Thioredoxin-like domains required for glucose regulatory protein 58–mediated reductive activation of mitomycin C leading to DNA cross-linking

Abstract

Glucose regulatory protein (GRP58) is known to mediate mitomycin C (MMC) -induced DNA cross-linking. However, the mechanism remains elusive. We hypothesized that thioredoxin-like domains, one at NH 2 terminus and another at COOH terminus, are required for GRP58-mediated MMC reductive activation leading to DNA cross-linking. Site-directed mutagenesis mutated cysteines in thioredoxin domains to serines. Wild-type (WT) and mutant GRP58 were cloned in pcDNA to produce GRP58 V5-tagged WT and mutant proteins on transfection in mammalian cells. Human colon carcinoma (HCT116) cells transiently expressing and Chinese hamster ovary cells stably expressing WT and mutant GRP58 were analyzed for MMC-induced DNA cross-linking. WT GRP58 was highly efficient in MMC-induced DNA cross-linking. However, both NH 2 -and COOH-terminal thioredoxin mutants showed significant reduction in MMC-induced DNA cross-linking. The coexpression of GRP58 with thioredoxin reductase 1 and/or treatment of cells with NADPH increased MMC-induced DNA crosslinking from the WT GRP58. In similar experiments, siRNA inhibition of thioredoxin reductase 1 led to decreased MMC-induced DNA cross-linking. Further experiments revealed that mutations in thioredoxin domains led to significant decrease in metabolic reductive activation of MMC. These results led to conclusion that GRP58, through its two thioredoxin-like domains, functions as a reductase leading to bioreductive drug MMC activation and DNA cross-linking.

RETRACTED: Overlapping signal sequences control nuclear localization and endoplasmic reticulum retention of GRP58

“…It has been shown earlier that mutations in thioredoxin like domains have no effect on subcellular localization of GRP58 [8]. There is very little information available on phosphorylation of GRP58 protein.…”

“…Disruption of GRP58 in mouse is lethal; however B cell specific deletion of GRP58 leads to impaired assembly of the major histocompatibility complex [3]. Besides, GRP58 is shown to play a role in gamete fusion [4], regulation of transcription factors like Ref1 [5], entry of SV40 in host cells [6] and in mitomycin C-induced DNA cross-linking [7–8]. …”

Curcumin reduced the side effects of mitomycin C by inhibiting GRP58‐mediated DNA cross‐linking in MCF‐7 breast cancer xenografts

“…It has been reported that MMC induces cytotoxicity via DNA cross‐linking and breaking ( 3,14 ) and GRP58 mediates MMC‐induced DNA cross‐linking. ( 12,14 ) In this study, we demonstrated that GRP58‐mediated cytotoxcity of MMC that led to DNA cross‐linking was inhibited by the combination treatment (Fig. 3).…”

Mitomycinoid Alkaloids: Mechanism of Action, Biosynthesis, Total Syntheses, and Synthetic Approaches

“…Recent studies have revealed that a glucose regulatory protein (GRP58) requires thioredoxin-like domains to catalyze metabolic reduction of MMC to 2,7- diaminomitosene, which then cross-links DNA. [30,47] Therefore, in contrast to performing a detoxification role, dithiols could generate cytotoxic metabolites by reducing MMC in close proximity to the nucleus. [43] The proposed mechanism of reduction of MMC by dithiols should help elucidate the specific interactions of analogous biomolecules with MMC.…”