe results may be useful for designing curative treatment strategies for dementia.

Cysteine 343 in the substrate binding domain is the primary S-Nitrosylated site in protein disulfide isomerase

Abnormal protein accumulations are typical pathological features for neurodegenerative diseases. Protein disulfide isomerase (PDI) is a critical enzyme in oxidative protein folding. S-nitrosylated PDI has been detected in the postmortem brain in neurodegenerative disease patients, but the effect of S-nitrosylation on PDI function and developing neurodegeneration was not clarified in detail. In this study, we identified that in vitro and in vivo S-nitrosylation of C343 in the b’ domain of PDI occurs. Reduced recombinant human PDI (hPDI) reacted quickly with S-nitrosocompounds, with an observed increase in the expected S-nitrosylated species and the appearance of the disulfide state of the active sites. Both Mononitrosylated and dinitrosylated were observed from the S-nitrosylation of hPDI. Dinitrosylated species were S-nitrosylated both cysteines at active site. But, at least in part, mononitrosylated species were S-nitrosylated on cysteine 343 in the substrate binding b’ domain. Although active site S-nitrosylation is reversible by reduced glutathione, S-nitrosylation of C343 is comparative stable. S-nitrosylation of PDI in SH-SY5Y cells was observed after the S-nitrosocysteine (SNOC) treatment and S-nitrosylated PDI was still detected 24 h after removing SNOC. While wild-type PDI was S-nitrosylated, the level of S-nitrosylation of the C343S mutant in over-expressed cells was substantially lower and only wild-type PDI of S-nitrosylation remained 24 h after removing SNOC in over-expressed cells. Both of in vitro and in vivo results suggested that S-nitrosylation of C343 in PDI may be the causative effect on physiological changes in neurodegerenative disease patients, and may be useful for the drug development for neurodegenerative diseases.

S-Nitrosylation at the active site decreases the ubiquitin-conjugating activity of ubiquitin-conjugating enzyme E2 D1 (UBE2D1), an ERAD-associated protein

S-Nitrosylation of protein cysteine thiol is a post-translational modification mediated by nitric oxide (NO). The overproduction of NO causes nitrosative stress, which is known to induce endoplasmic reticulum (ER) stress. We previously reported that S-nitrosylation of protein disulfide isomerase (PDI) and the ER stress sensor inositol-requiring enzyme 1 (IRE1) decreases their enzymatic activities. However, it remains unclear whether nitrosative stress affects ER-associated degradation (ERAD), a separate ER stress regulatory system responsible for the degradation of substrates via the ubiquitin-proteasomal pathway.In the present study, we found that the ubiquitination of a known ERAD substrate, serine/threonine-protein kinase 1 (SGK1), is attenuated by nitrosative stress. C-terminus of Hsc70-interacting protein (CHIP) together with ubiquitin-conjugating enzyme E2 D1 (UBE2D1) are involved in this modification. We detected that UBE2D1 is S-nitrosylated at its active site, Cys85 by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, in vitro and cell-based experiments revealed that S-nitrosylated UBE2D1 has decreased ubiquitin-conjugating activity.Our results suggested that nitrosative stress interferes with ERAD, leading to prolongation of ER stress by co-disruption of various pathways, including the molecular chaperone and ER stress sensor pathways. Given that nitrosative stress and ER stress are upregulated in the brains of patient with Parkinson’s disease (PD) and of those with Alzheimer’s disease (AD), our findings may provide further insights into the pathogenesis of these neurodegenerative disorders.

Bisphenol A and rotenone induce S-nitrosylation of protein disulfide isomerase (PDI) and inhibit neurite outgrowth of primary cultured cells of the rat hippocampus and PC12 cells.

