Protein disulfide isomerase a multifunctional protein with multiple physiological roles

Khan, Hyder Ali; Mutus, Bülent

doi:10.3389/fchem.2014.00070

Cited by 156 publications

(116 citation statements)

References 93 publications

(145 reference statements)

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“…Two broad categories of PDI assays have been employed [9,12,32,43,47,48]. The first involves the ability of PDI to catalyze the isomerization of mispaired disulfides in peptides and proteins.…”

Section: Resultsmentioning

confidence: 99%

Challenges in the evaluation of thiol-reactive inhibitors of human protein disulfide Isomerase

Foster

Thorpe

2017

Free Radical Biology and Medicine

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This paper addresses how to evaluate the efficacy of the growing inventory of thiol-reactive inhibitors of mammalian protein disulfide isomerase (PDI) enzymes under realistic concentrations of potentially competing thiol-containing peptides and proteins. For this purpose, we introduce a variant of the widely-used reductase assay by using a commercially-available cysteine derivative (BODIPY FL L-Cystine; BD-SS) that yields a 55-fold increase in fluorescence (excitation/emission; 490/513 nm) on scission of the disulfide bond. This plate reader-compatible method detects human PDI down to 5–10 nM, can utilize a range of thiol substrates (including 5 μM dithiothreitol, 10 μM reduced RNase thiols, and 5 mM glutathione; GSH), and can operate from pH 6–9.5 in a variety of buffers. PDI assays often employ low micromolar levels of substrates leading to ambiguities when thiol-directed inhibitors are evaluated. The present work utilizes 5 mM GSH for both pre-incubation and assay phases to more realistically reflect the high concentration of thiols that an inhibitor would encounter intracellularly. Extracellular PDI faces a much lower concentration of potentially competing thiols; to assess reductase activity under these conditions, the pre-reduced PDI is treated with inhibitor and then fluorescence increase upon reduction of BD-SS is followed in the absence of additional competing thiols. Both assay modes were tested with four mechanistically diverse PDI inhibitors. Two reversible reagents, 3,4-methylenedioxy-β-nitrostyrene (MNS) and the arsenical APAO, were found to be strong inhibitors of PDI in the absence of competing thiols, but were ineffective in the presence of 5 mM GSH. A further examination of the nitrostyrene showed that MNS not only forms facile Michael adducts with GSH, but also with the thiols of unfolded proteins (Kd values of 7 and <0.1 μM, respectively) suggesting the existence of multiple potential intracellular targets for this membrane-permeant reagent. The inhibition of PDI by the irreversible alkylating agent, the chloroacetamide 16F16, was found to be only modestly attenuated by 5 mM GSH. Finally, the thiol-independent flavonoid inhibitor quercetin-3-O-rutinoside was found to show equal efficacy in reoxidation and turnover assay types. This work provides a framework to evaluate inhibitors that may target the CxxC motifs of PDI and addresses some of the complexities in the interpretation of the behavior of thiol-directed reagents in vivo.

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Section: Resultsmentioning

confidence: 99%

Challenges in the evaluation of thiol-reactive inhibitors of human protein disulfide Isomerase

Foster

Thorpe

2017

Free Radical Biology and Medicine

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“…PDI is localized in the lumen of the endoplasmic reticulum (ER) as well as the cell surface and cytosol. Recent studies revealed that PDI, through its thiol redox activity, plays a role in pathogenesis, promotes increased ROS in cells and protein aggregation . It is known that covalent modification of PDI hinders its normal function in specific pathologies such as cancer, neurodegenerative disorders, cardiovascular and infectious diseases .…”

Section: Resultsmentioning

confidence: 99%

Identification of 4‐hydroxynonenal protein targets in rat, mouse and human liver microsomes by two‐dimensional liquid chromatography/tandem mass spectrometry

Golizeh

Geib

Sleno

2016

Rapid Comm Mass Spectrometry

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“…However, once nitrosylated, the chaperone and isomerase activities of these proteins are attenuated markedly, reducing the efficacy of this defensive strategy [78]. This has particularly serious consequences as misfolded proteins are a source of free radical damage in their own right, and hence, the loss of the protein disulfide isomerase defense leads to further exacerbation of O&NS within the cellular environment [113,114]. It is also clear that excessive nitrosylation of single proteins can lead to serious pathology.…”

Section: Hypernitrosylation and Pathologymentioning

confidence: 95%

Nitrosative Stress, Hypernitrosylation, and Autoimmune Responses to Nitrosylated Proteins: New Pathways in Neuroprogressive Disorders Including Depression and Chronic Fatigue Syndrome

Morris¹,

Berk

Klein

et al. 2016

Mol Neurobiol

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Nitric oxide plays an indispensable role in modulating cellular signaling and redox pathways. This role is mainly effected by the readily reversible nitrosylation of selective protein cysteine thiols. The reversibility and sophistication of this signaling system is enabled and regulated by a number of enzymes which form part of the thioredoxin, glutathione, and pyridoxine antioxidant systems. Increases in nitric oxide levels initially lead to a defensive increase in the number of nitrosylated proteins in an effort to preserve their function. However, in an environment of chronic oxidative and nitrosative stress (O&NS), nitrosylation of crucial cysteine groups within key enzymes of the thioredoxin, glutathione, and pyridoxine systems leads to their inactivation thereby disabling denitrosylation and transnitrosylation and subsequently a state described as "hypernitrosylation." This state leads to the development of pathology in multiple domains such as the inhibition of enzymes of the electron transport chain, decreased mitochondrial function, and altered conformation of proteins and amino acids leading to loss of immune tolerance and development of autoimmunity. Hypernitrosylation also leads to altered function or inactivation of proteins involved in the regulation of apoptosis, autophagy, proteomic degradation, transcription factor activity, immune-inflammatory pathways, energy production, and neural function and survival. Hypernitrosylation, as a consequence of chronically elevated O&NS and activated immune-inflammatory pathways, can explain many characteristic abnormalities observed in neuroprogressive disease including major depression and chronic fatigue syndrome/myalgic encephalomyelitis. In those disorders, increased bacterial translocation may drive hypernitrosylation and autoimmune responses against nitrosylated proteins.

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Protein disulfide isomerase a multifunctional protein with multiple physiological roles

Cited by 156 publications

References 93 publications

Challenges in the evaluation of thiol-reactive inhibitors of human protein disulfide Isomerase

Challenges in the evaluation of thiol-reactive inhibitors of human protein disulfide Isomerase

Identification of 4‐hydroxynonenal protein targets in rat, mouse and human liver microsomes by two‐dimensional liquid chromatography/tandem mass spectrometry

Nitrosative Stress, Hypernitrosylation, and Autoimmune Responses to Nitrosylated Proteins: New Pathways in Neuroprogressive Disorders Including Depression and Chronic Fatigue Syndrome

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