A Major Fraction of Endoplasmic Reticulum-located Glutathione Is Present as Mixed Disulfides with Protein

Bass, Rosemary; Ruddock, Lloyd W.; Klappa, Peter; Freedman, Robert B.

doi:10.1074/jbc.m304951200

Cited by 145 publications

(131 citation statements)

References 42 publications

(49 reference statements)

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“…This direct method has shown the global level of PSSG to be extremely low in both HEK and HeLa cells, consistent with results where basal levels of PSSG in platelets were undetectable by immunoblotting (29). However, in liver microsomes Ͼ50% of the glutathione equivalents in ER were found to be bound to protein (30). Although we cannot directly determine subcellular distributions of PSSG equivalents, we can estimate the maximal fraction of PSSG in the ER, assuming that all PSSG equivalents are found in the ER, and that the concentration of GS equivalents is the same in all compartments.…”

Section: Discussionsupporting

confidence: 62%

Quantifying the global cellular thiol–disulfide status

Hansen

Roth²,

Winther³

2009

Proc. Natl. Acad. Sci. U.S.A.

375

307

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It is widely accepted that the redox status of protein thiols is of central importance to protein structure and folding and that glutathione is an important low-molecular-mass redox regulator. However, the total cellular pools of thiols and disulfides and their relative abundance have never been determined. In this study, we have assembled a global picture of the cellular thiol-disulfide status in cultured mammalian cells. We have quantified the absolute levels of protein thiols, protein disulfides, and glutathionylated protein (PSSG) in all cellular protein, including membrane proteins. These data were combined with quantification of reduced and oxidized glutathione in the same cells. Of the total protein cysteines, 6% and 9.6% are engaged in disulfide bond formation in HEK and HeLa cells, respectively. Furthermore, the steady-state level of PSSG is <0.1% of the total protein cysteines in both cell types. However, when cells are exposed to a sublethal dose of the thiol-specific oxidant diamide, PSSG levels increase to >15% of all protein cysteine. Glutathione is typically characterized as the ''cellular redox buffer''; nevertheless, our data show that protein thiols represent a larger active redox pool than glutathione. Accordingly, protein thiols are likely to be directly involved in the cellular defense against oxidative stress.cysteine ͉ glutathione ͉ protein B ecause of the thiol group (SH), cysteine is the most chemically reactive natural amino acid found in cells. SH is mainly found in proteins (PSH) and in low-molecular-mass metabolites such as the highly abundant glutathione (GSH). Two SH groups can be oxidized to form a disulfide bond, and, in the cell, disulfides are mainly found in proteins (PSSP), in glutathione disulfide (GSSG), or as mixed disulfides between protein and glutathione (PSSG). In addition, SH groups can be reversibly oxidized by reactive oxygen species to nitrosothiols or sulfenic acids. Higher oxidation states, such as sulfinic and sulfonic acids, are generally considered to be irreversible.Disulfides are important for protein structure and folding, and formation of disulfide bonds can regulate protein function (1). Furthermore, glutathionylation, here used to denote the formation of PSSG, has been implicated as a mechanism for protection against irreversible protein oxidation during oxidative stress (2).The cytosolic glutathione pool is highly reducing (3) and consequently, disulfide bond formation is infrequent and typically only takes place transiently during catalysis of redox processes. In contrast, disulfide bond formation is favored in other compartments of the cell, such as the endoplasmic reticulum (ER) and the intermembrane space of mitochondria (4). The overall distribution of thiols and disulfides in different cellular pools defines the global thiol-disulfide redox status of the cell. Maintenance of thioldisulfide redox homeostasis in different subcellular compartments is important, and failure to do so can be detrimental to the structure, stability and activity of variou...

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Section: Discussionsupporting

confidence: 62%

Quantifying the global cellular thiol–disulfide status

Hansen

Roth²,

Winther³

2009

Proc. Natl. Acad. Sci. U.S.A.