Bisphenol A (BPA) interferes the function and development of the central nervous system (CNS), resulting in behavioral abnormalities and memory loss. S-nitrosylation of protein disulfide isomerase (PDI) is increased in brains with sporadic Alzheimer's disease and Parkinson's disease. The aim of the present study was to clarify the role of nitric oxide (NO) in BPA-induced neurotoxicity. Since rotenone induces NO-mediated neurodegeneration through S-nitrosylation of PDI, it was used as a positive control. First, rats were treated with BPA and rotenone, and S-nitrosylation of PDI was detected in rat brain microsomes. BPA and rotenone decreased RNase oxidation activity of PDI concomitant with S-nitrosylation of PDI. Next, to clarify S-nitrosylation of PDI by BPA and rotenone in rat brains, we treated the rat pheochromocytoma cell line PC12 and primary cultured neuron cells from the rat hippocampus with BPA (5 and 10 μM) and rotenone (100 or 200 nM). BPA induced S-nitrosylation of PDI, while NG-monomethyl-L-arginine (L-NMMA), a NOS inhibitor, exerted the opposite effects. Finally, to evaluate the toxicity of BPA in the CNS, we investigated its effects on neurite outgrowth of PC12 and primary cultured neuron cells. BPA inhibited neurite outgrowth of these cells, while L-NMMA reversed this inhibition. The involvement of PDI activity in neurite outgrowth was also examined, and bacitracin, a PDI inhibitor, is shown to decrease neurite outgrowth. Furthermore, the overexpression of PDI, but not a catalytically inactive PDI mutant, enhanced neurite outgrowth. These results suggested that S-nitrosylation of PDI induced by excessive NO caused BPA-induced neurotoxicity.

Protein disulfide isomerase regulation by nitric oxide maintains vascular quiescence and controls thrombus formation

Essentials Nitric oxide synthesis controls protein disulfide isomerase (PDI) function. Nitric oxide (NO) modulation of PDI controls endothelial thrombogenicity. S-nitrosylated PDI inhibits platelet function and thrombosis. Nitric oxide maintains vascular quiescence in part through inhibition of PDI. SUMMARY: Background Protein disulfide isomerase (PDI) plays an essential role in thrombus formation, and PDI inhibition is being evaluated clinically as a novel anticoagulant strategy. However, little is known about the regulation of PDI in the vasculature. Thiols within the catalytic motif of PDI are essential for its role in thrombosis. These same thiols bind nitric oxide (NO), which is a potent regulator of vessel function. To determine whether regulation of PDI represents a mechanism by which NO controls vascular quiescence, we evaluated the effect of NO on PDI function in endothelial cells and platelets, and thrombus formation in vivo. Aim To assess the effect of S-nitrosylation on the regulation of PDI and other thiol isomerases in the vasculature. Methods and results The role of endogenous NO in PDI activity was evaluated by incubating endothelium with an NO scavenger, which resulted in exposure of free thiols, increased thiol isomerase activity, and enhanced thrombin generation on the cell membrane. Conversely, exposure of endothelium to NO+ carriers or elevation of endogenous NO levels by induction of NO synthesis resulted in S-nitrosylation of PDI and decreased surface thiol reductase activity. S-nitrosylation of platelet PDI inhibited its reductase activity, and S-nitrosylated PDI interfered with platelet aggregation, α-granule release, and thrombin generation on platelets. S-nitrosylated PDI also blocked laser-induced thrombus formation when infused into mice. S-nitrosylated ERp5 and ERp57 were found to have similar inhibitory activity. Conclusions These studies identify NO as a critical regulator of vascular PDI, and show that regulation of PDI function is an important mechanism by which NO maintains vascular quiescence.

Decreased levels of PDI and P5 in oligodendrocytes in Alzheimer's disease.

Protein disulfide isomerase (PDI) is a chaperone protein located in the endoplasmic reticulum (ER). Nitric oxide-induced S-nitrosylation of PDI inhibits its enzymatic activity, leading to protein accumulation and activation of the unfolded protein response. Protein disulfide isomerase P5 (P5) is a member of the PDI family that mostly localizes to the ER lumen. Both S-nitrosylated PDI and S-nitrosylated P5 are found in Alzheimer's disease (AD) brain. Previously, we showed that expression of the ER stress marker, growth arrest, and DNA damage protein (GADD34) was significantly increased in neurons and oligodendrocytes in AD brain. In the present study, we showed that PDI and P5 levels were significantly decreased in oligodendrocytes in the brains of AD patients and an AD mouse model. Interestingly, these decreases were evident before the animals displayed typical AD pathology. Because we previously showed that small short interfering RNA knockdown of PDI or P5 could affect the viability of neuronal cells under ER stress, dysfunction of PDI and P5 under ER stress could cause apoptosis of neuronal cells. In summary, we showed that the levels of PDI and P5 were significantly decreased in the oligodendrocytes of AD patients. This phenomenon was also found in an AD mouse model before the animals displayed AD pathology. The overall findings suggest that oligodendrocytes may play important roles in AD pathogenesis.

