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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES004391-13
Application #
6055895
Study Section
Special Emphasis Panel (ZRG4-ALTX-1 (01))
Project Start
1997-09-01
Project End
2002-08-31
Budget Start
1999-09-01
Budget End
2000-08-31
Support Year
13
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Rochester
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
208469486
City
Rochester
State
NY
Country
United States
Zip Code
14627
Maines, Mahin D (2002) Heme oxygenase 1 transgenic mice as a model to study neuroprotection. Methods Enzymol 353:374-88
Ahmad, Zulfiqar; Salim, Mohammad; Maines, Mahin D (2002) Human biliverdin reductase is a leucine zipper-like DNA-binding protein and functions in transcriptional activation of heme oxygenase-1 by oxidative stress. J Biol Chem 277:9226-32
Salim, M; Brown-Kipphut, B A; Maines, M D (2001) Human biliverdin reductase is autophosphorylated, and phosphorylation is required for bilirubin formation. J Biol Chem 276:10929-34
Panahian, N; Maines, M D (2001) Site of injury-directed induction of heme oxygenase-1 and -2 in experimental spinal cord injury: differential functions in neuronal defense mechanisms? J Neurochem 76:539-54
Maines, M D; Panahian, N (2001) The heme oxygenase system and cellular defense mechanisms. Do HO-1 and HO-2 have different functions? Adv Exp Med Biol 502:249-72
Maines, M D; Ewing, J F; Huang, T J et al. (2001) Nuclear localization of biliverdin reductase in the rat kidney: response to nephrotoxins that induce heme oxygenase-1. J Pharmacol Exp Ther 296:1091-7
Huang, T J; McCoubrey Jr, W K; Maines, M D (2001) Heme oxygenase-2 interaction with metalloporphyrins: function of heme regulatory motifs. Antioxid Redox Signal 3:685-96
Chen, K; Gunter, K; Maines, M D (2000) Neurons overexpressing heme oxygenase-1 resist oxidative stress-mediated cell death. J Neurochem 75:304-13
Liu, N; Wang, X; McCoubrey, W K et al. (2000) Developmentally regulated expression of two transcripts for heme oxygenase-2 with a first exon unique to rat testis: control by corticosterone of the oxygenase protein expression. Gene 241:175-83
Ding, Y; McCoubrey Jr, W K; Maines, M D (1999) Interaction of heme oxygenase-2 with nitric oxide donors. Is the oxygenase an intracellular 'sink' for NO? Eur J Biochem 264:854-61

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