Hepatic Degradation of Cytochrome P450 Enzymes

描述（由申请人提供）：我们的研究工作的总体目标集中在天然和失活的肝细胞色素P450 （P450）作为整体内质网（ER）蛋白质降解模型的蛋白质水解降解特性。这些研究表明，所有通过蛋白血红素修饰而失活的p450 （CYPs 3A， 2B1和2C11）都被标记为通过细胞质ATP和泛素（Ub）依赖的26S蛋白酶体途径进行蛋白水解处理，这是一个典型的er相关降解（ERAD）过程。在这种情况下，CYP3A蛋白在丝氨酸/苏氨酸残基上被肝细胞质激酶磷酸化，泛素化，从其er锚点脱位并转移到细胞质26S蛋白酶体中进行处理。我们对酿酒酵母的研究表明，天然CYP3A4的周转与ERAD相似。虽然已经确定ub偶联酶（Ubc7p）和26S蛋白酶体参与CYP3A4 ERAD，但最近表征的ERAD参与者对CYP3A4泛素化(Cue1p？ub -连接酶)和蛋白酶体识别（Cdc48p/Ufd-1p/Hrd4p）是未知的，这是该提议的一个特定目标。同样，Thr264、Ser420和Ser478磷酸化（如果有的话）在天然CYP3A4 ERAD中的作用也不清楚，这是另一个特定的目标。当天然CYP3A4经历ERAD时，其er对应物天然cyp2b1和2C11被液泡/溶酶体降解。这种差异卫生分类的原因尚不清楚，但可能在于编码在单个P450序列中的结构/分子决定因素“degrons”，其表征也被提出。拟议的研究依赖于各种分析（高效液相色谱-肽定位/质谱分析，蛋白质化学，蛋白质组学），分子生物学（定点诱变，嵌合质粒）和细胞生物学（培养，表达，体内蛋白质交联）方法。这些研究集中在生理相关但被忽视的P450生物学方面。此外，他们将重点放在CYP3A4上，这是人类肝脏和肠道的主要酶，负责毒素、致癌物和超过60%的临床处方药的代谢，特别容易受到这种生物学命运的影响。最后，由于26S蛋白酶体负责抗原肽的产生，这些研究可能为慢性活动性和药物性肝炎及过敏反应患者血清中检测到的P450自身抗体提供见解。

DESCRIPTION (provided by applicant): The overall goals of our research effort have centered on the characterization of the proteolytic degradation of native and inactivated hepatic cytochromes P450 (P450s) as models of integral endoplasmic reticulum (ER) protein degradation. These studies have revealed that all P450s (CYPs 3A, 2B1 and 2C11) inactivated via heme-modification of the protein are branded for proteolytic disposal by the cytosolic ATP- and ubiquitin (Ub)-dependent 26S proteasomal pathway, a process typical of ER-associated degradation (ERAD). In this, the CYP3A protein is phosphorylated at Ser/Thr residues by liver cytosolic kinases, ubiquitinated, dislocated from its ER-anchor and translocated to the cytosolic 26S proteasome for disposal. Our studies in the yeast Saccharomyces cerevisiae reveal that the turnover of native CYP3A4 similarly involves ERAD. While the involvement of an Ub-conjugating enzyme (Ubc7p) and the 26S proteasome in CYP3A4 ERAD has been established, the contribution of additional, recently characterized ERAD participants to CYP3A4 ubiquitination (Cue1p? Ub-ligases?) and proteasomal recognition (Cdc48p/Ufd-1p/Hrd4p?) is unknown, and a specific goal of this proposal. Similarly, the role of Thr264, Ser420 and Ser478 phosphorylation, if any, in native CYP3A4 ERAD is also unclear and another specific aim. While native CYP3A4 undergoes ERAD, native CYPs 2B1 and 2C11, its ER-counterparts, are degraded by the vacuoles/lysosomes. The reason for this differential sanitary sorting is unknown, but may lie in structural/molecular determinants "degrons" encoded in the individual P450 sequence, whose characterization is also proposed. The proposed studies rely on various analytical (HPLC-peptide mapping/mass spectrometric analyses, protein chemistry, proteomics), molecular biological (site-directed mutagenesis, chimeric plasmids), and cell biological (culture, expression, in vivo protein cross-linking) approaches. These studies center on a physiologically relevant but neglected aspect of P450 biology. Furthermore, they are focused on CYP3A4, the major human liver and intestinal enzyme, that is responsible for the metabolism of toxins, carcinogens and over 60% of clinically prescribed drugs and is particularly susceptible to this biological fate. Finally, because the 26S proteasome is responsible for the generation of antigenic peptides, these studies may provide insight into the P450 autoantibodies detected in sera of patients with chronic active and drug induced hepatitis and hypersensitivity reactions.