Regulation of Protein Disulfide Isomerase By S-Nitrosylation Controls Its Function during Thrombus Formation

Protein disulfide isomerase (PDI) is an oxidoreductase that is essential for thrombus formation following vascular injury. Clinical trials testing the efficacy and safety of PDI inhibition in the setting of thrombotic disease are currently underway. Yet while preclinical and clinical trials of PDI in thrombosis have progressed rapidly, the mechanisms by which PDI is regulated in the vasculature and how it mediates thrombosis remain unknown. PDI has an a-b-b’-x-a’ domain structure, where the a and a’ domains contain a CGHC motif responsible for cleaving and forming disulfide bonds. The active site cysteines within the catalytic CGHC motif that perform oxidoreductive reactions can also undergo S-nitrosylation. We have evaluated the hypothesis that nitric oxide (NO) converts PDI into a nitrosylase and regulates PDI oxidoreductase activity in the vasculature during thrombus formation. Initial studies demonstrated that incubation of recombinant PDI with the NO donor, SNAP, resulted in an 83±1.4% decrease in its reductase activity. A transnitrosylase assay using the NO indicator DAF-FM showed that S-nitrosylated PDI (SNO-PDI) transferred NO into platelets and inhibited platelet aggregation. To define the molecular determinants of PDI nitrosylation activity, we evaluated mutant PDIs containing Cys -> Ala mutations of the CGHC (a domain)/CGHC (a’ domain) motifs in the platelet-based transnitrosylase assay. Wild-type PDI (CGHC/CGHC) demonstrated full reductase and nitrosylase activity and the enzymatically dead mutant (AGHA/AGHA) showed neither activity. In contrast, the CGHA/CGHA mutant maintained nitrosylase activity (41±0.23%), but had no reductase activity. This observation suggested that reductase and nitrosylase activities were separable. To further evaluate this supposition, we screened a series of PDI mutants in which intervening sequences of the CGHC domain had been modified. The screen identified CGPC/CGPC as a nitrosylase-biased mutant that showed a 59±2.31% decrease in reductase activity, but a 72±1.83% increase in nitrosylase activity compared to wild-type PDI. Another nitrosylase-biased mutant, CGRC/CGRC, showed a similar activity pattern. Since PDI is prothrombotic and SNO-PDI is antithrombotic, we compared the activity of nitrosylase-biased mutants with wild-type PDI in platelet aggregation studies in the presence of physiological concentrations of GSNO. While wild-type PDI had little effect on platelet aggregation, nitrosylase-biased PDIs such as the CGPC/CGPC and CGRC/CGRC mutant completely inhibited platelet aggregation. These studies show that the prothrombotic oxidoreductase activities of PDI are separable from their antithrombotic nitrosylase activities and that nitrosylase-biased PDI mutants have antiplatelet activity. We next evaluated the effect of PDI nitrosylation on thrombus formation in vivo. Infusion of SNO-PDI into mice inhibited thrombus formation following laser-induced vascular injury of cremaster arterioles. Mice deficient in glutathione-S-nitrosyl reductase (GSNOR) were used to assess the role of endogenous NO in thrombus formation. GSNOR enzymatically reduces GSNO, the main storage form of NO in cells. Platelet accumulation and fibrin formation were hardly detectable in GSNOR-/- mice. Infusion of recombinant WT PDI, but not an enzymatically dead PDI, reversed the defect in platelet accumulation and fibrin generation to levels of WT mice. In order to visualize NO during thrombus formation, the NO-sensitive dye DAF-FM was infused into mice and NO signal in endothelium monitored following laser-induced injury. DAF-FM signal decreased rapidly following laser injury of cremaster arterioles, indicating an activation-induced reduction in endothelial NO in vivo. In conclusion, our studies show that oxidoreductase and nitrosylase activities of PDI are separable and support a model whereby high endothelial NO levels maintain vascular quiescence in part by maintaining PDI as a nitrosylase and blocking its prothrombotic PDI activity. We propose that the reduction of NO levels that occurs with vascular injury or endothelial dysfunction contributes to the conversion of PDI from an anti-thrombotic nitrosylase to a prothrombotic reductase. DisclosuresNo relevant conflicts of interest to declare.

