Hepatic degradation of cytochrome P450 enzymes

 描述（由申请方提供）：肝细胞色素P450（P450 s）是代谢内源性和外源性物质（即药物、致癌物、毒素、天然和化学产品）的酶。这些药物通过增加形成或通过失活和/或蛋白水解降解减少来调节肝脏P450含量，导致临床显著的药物相互作用。这些通常源于由药物介导的P450稳定（即乙醇（EtOH））或增强的药物介导的P450降解（即葡萄柚汁）引起的P450转换的改变。P450如CYP 3A 4、主要的人肝/肠P450和EtOH代谢CYP 2 E1通过gp 78和CHIP E3泛素（Ub）连接酶和26 S蛋白酶体（UPD）的泛素化降解。我们发现，多位点蛋白磷酸化显着增强gp 78和CHIP介导的泛素化的P450赖氨酸残基内的带负电荷的酸性（天冬氨酸/谷氨酸）和磷酸化（丝氨酸/苏氨酸）集群。我们假设P450 S/T-磷酸化增加了这些簇的负电荷，增强了其分子E3-识别，并调节其UPD。我们通过最先进的化学交联/蛋白质组学分析、相关CYP 3A 4 D/E/S/T簇和每个带正电荷（K/R/H）gp 78补丁的定点诱变来验证这一假设，并证实其有效性。我们建议D/E/S/T簇和带正电荷的E3补丁之间的静电相互作用是E3底物识别的基础。因此，我们的目的1是通过类似的实验方法，通过检查其他肝P450和/或同源gp 78-和CHIP-底物（具有已确定的晶体结构）来验证这一假设。由于目前已知的底物非常少，我们的目的2是采用gp 78-/-和CHIP-/-以及相应的野生型小鼠肝细胞的SILAC偶联Ub-残基分析，以鉴定可能关键参与病理生理相关过程的其他肝E3-底物。CHIP-/-小鼠表现出年龄依赖性的肝脏氧化损伤，脂质过氧化和脂肪积累增加。我们已经表明，这主要是由于CYP 2 E1稳定，无效的氧化循环，蛋白毒性反应性O2-物种，并激活被CYP 2 E1功能抑制减弱的ASK 1/JNK 1信号通路。相比之下，据报道gp 78-/-小鼠表现出非常不同的表型。我们假设gp 78-/-肝脏不能稳定功能性CYP 2 E1，因为CHIP完全能够将其泛素化并将其转移到替代自噬-溶酶体降解（ALD）途径中，从而有效地防止其致病性积累。虽然gp 78和CHIP E3都可以独立地泛素化UPD所需的CYP 2 E1，但E4 gp 78也通过pro-UPD K48-Ub键延长Ub链。在其不存在的情况下，CHIP有效地泛素化CYP 2 E1，可能通过K63-Ub-连接将其路由到ALD。我们的目的3提出阐明这种病理生理学相关的CHIP-E3与gp 78-E4在CYP 2 E1降解和随后的肝氧化损伤、脂质过氧化和脂肪蓄积、酒精性肝病、糖尿病、肥胖和非酒精性脂肪性肝病（NAFLD）的临床标志中的作用。

 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).