Human biliverdin reductase is a leucine zipper-like DNA-binding protein and functions in transcriptional activation of heme oxygenase-1 by oxidative stress

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 (L1-L5), a basic domain, and a conserved alanine characterize the domain. In hBVR, a lysine replaces L3. The secondary structure model of hBVR predicts an α-helix-turn-β-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 ∼69 kDa. The protein on a denaturing gel separates into two anti-hBVR immuno-reactive proteins of ∼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 Lys143, Leu150, or Leu157 blocks both the formation of the ∼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 (O2.-) 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.