Characterization of the S-Denitrosation Activity of Protein Disulfide Isomerase

Abstract

S-Nitrosoglutathione (GSNO) denitrosation activity of recombinant human protein disulfide isomerase (PDI) has been kinetically characterized by monitoring the loss of the S-NO absorbance, using a NO electrode, and with the aid of the fluorogenic NO x probe 2,3-diaminonaphthalene. The initial rates of denitrosation as a function of [GSNO] displayed hyperbolic behavior irrespective of the method used to monitor denitrosation. The K m values estimated for GSNO were 65 ؎ 5 M and 40 ؎ 10 M for the loss in the S-NO bond and NO production (NO electrode or 2,3-diaminonaphthalene), respectively. Hemoglobin assay provided additional evidence that the final product of PDI-dependent GSNO denitrosation was NO ⅐ . A catalytic mechanism, involving a nitroxyl disulfide intermediate stabilized by imidazole (His 160 a-domain or His 589 a-domain), which after undergoing a one-electron oxidation decomposes to yield NO plus dithiyl radical, has been proposed. Evidence for the formation of thiyl/dithiyl radicals during PDI-catalyzed denitrosation was obtained with 4-((9-acridinecarbonyl)-amino)-2,2,6,6-tetramethylpiperidine-1-oxyl. Evidence has also been obtained showing that in a NO-and O 2 -rich environment, PDI can form N 2 O 3 in its hydrophobic domains. This "NO-charged PDI" can perform intra-and intermolecular S-nitrosation reactions similar to that proposed for serum albumin. Interestingly, reduced PDI was able to denitrosate S-nitrosated PDI (PDI-SNO) resulting in the release of NO. PDI-SNO, once formed, is stable at room temperature in the absence of reducing agent over the period of 2 h. It has been established that PDI is continuously secreted from cells that are net producers of NO-like endothelial cells. The present demonstration that PDI can be S-nitrosated and that PDI-SNO can be denitrosated by PDI suggests that this enzyme could be intimately involved in the transport of intracellular NO equivalents to the cell surface as well as the previous demonstration of PDI in the transfer of S-nitrosothiol-bound NO to the cytosol. Protein disulfide isomerase (PDI)1 was identified about 40 years ago (1). Although large levels of this enzyme are found in the endoplasmic reticulum, PDI is secreted from cells in which it associates electrostatically with the cell surface (2, 3). One of the most studied functions of PDI is its ability to catalyze isomerization and rearrangement of disulfide bonds in the endoplasmic reticulum, contributing to a proper folding of nascent proteins (4). Cell surface PDI was initially discovered in platelets (5), in which it plays a dual role in integrin-mediated adhesion and aggregation (6, 7), RSNO-mediated platelet inhibition, and GSNO denitrosation (8).S-Nitrosothiols (RSNOs) are known nitric oxide (NO) donors. RSNOs range in size from low molecular weight, such as cysteine-NO and homocysteine-NO, to high molecular weight nitrosated proteins, such as serum albumin-NO. They are known to prolong NO half-life (9) and to act as a NO transport system in a cellular environment (10 -12).An additional novel...

Redox-based regulation of signal transduction: Principles, pitfalls, and promises

“…PDI is also surface associated, where it may play a role in transport of NO from S-nitrosothiols across membranes (whether directly or by maintaining surfact thiols in the reduced state is not clear) [160,161]. Interestingly, S-nitrosylation of PDI [162,163] has been linked to neurodegenerative disease [162]. Interestingly, PDI itself can mediate its own denitrosylation [163].…”

“…Interestingly, S-nitrosylation of PDI [162,163] has been linked to neurodegenerative disease [162]. Interestingly, PDI itself can mediate its own denitrosylation [163].…”

S-Nitrosylation and uncompetitive/fast off-rate (UFO) drug therapy in neurodegenerative disorders of protein misfolding

“…6,8,9,12,14,16,17,[19][20][21][22]48 Over the past decade, accumulating evidence has suggested that S-nitrosylation can regulate the biological activity of a great variety of proteins, in some ways akin to phosphorylation. 10,[49][50][51][52][53][54] Chemically, NO is often a good 'leaving group,' facilitating further oxidation of critical thiol to disulfide bonds among neighboring (vicinal) cysteine residues or, via reaction with ROS, to sulfenic (ÀSOH), sulfinic (ÀSO 2 H), or sulfonic (ÀSO 3 H) acid derivatization of the protein. 19,20,22,55 Alternatively, S-nitrosylation may possibly produce a nitroxyl disulfide, in which the NO group is shared by close cysteine thiols.…”

“…Further evidence suggests that PDI may transport NO to the extracellular space, where it could conceivably exert additional adverse effects. 51 In addition to PDI, S-nitrosylation is likely to affect critical thiol groups on other chaperones, such as heat-shock protein (HSP) 90 in the cytoplasm 92 and possibly GRP in the ER. Normally, HSP90 stabilizes misfolded proteins and modulates the activity of cell signaling proteins, including NOS and calreticulin.…”

Nitrosative Stress in the ER: A New Role for S-Nitrosylation in Neurodegenerative Diseases

“…Earlier studies have indicated that PDI can break down S-nitrosoglutathione (32) and, when localized to the plasma membrane, may participate in cellular import of extracellular S-nitrosothiols (33,34). In both cases, SNO-PDI was a suggested (short-lived) intermediate.…”