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

P450 系统蛋白之间的相互作用及其在内质网微结构域中的分布

• 批准号: 9289536
• 负责人: WAYNE L BACKES
• 金额: $ 39.24万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9289536
• 关键词: Address; Affect; Affinity; Binding; Biochemical; Biological; Bioluminescence; CYP1A2 gene; CYP2B4 gene; CYP2E1 gene; Characteristics; Chemical Exposure; Chemicals; Chimera organism; Chimeric Proteins; Cholesterol; Complex; Complication; Crowding; Cytochrome P450; Cytochromes b5; DNA; Disease; Electron Microscopy; Electron Transport; Electrons; Endoplasmic Reticulum; Energy Transfer; Environment; Enzymes; Event; Generations; Goals; Grant; Heterogeneity; Imagery; Knowledge; Lipids; Measurement; Measures; Mediating; Membrane; Metabolic; Metabolism; Microsomes; Mixed Function Oxygenases; Monitor; Mutagenesis; NADP; NADPH-Ferrihemoprotein Reductase; Nature; Oxidation-Reduction; Phospholipids; Physiological; Protein Region; Proteins; RNA; Reaction; Role; Series; Site-Directed Mutagenesis; Source; Sphingomyelins; Structure; System; Testing; Toxic effect; Transfection; Water; Xenobiotic Metabolism; Xenobiotics; carcinogenesis; crosslink; enzyme activity; response; segregation

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.

抽象的 P450 系统包括多种参与消除多种外来化合物的酶 来自身体。 P450系统的目的是催化氧化反应，通常产生 更易溶于水且更容易排泄的产品。然而，在某些情况下，产品会发生反应 – 能够与 DNA、RNA 或关键蛋白质结合 – 并导致突变、致癌等 毒性。参与异生物质代谢的 P450 是电子传递链的末端成分 在内质网 (ER) 中发现 – P450 不能单独发挥作用，而是必须接收来自内质网的两个电子 它们的氧化还原伙伴是 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 心肺复苏。尽管该比率根据诱导状态而变化，但 P450 水平比 CPR 水平高出 7:1 至 20:1 的比例。由于 CPR 和 P450 形成 1:1 功能复合物，CPR 必须能够提供 电子到许多不同的 P450。如果特定的 P450 具有更高的关联亲和力 CPR，那么电子会优先流向那些 P450。因此，P450 的竞争能力较差 限制心肺复苏必须有允许它们接收电子的机制；否则他们就会 新陈代谢沉默。这些情况引发了有关 P450 系统组件如何工作的问题 组织在膜中。 P450 系统蛋白拥挤在 ER 中，提供了许多机会 相互影响。现在已确定 P450 酶可形成同聚复合物和异聚复合物，并且 这些复合物影响 P450 功能。作为一个额外的并发症，P450 系统蛋白驻留在 膜本质上是异质的，膜的特性会影响蛋白质 组织。因此，P450系统不能简单地被视为一系列单体 蛋白质与其氧化还原伙伴相互作用，但作为受影响的超分子复合物的组成部分 由其膜环境决定。该提案的目的是更好地了解蛋白质如何 P450单加氧酶系统在ER中组织以及P450-P450相互作用对其功能的作用 这些酶。这将通过解决以下具体目标来实现： 目标 1 – 表征 CPR 和多个 P450 之间的物理复合物并识别蛋白质区域 负责P450·P450复合物的形成；目标 2 – 确定 P450 系统蛋白的驻留位置 ER、ER 异质性如何影响 P450 系统蛋白定位以及哪些蛋白区域 负责特定膜域的定位。这些研究将加深我们对 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——这将对外源物质有毒代谢物的产生产生重大影响。