Interactions Among P450 System Proteins and Their Distribution into Endoplasmic Reticulum Microdomains

摘要 P450系统包括多种参与消除大量外来化合物的酶 从身体上。P450系统的目的是催化氧化反应，通常产生 更易溶于水且容易排出的产品。然而，在某些情况下，产物是反应性的。 -能够与DNA、RNA或关键蛋白质结合-并导致突变、致癌和其他 毒物。参与异体代谢的P450是电子转移链的末端组件 发现于内质网(ER)-P450不是单独起作用的，而是必须接受来自 他们的氧化还原伙伴NADPH-细胞色素P450还原酶(CPR)和/或细胞色素b5。虽然细胞色素 B5可以有效地转移第二电子，CPR被公认为第一电子源 并且可以单独支持单加氧酶反应。当存在于微粒体内时，P450处于大量过剩状态 因为心肺复苏术。尽管这一比例因诱导状态而异，但P450水平超过CPR的比例为7：1。 到20：1的比例。由于CPR和P450形成1：1的功能复合体，CPR必须能够提供 电子到许多不同的P450。在特定P450具有更高亲和力的情况下 CPR，那么电子将优先流向那些P450。因此，P450的竞争能力较弱 限制CPR必须具有允许它们接收电子的机制；否则它们将 新陈代谢方面的沉默。这些情况引发了关于P450系统的组件是如何 在膜中组织起来。P450系统蛋白在内质网中拥挤，提供了许多机会 互动。现已证实，P450酶既形成同聚体，也形成异构体复合体，并且 这些复合体影响P450的功能。作为一个额外的复杂因素，P450系统蛋白驻留在 性质不均一的膜，膜的特性可以影响蛋白质 组织。因此，P450系统需要考虑的不是简单的一系列单体 与氧化还原伙伴相互作用的蛋白质，但作为受影响的超分子复合体的组成部分 通过它的膜环境。这项提议的目的是更好地了解细胞内的蛋白质是如何 P450单加氧酶系统在内质网中的组织以及P450-P450相互作用对功能的影响 这些酶中。这将通过解决以下具体目标来实现：目标1-- 鉴定CPR和多个P450之间的物理复合体并鉴定其蛋白质区域 负责P450·P450复合体的形成；以及目标2-确定P450系统蛋白驻留在哪里 内质网，内质网异质性如何影响P450系统的蛋白质定位，以及蛋白质区域是什么 负责特定膜结构域的定位。这些研究将增加我们对 P450系统蛋白如何在生物膜中相互作用，以及膜如何影响这些 反应--这将对外源物质产生有毒代谢物产生重大影响。

Abstract The P450 system includes a variety of enzymes involved in the elimination of a host of foreign compounds from the body. The purpose of the P450 system is to catalyze oxygenation reactions, commonly generating products that are more water-soluble and readily excreted. In some cases, however, the products are reactive – capable of binding to DNA, RNA or critical proteins – and leading to mutagenesis, carcinogenesis and other toxicities. The P450s involved in xenobiotic metabolism are terminal components of an electron transfer chain found in the endoplasmic reticulum (ER) – P450 does not function alone but must receive two electrons from their redox partners NADPH-cytochrome P450 reductase (CPR) and/or cytochrome b5. Although cytochrome b5 can transfer the second electron efficiently, CPR is generally acknowledged as the primary electron source and can support the monooxygenase reaction alone. When present in microsomes, P450 is in a large excess over CPR. Although this ratio varies depending on induction status, P450 levels exceed those of CPR by a 7:1 to 20:1 ratio. Since CPR and P450 form a 1:1 functional complex, CPR must be capable of supplying electrons to many different P450s. In the event that particular P450s have a higher affinity for association with CPR, then electrons would preferentially flow to those P450s. Consequently, P450s less able to compete for limiting CPR must have mechanisms that allow them to receive electrons; otherwise they would be metabolically silent. These conditions raise questions regarding how the components of the P450 system are organized in the membrane. P450 system proteins are crowded in the ER, providing many opportunities to interact. It is now established that P450 enzymes form both homomeric and heteromeric complexes, and that these complexes affect P450 function. As an additional complication, the P450 system proteins reside in a membrane that is heterogeneous in nature, where the characteristics of the membranes can affect protein organization. Consequently, the P450 system needs to be considered not simply as a series of monomeric proteins interacting with their redox partners, but as components of a supramolecular complex that is affected by its membrane environment. The objective of this proposal is to better understand how the proteins of the P450 monooxygenase system are organized in the ER and the role of P450-P450 interactions on the function of these enzymes. This will be accomplished by addressing the following Specific Aims: Aim 1 – to characterize the physical complexes among CPR and multiple P450s and to identify the protein regions responsible for P450·P450 complex formation; and Aim 2 – to identify where P450 system proteins reside in the ER, how ER heterogeneity influences P450 system protein localization, and what protein regions are responsible for localization in specific membrane domains. These studies will increase our understanding of how the P450 system proteins interact in biological membranes, and how the membrane can affect these responses – which will have a significant impact on generation of toxic metabolites from xenobiotics.