Hepatic degradation of cytochrome P450 enzymes

 描述(申请人提供)：肝脏细胞色素P450(P450)是代谢内源性和外源性生物物质的酶，即药物、致癌物、毒素、天然和化学产品。这些药物通过失活和/或蛋白降解来增加形成或减少肝脏P450的含量，从而导致临床上显著的药物-药物相互作用。这些变化通常源于药物介导的P450稳定化，即乙醇(Etoh)或增强药物介导的P450降解(即西柚汁)引起的P450代谢的改变。P450如人体主要的肝脏/肠道P450和乙醇代谢的P450通过gp78、芯片E3泛素(Ub)连接酶和26S蛋白酶体(Upd)泛素化而被降解。我们发现，多位点蛋白磷酸化显著增强了gp78和ChIP介导的P450 Lys-残基的泛素化，这些残基位于带负电荷的酸性(Asp/Glu)和磷酸化(Ser/Thr)簇中。我们推测P450的S/T-磷酸化增强了这些簇的负电荷，增强了它的分子E3识别，并调节了它的UPD。我们通过最先进的化学交联/蛋白质组学分析、相关的CYP3A4D/E/S/T簇和每个带正电荷的(K/R/H)gp78补丁的定点突变来验证这一假说，并证实了它的有效性。我们认为，D/E/S/T团簇和带正电荷的E3斑块之间的这种静电相互作用是识别E3底物的基础。因此，我们的目标1是通过类似的实验方法检查其他具有已建立晶体结构的肝P450和/或同源gp78-和芯片底物来检验这一假设。由于目前已知的底物很少，我们的目标2是利用Gp78-/-和ChIP-/-以及相应的野生型小鼠肝细胞的SILAC耦合Ub-残留物分析来识别可能在病理生理相关过程中关键参与的额外的肝E3-底物。CHIP-/-小鼠表现出年龄依赖性的肝脏氧化损伤、脂质过氧化和脂肪堆积。我们已经证明，这在很大程度上是由于CYP2E1的稳定，无效的氧化循环，蛋白毒性的反应性O2-物种，以及激活的ASK1/JNK1信号通路，这些信号通路被CYP2E1功能抑制而减弱。相比之下，据报道，gp78-/-小鼠表现出非常不同的表型。我们假设gp78-/-肝脏不能稳定功能的CYP2E1，因为芯片完全能够泛素化它，并将它转移到另一条自噬-溶酶体降解(ALD)途径，从而有效地防止其病原体积累。虽然，gp78和芯片E3都可以独立地泛化其UPD所需的CYP2E1，因为E4 gp78也通过支持UPD的K48-Ub连接来延长Ub链。在没有它的情况下，CHIP可能通过K63-Ub连接将其泛化到ALD中，从而有效地泛化CYP2E1。我们的目标3旨在阐明这种病理生理学相关的芯片-E3和gp78-E4在CYP2E1降解和随之而来的肝脏氧化损伤、脂质过氧化和脂肪堆积、酒精性肝病、糖尿病、肥胖和非酒精性脂肪性肝病(NAFLD)的临床特征中的作用。

 DESCRIPTION (provided by applicant): Hepatic cytochromes P450 (P450s) are enzymes that metabolize endo- and xenobiotics i.e. drugs, carcinogens, toxins, natural and chemical products. These agents modulate liver P450 content via increased formation or reduction via inactivation and/or proteolytic degradation, resulting in clinically significant drug-drug interactions. These often stem from altered P450 turnover elicited by drug-mediated P450 stabilization i.e. ethanol (EtOH), or enhanced drug-mediated P450 degradation (i.e. grapefruit juice). P450s such as CYP3A4, the major human liver/intestinal P450, and the EtOH-metabolizing CYP2E1 are degraded via ubiquitination by gp78 and CHIP E3 ubiquitin (Ub)-ligases and the 26S proteasome (UPD). We found that multisite protein phosphorylation considerably enhances gp78- and CHIP-mediated ubiquitination of P450 Lys-residues residing within negatively charged acidic (Asp/Glu) and phosphorylated (Ser/Thr) clusters. We hypothesized that P450 S/T-phosphorylation boosts the negative charge of these clusters enhancing its molecular E3-recognition, and regulating its UPD. We tested this hypothesis via state-of-the-art chemical-crosslinking/proteomic analyses, site-directed mutagenesis of the relevant CYP3A4 D/E/S/T clusters and each positively charged (K/R/H) gp78 patch and confirmed its validity. We propose such electrostatic interactions between D/E/S/T clusters and positively charged E3 patches are fundamental to E3 substrate recognition. Thus, our Aim 1 is to test this hypothesis by examining other hepatic P450s and/or cognate gp78- and CHIP-substrates with established crystal structures through similar experimental approaches. Because very few substrates are currently known, our Aim 2 is to employ SILAC coupled Ub-remnant analyses of gp78-/- and CHIP-/- and corresponding wild type mouse hepatocytes to identify additional hepatic E3-substrates that may be critically involved in pathophysiological relevant processes. CHIP-/- mice exhibit age-dependent hepatic oxidative damage, elevated lipid peroxidation and fat accumulation. We have shown that this is largely due to CYP2E1 stabilization, futile oxidative cycling, proteotoxic reactive O2-species, and activation of ASK1/JNK1 signaling pathways that are attenuated by CYP2E1 functional inhibition. By contrast, gp78-/- mice reportedly exhibit a very different phenotype. We hypothesize that gp78-/- livers fail to stabilize functional CYP2E1, as CHIP is fully capable of ubiquitinating it and diverting it into the alternative autophagic-lysosomal degradation (ALD) pathway, thereby effectively preventing its pathogenic accumulation. Although, both gp78 and CHIP E3s can independently ubiquitinate CYP2E1 required for its UPD, as an E4 gp78 also elongates Ub-chains via pro-UPD K48-Ub linkages. In its absence, CHIP effectively ubiquitinates CYP2E1 possibly via K63-Ub-linkages that route it into ALD. Our Aim 3 proposes to elucidate this pathophysiologically relevant CHIP-E3 versus gp78-E4 role in CYP2E1 degradation and consequent hepatic oxidative damage, lipid peroxidation and fat accumulation, clinical hallmarks of alcoholic liver disease, diabetes, obesity and non-alcoholic fatty liver disease (NAFLD).