REGULATION OF HEME METABOLISM IN THE LIVER

肝脏血红素代谢的调节

• 批准号: 2137869
• 负责人: Maria Almira Correia
• 金额: $ 32.69万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-07-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2137869
• 关键词: SDS polyacrylamide gel electrophoresis; cytochrome P450; enzyme activity; enzyme induction /repression; enzyme substrate; heme; hemoprotein biosynthesis; high performance liquid chromatography; laboratory rabbit; laboratory rat; liver metabolism; nuclear runoff assay; phosphorylation; polymerase chain reaction; protein structure function; site directed mutagenesis; tissue /cell culture; tryptophan 2,3 dioxygenase

Heme is the prosthetic moiety of the hepatic hemoproteins Cytochromes P450 (P450) and tryptophan pyrrolase (TO), which are committed to metabolism/detoxification of endobiotics and xenobiotics (P450s), and oxidative tryptophan metabolism (TO). However, not all P450-mediated reactions are beneficial. Certain P450s through one electron oxidations, convert some substrates to acutely toxic radicals that escape the active site and elicit acute/chronic pathologies. Other P450s may favor non-radical pathways for the same substrates. Yet other P450s, in the course of such oxidations incur "suicide", mechanism-based inactivations, with destruction of prosthetic heme and/or apoprotein. Our studies indicate that not only the oxidative pathways favored by each P450 differ, but also that the mode of suicide inactivation by a particular substrate differs among P450s. Because the heme of all P450s is identical, they implicate the apoP450 active site structure in dictating their catalytic preferences. In this, P450IIIA1 is notorious both for its highly deviant catalytic behaviour and its overt digressions in otherwise highly conserved active site structure. Site-directed mutagenesis studies are proposed to determine the influence of these natural mutations on P450IIIA1-catalyses of radical-producing substrates, (DDEP and organic hydroperoxides), and its unusual suicide inactivation via irreversible heme binding to the apoprotein. Studies are also proposed to elucidate whether other presumed mechanism-based inactivations (P450IIIB1 by secobarbital, P450IIC11 by spironolactone), actually occur at the P450 active site and thus truly qualify as suicidal. Because after suicidal heme destruction, certain P450s (P450IIC11) incur immunochemically detectable loss, which is hemin-preventable and ATP-dependent, it is proposed that their heme-stripping and loss might promote their phosphorylation and/or ubiquitination as a prelude to their proteolytic disposal. Indeed, preliminary findings indicate that DDEP-inactivated P450IIC11 in isolated rat hepatocytes is phosphorylated. Studies are proposed to determine whether this is a feature of heme-stripped P450IIC11, to characterize its time-course, identify the phosphorylation sites and determine the heme-regulation of this process. Studies on heme-mediated regulation of rat hepatic TO will be extended: The marked functional TO loss resulting from immunochemically detectable structural loss after acute heme depletion, suggests that prosthetic heme loss destabilizes apoTO. However, not only TO loss persists in acute heme deficiency, but TO induction by glucocorticoids is also impaired. Studies are proposed to determine whether this impairment reflects instability of the newly synthesized apoTO in the absence of heme, or whether, in common with hepatic P450s, heme also regulates TO transcription/translation. It is believed, that collectively, these studies will provide insight into the structure-function relationships, the structural features that commit P450s to toxification/detoxification pathways and thus influence xenobiotic-mediated toxicity, and the ill-understood heme regulation of the turnover of these important hemoproteins.

血红素是肝脏血色素P450的修复体部分 (P450)和色氨酸吡咯酶(TO)，它们致力于 内生生物和外源生物的代谢/解毒(P450)，以及 氧化色氨酸代谢(TO)。然而，并不是所有的P450介导的 反应是有益的。某些P450通过一个电子的氧化， 将一些底物转化为剧毒的自由基，从而逃脱活性物质 并引起急性/慢性病理改变。其他P450可能会青睐 相同底物的非自由基途径。还有其他P450，在 这种氧化过程会导致“自杀”，即基于机制的失活， 随着假体血红素和/或载脂蛋白的破坏。我们的研究 这表明，不仅每个P450支持的氧化途径不同， 而且特定底物的自杀失活模式 在P450中是不同的。因为所有P450的血红素都是相同的，他们 ApoP450活性中心结构在决定其催化活性中的作用 偏好。在这一点上，P450IIIA1因其高度异常而臭名昭著 在其它高度保守的体系中的催化行为及其明显的离题 活动站点结构。建议进行定点突变研究以 确定这些自然突变对P450IIIA1催化的影响 产生自由基的底物(DDEP和有机过氧化氢)及其 通过不可逆的血红素结合使自杀性失活 载脂蛋白。还提出了一些研究，以阐明其他假设是否 基于机理的灭活(SECBARBILITE的P450IIIB1，P450IIC11 螺内酯)，实际上发生在P450活性部位，因此真正 有自杀行为的资格。因为在自杀性的亚铁血红素破坏之后， P450(P450IIC11)引起免疫化学可检测到的丢失，这是 氯化血红素是可预防的和依赖于三磷酸腺苷的，有人认为他们的 血红素的剥离和丢失可能促进它们的磷酸化和/或 泛素化作为其蛋白分解处置的前奏。的确， 初步研究结果表明，DDEP失活的P450IIC11在 大鼠肝细胞被磷酸化。建议进行研究以确定 这是否是去血红素的P450IIC11的特征，以表征其 时间-进程，确定磷酸化位点并确定 血红素--这一过程的调节。血红素介导的细胞免疫调节作用的研究 大鼠肝脏将扩大：明显的功能损失所致 急性血红素后免疫化学可检测到的结构丢失 耗竭，表明假体血红素丢失会破坏apoTO的稳定性。然而， 不仅在急性血红素缺乏症中持续丢失，而且在 糖皮质激素也受到损害。建议进行研究以确定 这种损害是否反映了新合成的apoTO的不稳定性 在没有血红素的情况下，或者是否与肝脏P450相同，血红素也 调节转录/翻译。人们相信，作为一个整体， 这些研究将提供对结构-功能的洞察 关系，承诺P450的结构特征 毒化/解毒途径及其对外源生物介导的影响 毒性，以及尚不清楚的血红素对这些物质周转的调节 重要的血球蛋白。