Alpha-synuclein oligomerization in manganese-induced nerve cell injury in brain slices: a role of NO-mediated S-nitrosylation of protein disulfide isomerase.

Over-exposure to manganese (Mn) has been known to induce endoplasmic reticulum (ER) stress involving protein misfolding. The proper maturation and folding of native proteins rely on the activity of protein disulfide isomerase (PDI). However, the exact mechanism of Mn-induced alpha-synuclein oligomerization is unclear. To explore whether alpha-synuclein oligomerization was associated with S-nitrosylation of PDI, we made the rat brain slice model of manganism and pretreated slices with L-Canavanine, a selective iNOS inhibitor. After slices were treated with Mn (0, 25, 100, and 400 μM) for 24 h, there were dose-dependent increases in apoptotic percentage of cells, lactate dehydrogenase (LDH) releases, production of NO, inducible nitric oxide synthase (iNOS) activity, the mRNA and protein expressions of iNOS, and PDI. Moreover, S-nitrosylated PDI and alpha-synuclein oligomerization also increased. However, there was a significant increase in the PDI activity of 25-μM Mn-treated slices. Then, PDI activity and the affinity between PDI and alpha-synuclein decreased significantly in response to Mn (100 and 400 μM), which was associated with S-nitrosylation of PDI. The results indicated that S-nitrosylated PDI could affect its activity. We use the L-Canavanine pretreatment brain slices to inhibit S-nitrosylation of PDI. The results showed that L-Canavanine pretreatment could reduce Mn-induced nerve cell injury and alpha-synuclein oligomerization. Additionally, there was a significant recovery in PDI activity in L-Canavanine-pretreated slices. The findings revealed that Mn induced nitrosative stress via the activation of iNOS and subsequent S-nitrosylation of PDI in cultured slices. Moreover, S-nitrosylation of PDI is an important signaling event in the Mn-induced alpha-synuclein oligomerization in brain slices.

Protein Disulfide Isomerase P5-Immunopositive Inclusions in Patients with Alzheimer's Disease

Amyloid plaques and neurofibrillary tangles (NFTs) are the major pathological characteristics of Alzheimer's disease (AD). NFTs are composed of tubular filaments and paired helical filaments containing polymerized hyperphosphorylated tau protein. Another feature of AD is excessive generation of nitric oxide (NO). Protein disulfide isomerase (PDI) is a chaperon protein located in the endoplasmic reticulum (ER). It was recently reported that NO-induced S-nitrosylation of PDI inhibits its enzymatic activity, leading to the accumulation of polyubiquitinated proteins, and activates the unfolded protein response. In addition, we previously reported the presence of PDI-immunopositive NFTs in AD. Here, we found that protein disulfide isomerase P5 (P5), which is a member of the PDI protein family, was co-localized with tau in NFTs. To our knowledge, this is the first report of P5-immunopositive inclusion in AD. Furthermore, we showed that S-nitrosylated P5 was present and the expression level of P5 was decreased in AD brains compared with that of control brains. We also demonstrated that the knock-down of PDI or P5 by siRNA could affect the viability of SH-SY5Y cells under ER stress. Previously, the observation of S-nitrosylated PDI in AD was reported. NO may inhibit P5 by inducing S-nitrosylation in the same manner as PDI, which inhibits its enzymatic activity allowing protein misfolding to occur in AD. The accumulation of misfolded proteins induces ER stress and may cause apoptosis of neuronal cells through S-nitrosylation and down-regulation of PDI and P5 in AD.

Potential Effect of S-Nitrosylated Protein Disulfide Isomerase on Mutant SOD1 Aggregation and Neuronal Cell Death in Amyotrophic Lateral Sclerosis

