Abstract
We reported previously that E-box and TATA-like elements repress human xanthine oxidoreductase gene (hXOR) expression. In the present investigation, we determined the means by which the E-box site functions in this basal repression. DNA affinity purification demonstrated that at least five proteins are involved in the nuclear protein complex binding to the E-box and adjacent Ku86-binding sites. Amino acid sequence analysis demonstrated that three proteins, DNA-PK catalytic subunit, Ku86, and Ku70 are components of DNA-dependent protein kinase (DNA-PK). By electrophoretic mobility shift assays, gel-shift, and site-directed mutagenesis, we confirmed Ku86 binding to the Ku86 site. Studies indicated that the other two proteins of the complex are AREB6-like proteins binding to the E-box. Pull-down and immunoprecipitation analyses demonstrated the binding of Ku86 to AREB6-like proteins. The functional loss of Ku86 increases hXOR promoter activity and transcript expression. Based on the findings, we propose that DNA-PK/AREB6-like proteins play a central role in repression of basal hXOR activity. AREB6-like proteins specifically bind to the E-box, whereas Ku86 binds an adjacent site and recruits DNA-PK catalytic subunit and Ku70 proteins. A working model is presented to account for the role of DNA-PK and AREB6-like proteins in regulating hXOR activity.
Highlights
Xanthine oxidoreductase (XOR)1 (EC 1.1.3.22), a member of molybdoflavoprotein hydroxylases, is a homodimer with each subunit of about 150 kDa containing four redox active centers: two iron-sulfur, one FAD, and one molybdopterin [1,2,3]
To determine mechanisms by which the E-box/Ku86 region regulates human XOR (hXOR) promoter activity, we examined the nuclear proteins binding to the E-box and Ku86 sites
Besides the E-box binding band, Fig. 1B demonstrated that two nuclear proteins binding to probe EG were competed for by the specific competitor and the probe containing the mutant E-box, but not by the probes containing a mutant Ku86 site, indicating that specific nuclear proteins bind to the Ku86 site
Summary
Xanthine oxidoreductase (XOR) (EC 1.1.3.22), a member of molybdoflavoprotein hydroxylases, is a homodimer with each subunit of about 150 kDa containing four redox active centers: two iron-sulfur, one FAD, and one molybdopterin [1,2,3]. More than just a rate-limiting enzyme in purine degradation, XOR plays important roles in physiological and pathological conditions as a producer of both urate and reactive oxygen species (ROS). In contrast to the role of urate as a protective antioxidant, XOR-derived superoxide anion and hydrogen peroxide lead to oxidative tissue injury in a variety of animal models that simulate several clinical disorders, including renal failure, endotoxin-induced mucosal injury, viral pneumonia, ischemiareperfusion injury, and cutaneous photosensitivity to hematoporphyrins [11,12,13,14,15,16,17,18]. To understand the physiological and pathophysiological role of XOR in humans, it is important to study the mechanisms underlying the regulation of its expression. We investigated the basis for the E-box/Ku86 repression of hXOR
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