Biliverdin reductase (BVR) catalyzes the last step in heme metabolism pathway, converting the product of heme oxygenase (HO) activity, biliverdin, to bilirubin. BVR is unique in having dual pH/dual cofactor requirements. The reductase is systolic-SH dependent enzyme and its activity is inhibited by SH reactive environmental agents and toxic metals. Recently, biliverdin has been identified as a liver tumor promotor; it also inhibits human herpes HHV-6 and HIV-1 replication. Bilirubin, on the other hand, is a potent cellular antioxidant that can protect human ventricular myocytes against oxidative damage and decreases risk of coronary artery disease. Oxygen radicals are suspected in the etiology of tissue injury in variety of diseases. High levels of bilirubin, however, are neurotoxic to the newborn and causes kernicterus. Because biliverdin is an inhibitor of HO activity in vivo, the compound may be the ultimate regulator of heme metabolism. The HO isozymes (HO-1 and HO-2), in turn, regulate cellular levels of heme, and hence control all activities that are hemoprotein dependent. Hemoproteins, such as NO synthase, guanylate cyclase, cyt P450, and others, carry out a wide range of vital unctions in the cell. Other heme degradation products, CO and Fe are also biologically active, with CO emerging as a neuronal signaling molecule for generation of cGMP, and Fe being a gene regulator for NO synthase. In comparison to their enzymes of heme metabolism little is known about BVR. We are the only laboratory who is actively involved in toxicological and molecular characterization of the enzyme. We have now discovered a second form of BVR in human and have observed nuclear localization of the enzyme in rat kidney treated with the nephrotoxin, bromobenzene. Moreover, we have found biliverdin to be a potent inhibitor of NO synthase; NO influences a host of cellular functions, including neurotransmission and macrophage cell killing. And, nuclear localization of proteins is associated with gene regulation among other functions. Collectively, these developments suggest vital and previously unsuspected functions of BVR and dictate a need for further understanding of function and characterization of the reductases. This is the overall objective f the present proposal, and the Specific Aims are: 1) To further characterize at biochemical, molecular, and toxicological levels the 2 forms of human BVR proteins and gene(s), and to examine their transcriptional regulation by metals, oxidizing gents and thermal stress in cell culture systems. 2) To analyze the promotor region of rat BVR-1 gene for function o regulatory elements, and to examine the effect of changes caused by exposure to metal compounds (Hg, Cu, cis-Pt) on serum steroid (adrenal and testicular) profiles, and by chemical manipulations on BVR expression in the brain. Developmental regulation of brain BVR will also be examined. 3) To explore biological significance of nuclear localization f BVR as it pertains to interaction with heme and HO isozymes, and examine whether other nephrotoxins and metal complexes (e.g. Hg, cis-Pt) also cause nuclear localization of the protein. Also, the possibility that a second form of BVR is also present in rat will be investigated. 4) To examine the role of BVR-1 and its product bilirubin against lipid peroxidation and DNA damage; in vitro gene knockout and over expression systems will be used. 5) To elucidate the mechanism by which biliverdin inhibits NO synthase activity and consequences of the inhibition to cGMP production and NO-mediated cellular events. Isolated protein, macrophages, hippocampal slices, and intact rats will be used.
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