375

307

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“…The calculated redox potential values were between −170 and −185 mV in the ER lumen in contrast to an approximately −230 mV in the cytosol. Similar GSH/GSSG ratios (3:1-6:1) were observed by using ER-derived microsomal vesicles [13,14].…”

Section: Glutathione and Its Disulfide The Main Redox Buffer In The supporting

confidence: 52%

Composition of the redox environment of the endoplasmic reticulum and sources of hydrogen peroxide

Margittai

Enyedi

Csala

et al. 2015

Free Radical Biology and Medicine

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“…The redox potential calculated from these values is Ϫ0.24 and Ϫ0.18 V, respectively. Recent observations suggest an even larger difference between the redox potentials of the two compartments (3).…”

mentioning

confidence: 99%

“…The lumen of the endoplasmic reticulum (ER) 3 is a separate metabolic compartment of the eukaryotic cell (1). Because of the limited and selective permeability of the ER membrane and because of special intraluminal reactions, it differs from the cytosol in numerous parameters.…”

mentioning

confidence: 99%

Uncoupled Redox Systems in the Lumen of the Endoplasmic Reticulum

Piccirella

Czegle

Lizák

et al. 2006

Journal of Biological Chemistry

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The redox state of the intraluminal pyridine nucleotide pool was investigated in rat liver microsomal vesicles. The vesicles showed cortisone reductase activity in the absence of added reductants, which was dependent on the integrity of the membrane. The intraluminal pyridine nucleotide pool could be oxidized by the addition of cortisone or metyrapone but not of glutathione. On the other hand, intraluminal pyridine nucleotides were slightly reduced by cortisol or glucose 6-phosphate, although glutathione was completely ineffective. Redox state of microsomal protein thiols/disulfides was not altered either by manipulations affecting the redox state of pyridine nucleotides or by the addition of NAD(P) ؉ or NAD(P)H. The uncoupling of the thiol/disulfide and NAD(P) ؉ / NAD(P)H redox couples was not because of their subcompartmentation, because enzymes responsible for the intraluminal oxidoreduction of pyridine nucleotides were distributed equally in smooth and rough microsomal subfractions. Instead, the phenomenon can be explained by the negligible representation of glutathione reductase in the endoplasmic reticulum lumen. The results demonstrated the separate existence of two redox systems in the endoplasmic reticulum lumen, which explains the contemporary functioning of oxidative folding and of powerful reductive reactions.The lumen of the endoplasmic reticulum (ER) 3 is a separate metabolic compartment of the eukaryotic cell (1). Because of the limited and selective permeability of the ER membrane and because of special intraluminal reactions, it differs from the cytosol in numerous parameters. One of the most characteristic differences is that luminal thiols (including protein thiols and glutathione) are present in a more oxidized state than the cytosolic ones. Although the ratio between reduced and oxidized glutathione is 100:1 in the cytosol, this value is around 1-2:1 in the ER lumen (2). The redox potential calculated from these values is Ϫ0.24 and Ϫ0.18 V, respectively. Recent observations suggest an even larger difference between the redox potentials of the two compartments (3).The typical redox potential of a given compartment is a major determinant of rate and direction of redox reactions. The oxidizing environment in the ER lumen is both a prerequisite and a consequence of the oxidative folding of secretory and membrane proteins (4 -6). By generalizing the observations related to the redox state of thiols found in the ER of cells engaged in protein secretion, it is now supposed that the redox conditions in the ER lumen are uniformly oxidizing. However, several intraluminal reactions have been described, which require reducing equivalents. Such reactions involve the isomerization of disulfide bonds (7), the steps of the vitamin K cycle (8), the biotransformation of several endogenous ketones and aldehydes (9), and the reactivation of steroids. One of the main sources of intraluminal reducing equivalents is hexose-6-phosphate dehydrogenase (H6PDH), which generates NADPH at the expense of the oxidation of ...

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A Major Fraction of Endoplasmic Reticulum-located Glutathione Is Present as Mixed Disulfides with Protein

Cited by 145 publications

References 42 publications

Quantifying the global cellular thiol–disulfide status

Quantifying the global cellular thiol–disulfide status

Composition of the redox environment of the endoplasmic reticulum and sources of hydrogen peroxide

Uncoupled Redox Systems in the Lumen of the Endoplasmic Reticulum

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