Uncoupled Redox Systems in the Lumen of the Endoplasmic Reticulum

Abstract

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 ...

Deletion of Hexose-6-phosphate Dehydrogenase Activates the Unfolded Protein Response Pathway and Induces Skeletal Myopathy

“…It is distinct from its cytosolic homolog, glucose-6-phosphate dehydrogenase, in being localized exclusively to the lumen of the endoplasmic reticulum (ER). H6PD converts glucose 6-phosphate to 6-phosphogluconolactonate with the concomitant production of NADPH, thereby maintaining adequate levels of reductive cofactors in the oxidizing environment of the ER (2,3).…”

Contrasting Influence of NADPH and a NADPH-Regenerating System on the Metabolism of Carbonyl-Containing Compounds in Hepatic Microsomes

“…In brief, metabolism assays were initiated by the addition of either 250 l of NRS or NADPH to the microsomal suspension. All alamethicin assays were conducted at 50 g/mg microsomal protein and were incubated for 10 min before initiation of the reaction (Piccirella et al, 2006). Abiotic microsomal controls were conducted with addition of the parent compound and omission of either NADPH or G6P from the NRS.…”

“…G6P entering the lumen is used as a substrate by intraluminal hexose-6-phosphate dehydrogenase (H6PDH) for NADPH production from a separate NADP ϩ pool enclosed within the ER. The intraluminal production of NADPH is then directly coupled to 11␤-HSD1 activity Piccirella et al, 2006).We recently reported on the 11␤-HSD1-mediated carbonyl reduction of the broad spectrum 1,2,4-triazole fungicide triadimefon to triadimenol (Kenneke et al, 2008). The current study focuses on the contrasting effects of NADPH and NRS on the kinetic transformation Article, publication date, and citation information can be found at…”

“…G6P entering the lumen is used as a substrate by intraluminal hexose-6-phosphate dehydrogenase (H6PDH) for NADPH production from a separate NADP ϩ pool enclosed within the ER. The intraluminal production of NADPH is then directly coupled to 11␤-HSD1 activity Piccirella et al, 2006).…”

Lipotoxicity in the liver

“…It also favors cell death through activation of certain stress-sensitive signaling pathways, such as nuclear factor κB, p38MAPK, and c-Jun N-terminal kinase (JNK) [51,52] . In addition, a general cellular oxidative stress inevitably affects the intricate redox homeostasis in the ER lumen [53][54][55] . The controlled maintenance of an oxidized thiol-disulfide redox system in this compartment is the prerequisite of appropriate protein maturation [56] .…”