Aggregation of misfolded protein and resultant intracellular inclusion body formation are common hallmarks of mutant superoxide dismutase (mSOD1)-linked familial amyotrophic lateral sclerosis (FALS) and have been associated with the selective neuronal death. Protein disulfide isomerase (PDI) represents a family of enzymatic chaperones that can fold nascent and aberrant proteins in the endoplasmic reticulum (ER) lumen. Recently, our group found that S-nitrosylated PDI could contribute to protein misfolding and subsequent neuronal cell death. However, the exact role of PDI in the pathogenesis of ALS remains unclear. In this study, we propose that PDI attenuates aggregation of mutant/misfolded SOD1 and resultant neurotoxicity associated with ER stress. ER stress resulting in PDI dysfunction therefore provides a mechanistic link between deficits in molecular chaperones, accumulation of misfolded proteins, and neuronal death in neurodegenerative diseases. In contrast, S-nitrosylation of PDI inhibits its activity, increases mSOD1 aggregation, and increases neuronal cell death. Specifically, our data show that S-nitrosylation abrogates PDI-mediated attenuation of neuronal cell death triggered by thapsigargin. Biotin switch assays demonstrate S-nitrosylated PDI both in the spinal cords of SOD1 (G93A) mice and human patients with sporadic ALS. Therefore, denitrosylation of PDI may have therapeutic implications. Taken together, our results suggest a novel strategy involving PDI as a therapy to prevent mSOD1 aggregation and neuronal degeneration. Moreover, the data demonstrate that inactivation of PDI by S-nitrosylation occurs in both mSOD1-linked and sporadic forms of ALS in humans as well as mice.

S‐nitrosylated protein disulfide isomerase contributes to mutant SOD1 aggregates in amyotrophic lateral sclerosis

A major hallmark of mutant superoxide dismutase (SOD1)-linked familial amyotrophic lateral sclerosis is SOD1-immunopositive inclusions found within motor neurons. The mechanism by which SOD1 becomes aggregated, however, remains unclear. In this study, we aimed to investigate the role of nitrosative stress and S-nitrosylation of protein disulfide isomerase (PDI) in the formation of SOD1 aggregates. Our data show that with disease progression inducible nitric oxide synthase (iNOS) was up-regulated, which generated high levels of nitric oxide (NO) and subsequently induced S-nitrosylation of PDI in the spinal cord of mutant SOD1 transgenic mice. This was further confirmed by in vitro observation that treating SH-SY5Y cells with NO donor S-nitrosocysteine triggered a dose-dependent formation of S-nitrosylated PDI. When mutant SOD1 was over-expressed in SH-SY5Y cells, the iNOS expression was up-regulated, and NO generation was consequently increased. Furthermore, both S-nitrosylation of PDI and the formation of mutant SOD1 aggregates were detected in the cells expressing mutant SOD1(G93A). Blocking NO generation with the NOS inhibitor N-nitro-L-arginine attenuated the S-nitrosylation of PDI and inhibited the formation of mutant SOD1 aggregates. We conclude that NO-mediated S-nitrosylation of PDI is a contributing factor to the accumulation of mutant SOD1 aggregates in amyotrophic lateral sclerosis.

SOD1 aggregation in astrocytes following ischemia/reperfusion injury: a role of NO-mediated S-nitrosylation of protein disulfide isomerase (PDI)

BackgroundUbiquitinated-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.MethodsTo 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.ResultsOur 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.ConclusionsNO-mediated S-nitrosylation of PDI may be involved in the formation of the SOD1-linked ubiquitinated-protein aggregates in cerebral ischemia/reperfusion injury.

Protein Disulfide Isomerase Immunopositive Glial Cytoplasmic Inclusions in Patients With Multiple System Atrophy

ABSTRACTBackground: Glial cytoplasmic inclusions (GCIs) are the pathological hallmarks of multiple system atrophy (MSA) and α-synuclein is abnormally deposited in GCIs. Protein disulfide isomerase (PDI) is a member of the thioredoxin superfamily and is believed to accelerate the folding of disulfide-bonded proteins by catalyzing the disulfide interchange reaction, which is the rate-limiting step during protein folding in the luminal space of the endoplasmic reticulum (ER). Nitric-oxide-induced (NO-induced) S-nitrosylation of PDI inhibits its enzymatic activity, leading to the accumulation of polyubiquitinated proteins, and activates the unfolded protein response in neurodegenerative diseases. Materials and Methods: Postmortem brain specimens from five patients with MSA and five normal control brains were utilized in this immunohistochemical study. Results: We found GCIs positive for anti-PDI antibody in the brain of patients with MSA. In addition, we observed colocalization of α-synuclein and leucine-rich repeat kinase 2 (LRRK2) with PDI in GCIs. As LRRK2 immunoreactivity is associated with one of the earliest oligodendrocytic abnormalities in MSA, colocalization of LRRK2 and PDI in GCIs may be a link to the ER stress of glial cells in the early stages of MSA. Conclusions: In MSA, NO may inhibit PDI by inducing S-nitrosylation, which inhibits its enzymatic activity and thus allows protein misfolding to occur.

