MULTIPLE FORMS OF HEME OXYGENASE--REGULATION BY TOXINS

描述(改编自申请者的摘要)：血红素加氧酶 (HO)系统调节细胞内的血红素水平，从而控制所有 依赖于血球蛋白的活动。血液蛋白，如NO 合酶、鸟苷环化酶、细胞色素P450等对 细胞功能。HO产品：一氧化碳、胆红素和铁，具有生物学意义 激活。一氧化碳起信号分子的作用，铁是基因调节因子，而 胆红素既是新生儿的一种神经毒素，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。 胆汁色素还显示出抗艾滋病毒和疱疹病毒的活性。 我们之前已经鉴定了两种形式的HO酶：HO-1，它是 现在被认为是一种应激蛋白(HSP32)，并可由一系列 有毒环境物质；以及由肾上腺诱导的HO-2 糖皮质激素GCs。我们现在发现了第三种形式的Ho，我们称之为 HO-3可由细菌内毒素诱导，是半胱氨酸-和 富含组氨酸，并可能在其血红素调节基序(HFM)上结合血红素。 此外，我们还发现了HO-2基因调控的新方面，包括 存在2个高亲和力HRMS；5个不同的发育调节 以及一种HO-2免疫反应蛋白，它是 在GC处理的大鼠睾丸中有明显的诱导作用。这些新发现加上 HO活性的重要功能提示需要进一步研究 这个系统。本申请的具体目的是：1)表征 纯化的HO-3天然、半胱氨酸或组氨酸-丙氨酸突变体的动力学性质，以及 与血红素和金属离子的相互作用。2)检查细胞/组织 HO-3的分布及其受氧化应激、金属离子、 荷尔蒙调节和成熟。3)鉴定和分离 大鼠HO-3基因及其在包括人类在内的其他物种中的存在 纸巾。同时，对该基因的启动子区域进行测序和分析。 对于监管要素。4)分析HO-2作为血红素结合/调节因子 蛋白质，重点是HRMS的特征和3‘端非编码区 有两份“氧气感应”的主题。具体来说，HO-2的活性 作为血红素/氧传感器；交替使用Polya+信号对 消息稳定性和翻译效率；以及 Polya+信号的不同用法及其老化和调制 将对缺血进行检查。5)进一步鉴定HO-2基因和 成绩单。研究将包括：细胞/组织特异性表达 POLYA+信号的三个5‘UTRs及其用法； 5‘UTRs；调控元件启动子的特征； 脑和睾丸转录本对化学物质、激素因素的反应 和成熟；以及交替的5‘和3’UTRs对翻译的影响 效率。第二个HO-2免疫反应蛋白的性质 GC处理的大鼠睾丸也将被调查。

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.