HEPATIC DEGRADATION OF CYTOCHROME P450 ENZYMES

肝脏内质网(ER)锚定的血红蛋白，细胞色素P450(P450)，在生理相关的内生生物和外源生物(临床相关药物、环境毒素和致癌物)的代谢中起着重要作用。然而，在这种新陈代谢的过程中，参与的酶被牺牲在基于机制的“自杀失活”中。这种失活的一种形式需要将P450假体血红素破坏成片段，这些片段不可逆转地修改其活性部位的蛋白质。在体内，这种异常的修饰会触发P450的降解。这项提议的长期目标集中在这样一个假设上，即P450蛋白的血红素修饰使其易于降解。因此，他们集中在血红素修饰的蛋白质的结构特征，以及阐明其蛋白质降解的机制。到目前为止的研究结果表明，血红素修饰的P450 3A4蛋白被胞浆26S蛋白酶体磷酸化、泛素化和降解，提出了一些有趣的基本问题，这些问题是本提案的主题：(I)内质网结合的P450蛋白是如何传递到胞浆蛋白酶体的？(Ii)针对26S蛋白酶体的P450需要哪些精确的位点(通常是LySepsilon-NH2)泛素化？(Iii)哪些特定的泛素结合酶参与了P450的靶向？更重要的是，从生理上讲，这种天然的酶是否与血红素修饰的蛋白质一样被处理？虽然泛素化和降解是相关的，但目前还不清楚这些事件是否需要磷酸化。因此，最终目标(Iv)是通过表征所涉及的细胞激酶、被磷酸化的蛋白质位点以及使用已识别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、定点突变、生化、蛋白质化学和质谱学方法将被用来阐明这些问题，以完整的新鲜分离的大鼠肝细胞、COS-7细胞和酿酒酵母为模型。拟议的研究集中在生理上相关但被忽视的P450生物学方面。由于P450是一种完整的内质网膜蛋白，对其代谢的研究将为其他内质网居住者提供一个生物学原型。此外，这些研究的重点是P450 3A4，这是人类肝脏和肠道的主要酶及其大鼠的同源物，负责超过60%的临床处方药物的代谢，特别容易受到这种生物命运的影响。最后，由于26S蛋白酶体亚基负责抗原肽的产生，这些研究可能为在慢性活动性肝炎和药物诱导的肝炎和过敏反应患者的血清中检测到P450自身抗体的产生提供帮助。

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.