Abstract
Human biliverdin reductase (hBVR) is a serine/threonine kinase that catalyzes reduction of the heme oxygenase (HO) activity product, biliverdin, to bilirubin. A domain of biliverdin reductase (BVR) has primary structural features that resemble leucine zipper proteins. A heptad repeat of five leucines (L(1)--L(5)), a basic domain, and a conserved alanine characterize the domain. In hBVR, a lysine replaces L(3). The secondary structure model of hBVR predicts an alpha-helix-turn-beta-sheet for this domain. hBVR translated by the rabbit reticulocyte lysate system appears on a nondenaturing gel as a single band with molecular mass of approximately 69 kDa. The protein on a denaturing gel separates into two anti-hBVR immunoreactive proteins of approximately 39.9 + 34.6 kDa. The dimeric form, but not purified hBVR, binds to a 100-mer DNA fragment corresponding to the mouse HO-1 (hsp32) promoter region encompassing two activator protein (AP-1) sites. The specificity of DNA binding is suggested by the following: (a) hBVR does not bind to the same DNA fragment with one or zero AP-1 sites; (b) a 56-bp random DNA with one AP-1 site does not form a complex with hBVR; (c) in vitro translated HO-1 does not interact with the 100-mer DNA fragment with two AP-1 sites; (d) mutation of Lys(143), Leu(150), or Leu(157) blocks both the formation of the approximately 69-kDa specimens and hBVR DNA complex formation; and (e) purified preparations of hBVR or hHO-1 do not bind to DNA with two AP-1 sites. The potential significance of the AP-1 binding is suggested by the finding that the response of HO-1, in COS cells stably transfected with antisense hBVR, with 66% reduced BVR activity, to superoxide anion (O(2)()) formed by menadione is attenuated, whereas induction by heme is not affected. We propose a role for BVR in the signaling cascade for AP-1 complex activation necessary for HO-1 oxidative stress response.
Highlights
IX␣ at the ␥ meso bridge to produce bilirubin
The specificity of DNA binding is suggested by the following: (a) Human biliverdin reductase (hBVR) does not bind to the same DNA fragment with one or zero AP-1 sites; (b) a 56-bp random DNA with one AP-1 site does not form a complex with hBVR; (c) in vitro translated heme oxygenase (HO)-1 does not interact with the 100-mer DNA fragment with two AP-1 sites; (d) mutation of Lys143, Leu150, or Leu157 blocks both the formation of the ϳ69-kDa specimens and hBVR DNA complex formation; and (e) purified preparations of hBVR or hHO-1 do not bind to DNA with two AP-1 sites
The potential significance of the AP-1 binding is suggested by the finding that the response of HO-1, in COS cells stably transfected with antisense hBVR, with 66% reduced biliverdin reductase (BVR) activity, to superoxide anion (O2.) formed by menadione is attenuated, whereas induction by heme is not affected
Summary
IX␣ at the ␥ meso bridge to produce bilirubin. Biliverdin is the product of heme (Fe-protoporphyrin IX) oxidation by the heme oxygenase (HO) system. Human BVR (hBVR) is a 296-residue-long polypeptide that, based on its predicted amino acid sequence, has a region with certain key residues that are conserved in proteins that have a leucine zipper dimerization domain, such as human Shaker, human c-Myc, Saccharomyces GCN4, human c-Jun, human CREB, human c-Fos, and Saccharomyces YAP-1 (Fig. 1). This motif is found in the rat enzyme (Fig. 1). We present data that show specific binding of native hBVR to DNA and suggest a role for BVR in regulation of HO-1 oxidative stress response
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