Redox modulation by S-nitrosylation contributes to protein misfolding, mitochondrial dynamics, and neuronal synaptic damage in neurodegenerative diseases

The pathological processes of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases engender synaptic and neuronal cell damage. While mild oxidative and nitrosative (nitric oxide (NO)-related) stress mediates normal neuronal signaling, excessive accumulation of these free radicals is linked to neuronal cell injury or death. In neurons, N-methyl-D-aspartate (NMDA) receptor (NMDAR) activation and subsequent Ca(2+) influx can induce the generation of NO via neuronal NO synthase. Emerging evidence has demonstrated that S-nitrosylation, representing covalent reaction of an NO group with a critical protein thiol, mediates the vast majority of NO signaling. Analogous to phosphorylation and other posttranslational modifications, S-nitrosylation can regulate the biological activity of many proteins. Here, we discuss recent studies that implicate neuropathogenic roles of S-nitrosylation in protein misfolding, mitochondrial dysfunction, synaptic injury, and eventual neuronal loss. Among a growing number of S-nitrosylated proteins that contribute to disease pathogenesis, in this review we focus on S-nitrosylated protein-disulfide isomerase (forming SNO-PDI) and dynamin-related protein 1 (forming SNO-Drp1). Furthermore, we describe drugs, such as memantine and newer derivatives of this compound that can prevent both hyperactivation of extrasynaptic NMDARs as well as downstream pathways that lead to nitrosative stress, synaptic damage, and neuronal loss.

S-Nitrosylation of Critical Protein Thiols Mediates Protein Misfolding and Mitochondrial Dysfunction in Neurodegenerative Diseases

Excessive nitrosative and oxidative stress is thought to trigger cellular signaling pathways leading to neurodegenerative conditions. Such redox dysregulation can result from many cellular events, including hyperactivation of the N-methyl-D-aspartate-type glutamate receptor, mitochondrial dysfunction, and cellular aging. Recently, we and our colleagues have shown that excessive generation of free radicals and related molecules, in particular nitric oxide species (NO), can trigger pathological production of misfolded proteins, abnormal mitochondrial dynamics (comprised of mitochondrial fission and fusion events), and apoptotic pathways in neuronal cells. Emerging evidence suggests that excessive NO production can contribute to these pathological processes, specifically by S-nitrosylation of specific target proteins. Here, we highlight examples of S-nitrosylated proteins that regulate misfolded protein accumulation and mitochondrial dynamics. For instance, in models of Parkinson's disease, these S-nitrosylation targets include parkin, a ubiquitin E3 ligase and neuroprotective molecule, and protein-disulfide isomerase, a chaperone enzyme for nascent protein folding. S-Nitrosylation of protein-disulfide isomerase may also be associated with mutant Cu/Zn superoxide dismutase toxicity in amyotrophic lateral sclerosis. Additionally, in models of Alzheimer's disease, excessive NO generation leads to the formation of S-nitrosylated dynamin-related protein 1 (forming SNO-Drp1), which contributes to abnormal mitochondrial fragmentation and resultant synaptic damage.

Accumulation of Misfolded Protein Through Nitrosative Stress Linked to Neurodegenerative Disorders

Protein quality control is a critical feature of intracellular homeostasis. In particular, unfolded or misfolded proteins resulting from environmental stresses or free radicals are rapidly degraded via the ubiquitin-proteasome pathway. Nitric oxide (NO), a free radical gas, has been reported to be involved in such processes as vasorelaxation and neurotransmission. Conversely, NO also is implicated in neuronal cell death or neurodegeneration. Recent reports suggest that S-nitrosylation of proteins is a significant cause of neural dysfunction leading to neurodegenerative disorders. Specifically, S-nitrosylation of parkin eventually leads to the accumulation of unfolded proteins and subsequent neuronal death. The focus of this review is the identity of the target of NO. Nitrosative stress prevents normal functioning of the endoplasmic reticulum (ER) via S-nitrosylation of protein-disulfide isomerase (PDI), which is located in the ER lumen. This may contribute to the accumulation of misfolded proteins, as well as sustained activation of the unfolded protein response (UPR) pathway. These phenomena may be linked to the development of sporadic neurodegenerative diseases.