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

摘要 P450系统包括参与消除大量外来化合物的多种酶 从尸体上。P450系统的目的是催化氧化反应，通常产生 更易溶于水且易于排泄的产品。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，并导致诱变、致癌等 毒性P450参与异生物质代谢，是电子传递链的末端组分 在内质网（ER）中发现的- P450并不单独起作用，但必须接受两个电子， 它们的氧化还原伙伴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通常被认为是初级电子源 并且可以单独支持单加氧酶反应。当存在于微粒体中时，P450大量过量 而不是心肺复苏虽然这个比例因诱导状态而异，但P450水平超过CPR水平7：1。 20：1的比例。由于CPR和P450形成1：1的功能复合体，因此CPR必须能够提供 不同的P450电子。如果特定的P450具有更高的亲和力， CPR，那么电子会优先流向那些P450。因此，P450的竞争能力较低， 限制CPR必须具有允许它们接收电子的机制;否则它们将被 代谢沉默。这些情况提出了关于P450系统的组件如何 组织在膜上。P450系统蛋白在ER中拥挤，提供了许多机会， 互动.现在已经确定P450酶形成同聚和异聚复合物， 这些复合物影响P450功能。作为一个额外的复杂性，P450系统蛋白存在于一个细胞中。 本质上是异质性的膜，其中膜的特性可以影响蛋白质 organization.因此，P450系统不需要简单地被认为是一系列单体聚合物。 蛋白质与其氧化还原伙伴相互作用，但作为超分子复合物的组分， 由它的膜环境决定。这项建议的目的是更好地了解蛋白质的 P450单加氧酶系统在内质网中的作用及P450-P450相互作用对功能的影响 这些酶。这将通过实现以下具体目标来实现： 表征CPR和多个P450之间的物理复合物，并鉴定蛋白质区域 目的2 -鉴定P450系统蛋白在细胞中的位置， ER，ER异质性如何影响P450系统蛋白定位， 负责定位在特定的膜结构域。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-这将对外源性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.