Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94158-2517, USA.
J Biol Chem. 2011 Mar 18;286(11):9443-56. doi: 10.1074/jbc.M110.176685. Epub 2011 Jan 5.
Human liver CYP2E1 is a monotopic, endoplasmic reticulum-anchored cytochrome P450 responsible for the biotransformation of clinically relevant drugs, low molecular weight xenobiotics, carcinogens, and endogenous ketones. CYP2E1 substrate complexation converts it into a stable slow-turnover species degraded largely via autophagic lysosomal degradation. Substrate decomplexation/withdrawal results in a fast turnover CYP2E1 species, putatively generated through its futile oxidative cycling, that incurs endoplasmic reticulum-associated ubiquitin-dependent proteasomal degradation (UPD). CYP2E1 thus exhibits biphasic turnover in the mammalian liver. We now show upon heterologous expression of human CYP2E1 in Saccharomyces cerevisiae that its autophagic lysosomal degradation and UPD pathways are evolutionarily conserved, even though its potential for futile catalytic cycling is low due to its sluggish catalytic activity in yeast. This suggested that other factors (i.e. post-translational modifications or "degrons") contribute to its UPD. Indeed, in cultured human hepatocytes, CYP2E1 is detectably ubiquitinated, and this is enhanced on its mechanism-based inactivation. Studies in Ubc7p and Ubc5p genetically deficient yeast strains versus corresponding isogenic wild types identified these ubiquitin-conjugating E2 enzymes as relevant to CYP2E1 UPD. Consistent with this, in vitro functional reconstitution analyses revealed that mammalian UBC7/gp78 and UbcH5a/CHIP E2-E3 ubiquitin ligases were capable of ubiquitinating CYP2E1, a process enhanced by protein kinase (PK) A and/or PKC inclusion. Inhibition of PKA or PKC blocked intracellular CYP2E1 ubiquitination and turnover. Here, through mass spectrometric analyses, we identify some CYP2E1 phosphorylation/ubiquitination sites in spatially associated clusters. We propose that these CYP2E1 phosphorylation clusters may serve to engage each E2-E3 ubiquitination complex in vitro and intracellularly.
人肝 CYP2E1 是一种单域、内质网锚定的细胞色素 P450，负责将临床相关药物、低分子量外源化学物质、致癌物和内源性酮类物质进行生物转化。CYP2E1 底物的络合将其转化为稳定的慢转化物种，主要通过自噬溶酶体降解进行降解。底物去络合/去除导致快速转化的 CYP2E1 物种，推测是通过其无效的氧化循环产生的，这会导致内质网相关的泛素依赖性蛋白酶体降解 (UPD)。因此，CYP2E1 在哺乳动物肝脏中表现出两相转化。我们现在在酿酒酵母中异源表达人 CYP2E1 时发现，即使其在酵母中的催化活性缓慢导致无效催化循环的潜力较低，但它的自噬溶酶体降解和 UPD 途径在进化上是保守的。这表明其他因素（即翻译后修饰或“降解子”）有助于其 UPD。事实上，在培养的人肝细胞中，CYP2E1 可检测到泛素化，并且在其基于机制的失活时会增强。在 Ubc7p 和 Ubc5p 基因缺陷酵母菌株与相应的同基因野生型的研究中，这些泛素结合 E2 酶被鉴定为与 CYP2E1 UPD 相关。与此一致，在体外功能重建分析中发现，哺乳动物 UBC7/gp78 和 UbcH5a/CHIP E2-E3 泛素连接酶能够泛素化 CYP2E1，该过程通过蛋白激酶 (PK) A 和/或 PKC 包含而增强。PKA 或 PKC 的抑制阻断了细胞内 CYP2E1 的泛素化和周转率。在这里，通过质谱分析，我们在空间相关的簇中鉴定了一些 CYP2E1 磷酸化/泛素化位点。我们提出这些 CYP2E1 磷酸化簇可能用于在体外和细胞内结合每个 E2-E3 泛素化复合物。
