MULTIPLE FORMS OF HEME OXYGENASE--REGULATION BY TOXINS

描述（摘自申请人摘要）：血红素加氧酶 (HO)系统调节血红素的细胞水平，因此控制所有 依赖于血蛋白的活动。 血红素蛋白，如NO 合成酶，鸟苷酸环化酶，细胞色素P450和其他酶对 细胞功能。 HO产物：CO、胆红素和铁，在生物学上是 活跃 一氧化碳作为信号分子发挥作用，铁是基因调节因子， 胆红素既是新生儿的神经毒素，也是一种有效的抗氧化剂： 氧自由基被怀疑是多种疾病的病因。 胆色素还显示出抗HIV和疱疹病毒的抗病毒活性。 我们先前已经表征了两种形式的HO酶：HO-1，其是 现在被认为是一种应激蛋白（HSP 32）， 有毒环境因子;和HO-2，其由肾上腺素诱导 糖皮质激素GC。 我们现在已经发现了HO的第三种形式，我们称之为 HO-3可被细菌内毒素诱导，是半胱氨酸， 富含组氨酸且可能在其血红素调节基序（HFM）处结合血红素。 此外，我们还发现了HO-2基因调控的新方面，包括 存在2种高亲和力HRM; 5种不同的发育调节 睾丸中的HO-2免疫反应蛋白， 在GC处理的大鼠睾丸中显著诱导。 这些新发现加上 HO活动的重要功能表明需要进一步研究 the system. 本申请的具体目的是：1）表征 用于动力学性质的HO-3的纯化的天然、Cys或His-Ala突变体，和 与血红素和金属离子相互作用。 2)检查细胞/组织 HO-3的分布;以及氧化应激，金属离子， 荷尔蒙控制和成熟 3)为了表征和分离 大鼠HO-3基因，并检查其在其他物种，包括人类中的存在 组织中 另外，对该基因的启动子区进行测序并分析其 监管元素。 4)为了分析HO-2作为血红素结合/调节 蛋白质，重点是表征HRM和3 'UTR， 有两个“氧气感应”基序的拷贝。 具体而言，HO-2的活性 作为血红素/氧传感器;使用交替polyA +信号对 信息稳定性和翻译效率;以及 polyA +信号的不同使用，以及其通过老化和 将检查局部缺血。 5)为了进一步研究HO-2基因， 成绩单 研究将包括：细胞/组织特异性表达 三个5 'UTR和polyA +信号的使用; 5 'UTR;调控元件启动子的表征; 大脑和睾丸中的转录本对化学物质、激素因子的反应 以及，交替的5'和3' UTR对翻译和成熟的影响。 效率 第二HO-2免疫反应蛋白的性质， 还将研究GC处理的大鼠睾丸。

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.