Quercetin-3-rutinoside Inhibits Protein Disulfide Isomerase by Binding to Its b′x Domain

Lin, Lin; Gopal, Srila; Sharda, Anish V.; Passam, Freda; Bowley, Sheryl R.; Stopa, Jack D.; Xue, Guangpu; Yuan, Cai; Furie, Barbara C.; Flaumenhaft, Robert; Huang, Mingdong; Furie, Bruce

doi:10.1074/jbc.m115.666180

Cited by 87 publications

(111 citation statements)

References 26 publications

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“…Fig. 10 shows the effect of 1 and 10 μM rutin on the activity of PDI red , demonstrating attenuated reoxidation after 30 s. When challenged with 5 mM GSH, rutin succeeded in inhibiting PDI turnover activity, supporting a non-thiol based method of inhibition [26]. …”

Section: Resultsmentioning

confidence: 98%

“…1D) as a reversible inhibitor of PDI [25]. Inhibition reflects binding of the flavonoid to the b'x domain of PDI rather than a covalent interaction with the CxxC motif of the a and a' domains of PDI red [26]. Since these studies used the less sensitive insulin reductase assay, we were interested in applying the BD-SS reoxidation and turnover assays to assess their suitability for high throughput applications.…”

Section: Resultsmentioning

confidence: 99%

“…abb'x or bb'xa' ) [26]. We sought to extend this observation by exploring the effect of rutin on full-length mutants of PDI in which either the a or a' domains were rendered inactive by replacing the catalytic disulfide CxxC with an SxxS sequence (See Materials and Methods).…”

Section: Resultsmentioning

confidence: 99%

“…While the enone functionality of rutin (Fig. 1D) is a potential weak Michael acceptor, its mode of inhibition appears to be unrelated to the CxxC motifs of PDI [26]. The observation that a sizable proportion of PDI inhibitors are thiol-reactive raises important issues concerning their mode of action and specificity in vivo , and how PDI inhibition should be best assessed in vitro .…”

Section: Introductionmentioning

confidence: 99%

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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: 98%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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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“…The administration of quercetin-3-rutinoside similarly inhibited thrombus formation following vascular injury in mouse models of thrombosis (6). Quercetin-3-rutinoside blocks PDI activity by binding to the substrate-binding pocket on PDI and inducing a conformational change in the enzyme, which results in a more compact molecular envelope and reduces substrate binding (7). Structure-activity relationship assays showed that all quercetins tested that possessed a glycoside at the third position on the C ring inhibited PDI, including isoquercetin (6) (also known as quercetin-3-glucoside), which has improved bioavailability in humans compared with quercetin-3-rutinoside (8)(9)(10)(11).…”

Section: L I N I C a L M E D I C I N Ementioning

confidence: 99%

Protein disulfide isomerase inhibition blocks thrombin generation in humans by interfering with platelet factor V activation

Stopa¹,

et al. 2017

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Protein disulfide isomerase is regulated in multiple ways: Consequences for conformation, activities, and pathophysiological functions

Wang

2020

BioEssays

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Protein disulfide isomerase (PDI) is one of the most abundant and critical protein folding catalysts in the endoplasmic reticulum of eukaryotic cells. PDI consists of four thioredoxin domains and interacts with a wide range of substrate and partner proteins due to its intrinsic conformational flexibility. PDI plays multifunctional roles in a variety of pathophysiological events, both as an oxidoreductase and a molecular chaperone. Recent studies have revealed that the conformation and activity of PDI can be regulated in multiple ways, including posttranslational modification and substrate/ligand binding. Here, we summarize recent advances in understanding the function and regulation of PDI in different pathological and physiological events. We propose that the multifunctional roles of PDI are regulated by multiple mechanisms. Furthermore, we discuss future directions for the study of PDI, emphasizing how different regulatory modes are linked to the conformational changes and biological functions of PDI in the context of diverse pathophysiologies.

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Quercetin-3-rutinoside Inhibits Protein Disulfide Isomerase by Binding to Its b′x Domain

Cited by 87 publications

References 26 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

Protein disulfide isomerase inhibition blocks thrombin generation in humans by interfering with platelet factor V activation

Protein disulfide isomerase is regulated in multiple ways: Consequences for conformation, activities, and pathophysiological functions

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