MULTIPLE FORMS OF HEME OXYGENASE--REGULATION BY TOXINS

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DESCRIPTION (Adapted from the APPLICANT'S ABSTRACT): The heme oxygenase (HO) system regulates cellular levels of heme, and hence controls all activities that are hemoprotein-dependent. Hemoproteins, such as NO synthase, guanylate cyclase, cytochrome P450s, and others are vital to cellular functions. HO products: CO, bilirubin, and iron, are biologically active. CO functions as a signal molecule, iron is a gene regulator, and bilirubin is both a neurotoxin in the newborn and a potent antioxidant: oxygen radicals are suspected in the etiology of a variety of diseases. Bile pigments also display antiviral activity against HIV and herpes virus. We have previously characterized two forms of HO enzymes: HO-1, which is now recognized as a stress protein (HSP32) and is inducible by a host of toxic environmental agents; and HO-2, which is induced by adrenal glucocorticoids GCs. We have now discovered a third form of HO, we call HO-3, which is inducible by bacterial endotoxins, is cysteine-and histidine-rich and likely to bind heme at its heme regulatory motifs (HFMs). Also, we have uncovered new aspects of HO-2 gene regulation, including the presence of 2 high affinity HRMs; 5 different developmentally regulated transcripts in the testis; and an HO-2 immunoreactive protein that is greatly induced in GC- treated rat testis. These new findings plus the vital functions of HO activity indicate need for further investigation of the system. The Specific Aims of this application are: 1) To characterize purified native, Cys or His-Ala mutants of HO-3 for kinetic properties, and interaction with heme and metal ions. 2) To examine cellular/tissue distribution of HO-3; and its regulation by oxidative stress, metal ions, hormonal manipulation, and maturation. 3) To characterize and isolate the rat HO-3 gene and examine its presence in other species, including human tissues. Also, to sequence the promoter region of the gene and analyze it for regulatory elements. 4) To analyze HO-2 as a heme binding/regulatory protein, with emphasis on the characterization of HRMs and the 3'UTR that has 2 copies of the "oxygen sensing" motif. Specifically, activity of HO-2 as a heme/oxygen sensor; effects of the use of alternate polyA + signals on message stability and translational efficiency; and, the mechanism for differential usage of polyA + signals, and its modulation by aging and ischemia will be examined. 5) To further characterize HO-2 gene and transcripts. Studies will include: cell/tissue specific expression of three 5'UTRs and usage of polyA + signals; the structural organization of the 5'UTRs; characterization of the promoters for regulatory elements; response of transcripts in brain and testis to chemicals, hormonal factors and maturation; and, the effects of alternate 5' and 3'UTRs on translational efficiency. The nature of the second HO-2 immunoreactive protein in GC-treated rat testis also will be investigated.