HEPATIC DEGRADATION OF CYTOCHROME P450 ENZYMES

肝内质网（ER）锚定的血红素蛋白，细胞色素P450（P450），有助于生理相关的内源性和外源性物质（临床相关药物，环境毒素和致癌物）的代谢。 然而，在这种代谢过程中，参与的酶在基于机制的“自杀失活”中被牺牲。 这种失活的一种形式需要将P450假体血红素破坏成片段，这些片段在其活性位点不可逆地修饰其蛋白质。 这种异常修饰在体内触发P450降解。 该提案的长期目标集中在P450蛋白的血红素修饰使其易于降解的假设上。 因此，他们集中在血红素修饰的蛋白质的结构表征，并阐明其蛋白水解降解的机制。迄今为止的研究结果表明，血红素修饰的P450 3A 4蛋白被磷酸化，泛素化和降解的胞质26 S蛋白酶体，提出了一些有趣的基本问题，这是本建议的主题：（i）如何ER结合的P450蛋白交付到胞质蛋白酶体？(ii)P450靶向26 S蛋白酶体需要什么样的精确位点（通常是Lysepsilon-NH 2）被泛素化？(iii)哪些特定的泛素结合酶参与P450靶向？ 更重要的是，在生理学上，天然酶是否与血红素修饰的蛋白质类似？ 虽然泛素化和降解是相关的，但还不清楚磷酸化是否是这些事件所必需的。 因此，最终的目的（iv）是通过表征所涉及的细胞激酶、磷酸化的蛋白质位点和使用所鉴定的激酶的选择性抑制剂作为探针来确定磷酸化在P450降解中的作用。 免疫学、形态学、定点诱变、生物化学、蛋白质化学和质谱方法将用于阐明这些问题，使用完整的新鲜分离的大鼠肝细胞、COS-7细胞和S.酿酒酵母作为模型。 拟议的研究集中在P450生物学的生理相关但被忽视的方面。 由于P450是整合ER膜蛋白，阐明其营业额将提供其他ER居民的生物原型。此外，这些研究主要集中在P450 3A 4上，这是主要的人类肝脏和肠道酶及其大鼠直系同源物，负责超过60%的临床处方药物的代谢，并且特别容易受到这种生物命运的影响。最后，由于26 S蛋白酶体亚基负责抗原肽的产生，这些研究可能会提供深入了解在慢性活动性和药物诱导的肝炎和超敏反应患者血清中检测到的P450自身抗体的产生。

The hepatic endoplasmic reticulum (ER)-anchored hemoproteins, cytochromes P450 (P450s), are instrumental in the metabolism of physiologically relevant endobiotics and xenobiotics (clinically relevant drugs, environmental toxins and carcinogens). In the course of this metabolism, however, the participating enzyme is sacrificed in a mechanism-based "suicide-inactivation". One form of this inactivation entails destruction of P450 prosthetic heme into fragments that irreversibly modify its protein at its active site. Such abnormal modification in vivo, triggers P450 degradation. The long-term goals of this proposal are centered on the hypothesis that heme-modification of the P450 protein predisposes it for degradation. Thus, they have centered on structural characterization of the heme-modified protein, and elucidation of the mechanism of its proteolytic degradation. Findings to date reveal that the heme-modified P450 3A4 protein is phosphorylated, ubiquitinated and degraded by the cytosolic 26S proteasome, raising some intriguing fundamental questions which are the subject of the present proposal: (i) How are the ER-bound P450 proteins delivered to the cytosolic proteasome? (ii) What precise sites (usually Lysepsilon-NH2) are required to be ubiquitinated for such P450 targeting to the 26S proteasome? (iii) Which particular ubiquitin conjugating enzymes are involved in this P450 targeting? And more importantly, is physiologically the native enzyme similarly disposed of as the heme-modified protein? While the ubiquitination and degradation are related, it is unclear whether phosphorylation is necessary for these events. Thus, the final aim (iv) is to determine the role of phosphorylation in P450 degradation by characterizing the cellular kinases involved, the protein sites phosphorylated and the use of selective inhibitors of the identified kinases as probes. Immunological, morphological, site-directed mutagenesis, biochemical, protein chemistry and mass spectrometric approaches will be used in elucidating these issues, using intact freshly isolated rat hepatocytes, COS-7 cells, and S. cerevisiae as models. The proposed studies center on a physiologically relevant but neglected aspect of P450 biology. Because P450s are integral ER-membrane proteins, elucidation of its turnover will provide a biological prototype for other ER-residents. Furthermore, these studies are focused on P450 3A4, the major human liver and intestinal enzyme and its rat orthologs, which are responsible for the metabolism of over 60 percent of clinically prescribed drugs, and are particularly susceptible to this biological fate. Finally, because the 26S proteasomal subunits are responsible for the generation of antigenic peptides, these studies may provide insight into the generation of P450 autoantibodies detected in sera of patients with chronic active and drug-induced hepatitis and hypersensitivity reactions.